Structure creates Function

Artefii incipit liber qui clavis maioris sapientiae dicitur.

2011-11-01T04:57:00.000-07:00

capitulum secundum, de generatione mineralium

dicamus ergo de generatione mineralium: dixerunt autem quidam quod natura mineralium omnium est argentu vivum cum sulphure, & dixerunt quod ex quo sive radix ipsorum mineralium est argentum vivum cum sulphure.

2010-08-25T16:30:00.000-07:00

http://docsouth.unc.edu/imls/lecontesalt/leconte.html

"
Gunpowder is a mixture of saltpetre (potassium nitrate), charcoal and sulphur. The most critical ingredient is the saltpetre, says Smith, not least because it is by far the largest single constituent. By 1801, saltpetre was being supplied from natural nitrate deposits in India and south-east Asia. But from the mid-14th century until the 1620s — at least 15 years after the plot to blow up parliament — saltpetre was still made in the traditional medieval way, from manure.
"
Blast from the past. By: Hamer, Mick, New Scientist, 02624079, 11/5/2005, Vol. 188, Issue 2524

मक-Fold

2010-08-05T21:01:00.001-07:00

http://www.major.iric.ca/MC-Fold/

The Shocking Truth About Running Shoes

2010-01-31T18:03:00.000-08:00

The Shocking Truth About Running Shoes

By Gisela Telis
ScienceNOW Daily News
http://sciencenow.sciencemag.org
27 January 2010

Haile Gebrselassie, the world's fastest marathoner, once said of his early career, "When I wore shoes, it was difficult." A new study reveals why: Humans run differently in bare feet. Researchers have discovered that sneakers and other sports shoes alter our natural gait, which normally protects us from the impact of running. The finding offers new insight on how early humans ran and raises concerns that sports shoes may promote more injuries than they prevent.

About 2 million years ago, the ancestors of modern humans evolved the physiological "equipment" for running--long legs, large buttocks, and springy structures in the feet, among other features. Athletic shoes weren't invented until the early 1900s, and it wasn't until the 1970s that they found widespread popularity. So how did humans manage to run comfortably before the invention of purpose-built footwear?

Daniel Lieberman, a human evolutionary biologist at Harvard University--and an avid runner--decided to find out. He and colleagues looked at more than 200 shod and unshod runners in the United States and the Rift Valley Province of Kenya, which is known for its great endurance runners. The volunteers represented a spectrum of shoe experience, including adults who had grown up wearing shoes, those who had grown up running shoeless but who now wore shoes, and those who had never worn shoes at all. Lieberman's team arranged a trial in which each group ran shod (either in ASICS GEL-Cumulus 10s or in their own shoes) and bare and measured their running gait and the impact on their bodies.

The researchers noticed a difference right away. Whereas shod runners tended to land on the heel of the foot, barefoot runners landed on the ball of the foot or with a flat foot. The unshod runners' style causes more flex in the foot's springlike arch, ankle, and knee and engages more foot and calf muscles, blunting the impact on the body and making for a more comfortable "ride." As their feet collide with the ground--in this case, a running track--barefoot runners experience a shock of only 0.5 to 0.7 times their body weight, whereas shod heel strikers experience 1.5 to two times their body weight--a threefold to fourfold difference.

"I always assumed it was painful and crazy to run barefoot," says a surprised Lieberman. Instead, the findings--published tomorrow in Nature--suggest that going barefoot can reduce the likelihood of pain and damage, because many running injuries, like shin splints and plantar fasciitis, are stress- and impact-induced.

"This is an excellent study," says Dennis Bramble, an evolutionary morphologist at the University of Utah in Salt Lake City. "Heel strikes don't allow you to use these really nifty springs that are unique to human beings, so we're being less efficient than we could be," he says. "It confirms what we should have known all along: We're built to run barefoot."

That confirmation will stoke an ongoing debate. As a glance at this month's Runner's World magazine and a recent book on shoeless running called Born to Run attest, barefoot running has gained a small but devoted following in the past decade, prompting controversy in the running community over whether it is best to run shod or unshod.

So should sporty types shed their shoes and jump on the barefooted bandwagon? "Not at all," says Lieberman. "Shoes are comfortable, and they protect the foot" from glass, asphalt, and other harsh realities of urban running, he notes. Instead, Lieberman (who has since taken up occasional barefoot running himself) recommends a gradual transition for the bare-curious, one that allows the feet and calves to strengthen slowly and avoid injury.

नोट्स ओं senescence

2009-12-17T14:29:00.000-08:00

Cell death and organ development in plants

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Author(s): Rogers HJ
Source: CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 71 Book Series: CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY Volume: 71 Pages: 225-+ Published: 2005
Times Cited: 19 References: 167 Citation MapCitation Map
Abstract: Programmed cell death (PCD) is an important feature of plant development; however, the mechanisms responsible for its regulation in plants are far less well understood than those operating in animals. In this review data from a wide variety of plant PCD systems is analyzed to compare what is known about the underlying mechanisms. Although senescence is clearly an important part of plant development, only what is known about PCD during senescence is dealt with here. In each PCD system the extracellular and intracellular signals triggering PCD are considered and both cytological and molecular data are discussed to determine whether a unique model for plant PCD can be derived. In the majority of cases reviewed, PCD is accompanied by the formation of a large vacuole, which ruptures to release hydrolytic enzymes that degrade the cell contents, although this model is clearly not universal. DNA degradation and the activation of proteases is also common to most plant PCD systems, where they have been studied; however, breakdown of DNA into nucleosomal units (DNA laddering) is not observed in all systems. Caspase-like activity has also been reported in several systems, but the extent to which it is a necessary feature of all plant PCD has not yet been established. The trigger for tonoplast rupture is not fully understood, although active oxygen species (AOS) have been implicated in several systems. In. two systems, self incompatibility and tapetal breakdown as a result of cytoplasmic male sterility, there is convincing evidence for the involvement of mitochondria including release of cytochrome c. However, in other systems, the role of the mitochondrion is not clear-cut. How cells surrounding the cell undergoing PCD protect themselves against death is also discussed as well as whether there is a link between the eventual fate of the cell corpse and the mechanism of its death. (c) 2005, Elsevier Inc.
Document Type: Review
Language: English
KeyWords Plus: TRACHEARY-ELEMENT DIFFERENTIATION; CASTOR BEAN ENDOSPERM; SENESCENCE-ASSOCIATED GENES; CYTOPLASMIC MALE-STERILITY; CYSTEINE PROTEASE GENE; WHEAT ALEURONE CELLS; ZEA-MAYS L.; LEAF SENESCENCE; AERENCHYMA FORMATION; BARLEY ALEURONE
Reprint Address: Rogers, HJ (reprint author), Cardiff Univ, Sch Biosci, Cardiff CF10 3TL, Wales
Addresses:
1. Cardiff Univ, Sch Biosci, Cardiff CF10 3TL, Wales
Publisher: ELSEVIER ACADEMIC PRESS INC, 525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495 USA
Subject Category: Developmental Biology
IDS Number: BDI97
ISSN: 0070-2153
DOI: 10.1016/S0070-2153(05)71007-3

The molecular and genetic control of leaf senescence and longevity in Arabidopsis

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Author(s): Lim PO, Nam HG
Source: CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 67 Book Series: CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY Volume: 67 Pages: 49-83 Published: 2005
Times Cited: 18 References: 111 Citation MapCitation Map
Abstract: The life of a leaf initiated from a leaf primordium ends with senescence, the final step of leaf development. Leaf senescence is a developmentally programmed degeneration process that is controlled by multiple developmental and environmental signals. It is a highly regulated and complex process that involves orderly, sequential changes in cellular physiology, biochemistry, and gene expression. Elucidating molecular mechanisms underlying such a complex, yet delicate process of leaf senescence is a challenging and important biological task. For the past decade, impressive progress has been achieved on the molecular processes of leaf senescence through identification of genes that show enhanced expression during senescence. In addition, Arabidopsis has been established as a model plant for genetic analysis of leaf senescence. The progress on the characterization of genetic mutants of leaf senescence in Arabidopsis has firmly shown that leaf senescence is a genetically controlled developmental phenomenon involving numerous regulatory elements. Especially, employment of global expression analysis as well as genomic resources in Arabidopsis has been very fruitful in revealing the molecular genetic nature and mechanisms underlying leaf senescence. This progress, including molecular characterization of some of the genetic regulatory elements, are revealing that senescence is composed of a complex regulatory network. In this review, we will present current understanding of the molecular genetic mechanisms by which leaf senescence is regulated and processed, focusing mostly on the regulatory factors of senescence in Arabidopsis. We also present a potential biotechnological implication of leaf senescence studies on the improvement of important agronomic traits such as crop yield and post-harvest shelf life. We further provide future research prospects to better understand the complex regulatory network of senescence. (c) 2005, Elsevier Inc.
Document Type: Review
Language: English
KeyWords Plus: END RULE PATHWAY; CAENORHABDITIS-ELEGANS; PROTEIN-DEGRADATION; PLANT SENESCENCE; PATHOGEN-DEFENSE; PHOSPHOLIPASE-D; MESSENGER-RNAS; TOMATO PLANTS; JASMONIC ACID; LARGE SUBUNIT
Reprint Address: Lim, PO (reprint author), Cheju Natl Univ, Dept Sci Educ, Cheju 690756, South Korea
Addresses:
1. Pohang Univ Sci & Technol, Natl Res Lab Plant Mol Genet, Div Mol & Life Sci, Pohang 790784, Kyungbuk South Korea
Publisher: ELSEVIER ACADEMIC PRESS INC, 525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495 USA
Subject Category: Developmental Biology
IDS Number: BCM26
ISSN: 0070-2153
DOI: 10.1016/S0070-2153(04)67002-5

The molecular analysis of leaf senescence - a genomics approach

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Author(s): Buchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S, Page T, Pink D
Source: PLANT BIOTECHNOLOGY JOURNAL Volume: 1 Issue: 1 Pages: 3-22 Published: JAN 2003
Times Cited: 155 References: 140 Citation MapCitation Map
Abstract: Senescence in green plants is a complex and highly regulated process that occurs as part of plant development or can be prematurely induced by stress. In the last decade, the main focus of research has been on the identification of senescence mutants, as well as on genes that show enhanced expression during senescence. Analysis of these is beginning to expand our understanding of the processes by which senescence functions. Recent rapid advances in genomics resources, especially for the model plant species Arabidopsis, are providing scientists with a dazzling array of tools for the identification and functional analysis of the genes and pathways involved in senescence. In this review, we present the current understanding of the mechanisms by which plants control senescence and the processes that are involved.
Document Type: Review
Language: English
Author Keywords: Arabidopsis; cell death; post-harvest; senescence; signalling pathways; stress
KeyWords Plus: PROGRAMMED CELL-DEATH; CYTOSOLIC GLUTAMINE-SYNTHETASE; PATHOGENESIS-RELATED PROTEINS; SENESCING ARABIDOPSIS LEAVES; ATP-DEPENDENT PROTEASE; DEFENSE-RELATED GENES; STAY-GREEN; DIFFERENTIAL EXPRESSION; FESTUCA-PRATENSIS; PLANT SENESCENCE
Reprint Address: Buchanan-Wollaston, V (reprint author), Hort Res Int, Prod Qual Team, Wellesbourne CV35 9EF, Warwick England
Addresses:
1. Hort Res Int, Prod Qual Team, Wellesbourne CV35 9EF, Warwick England
E-mail Addresses: vicky.b-wollaston@hri.ac.uk
Publisher: BLACKWELL PUBLISHING LTD, 9600 GARSINGTON RD, OXFORD OX4 2DG, OXON, ENGLAND
Subject Category: Biotechnology & Applied Microbiology; Plant Sciences
IDS Number: 774QE
ISSN: 1467-7644

F-box proteins in flowering plants

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Author(s): Wang HY, Huang J, Lai Z, Xue YB
Source: CHINESE SCIENCE BULLETIN Volume: 47 Issue: 18 Pages: 1497-1501 Published: SEP 2002
Times Cited: 1 References: 45 Citation MapCitation Map
Abstract: In eukaryotes, the ubiquitin-mediated protein degradation pathway has been shown to control several key biological processes such as cell division, development, metabolism and immune response. F-box proteins, as a part of SCF (Skp1-Cullin (or Cdc53)-F-box) complex, functioned by interacting with substrate proteins, leading to their subsequent degradation by the 26S proteasome. To date, several F-box proteins identified in Arabidopsis and Antirrhinum have been shown to play important roles in auxin signal transduction, floral organ formation, flowering and leaf senescence. Arabidopsis genome sequence analysis revealed that it encodes over 1000 predicted F-box proteins accounting for about 5% of total predicted proteins. These results indicate that the ubiquitin-mediated protein degradation involving the F-box proteins is an important mechanism controlling plant gene expression. Here, we review the known F-box proteins and their functions in flowering plants.
Document Type: Review
Language: English
Author Keywords: SCF complex; F-box protein; proteolysis; auxin signal transduction
KeyWords Plus: UBIQUITIN-DEPENDENT PROTEOLYSIS; AUXIN RESPONSE; CELL-CYCLE; SACCHAROMYCES-CEREVISIAE; ARABIDOPSIS-THALIANA; AUX/IAA PROTEINS; COP9 SIGNALOSOME; LIGASE COMPLEX; GENE; DEGRADATION
Reprint Address: Xue, YB (reprint author), Chinese Acad Sci, Inst Genet & Dev Biol, Beijing 100080, Peoples R China
Addresses:
1. Chinese Acad Sci, Inst Genet & Dev Biol, Beijing 100080, Peoples R China
E-mail Addresses: ybxue@genetics.ac.cn
Publisher: SCIENCE CHINA PRESS, 16 DONGHUANGCHENGGEN NORTH ST, BEIJING 100717, PEOPLES R CHINA
Subject Category: Multidisciplinary Sciences
IDS Number: 595TD
ISSN: 1001-6538

Current molecular understanding of the genetically programmed process of leaf senescence

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Author(s): Chandlee JM
Source: PHYSIOLOGIA PLANTARUM Volume: 113 Issue: 1 Pages: 1-8 Published: SEP 2001
Times Cited: 33 References: 47 Citation MapCitation Map
Abstract: Natural leaf senescence proceeds through an orderly program of events referred to, generally, as the 'senescence syndrome'. Leaf senescence consists of primarily, but not exclusively, a set of degradative and remobilization activities that salvage valuable nutrients by reallocation to the seeds or other viable parts of the plant. The program requires changes in gene expression and eventually culminates in death of the leaf or whole plant. Leaf/whole plant senescence has now been scrutinized extensively using molecular genetic approaches and a clearer picture of the events that comprise the developmental program is beginning to emerge. However, while understandings of the phenomenological aspects of the program have become apparent, the mechanistic aspects, particularly with regard to the processes required for induction and regulation of the program, are still far from clear. Molecular evidence suggests the process is complex in terms of the wide array of genes and activities expressed, and in terms of the overall regulation of progression of the events of the syndrome. This article attempts to review our current understanding of leaf senescence and includes a brief discussion of aspects of the process that require clarification if we are to more fully understand this complex developmental program.
Document Type: Review
Language: English
KeyWords Plus: GLUTAMINE-SYNTHETASE GENES; ARABIDOPSIS-THALIANA; BRASSICA-NAPUS; DIFFERENTIAL EXPRESSION; STAY-GREEN; TRANSGENIC PLANTS; TOMATO PLANTS; IDENTIFICATION; ETHYLENE; PROTEINS
Reprint Address: Chandlee, JM (reprint author), Univ Rhode Isl, Dept BIochem Microbiol & Mol Genet, Morrill Hall, Kingston, RI 02881 USA
Addresses:
1. Univ Rhode Isl, Dept BIochem Microbiol & Mol Genet, Kingston, RI 02881 USA
Publisher: MUNKSGAARD INT PUBL LTD, 35 NORRE SOGADE, PO BOX 2148, DK-1016 COPENHAGEN, DENMARK
Subject Category: Plant Sciences
IDS Number: 467TZ
ISSN: 0031-9317

Plant programmed cell death: A common way to die

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Author(s): Danon A, Delorme V, Mailhac N, Gallois P
Source: PLANT PHYSIOLOGY AND BIOCHEMISTRY Volume: 38 Issue: 9 Pages: 647-655 Published: SEP 2000
Times Cited: 91 References: 62 Citation MapCitation Map
Abstract: In the last few years programmed cell death in plants inspired many studies in development and environmental stresses. Some of these studies showed that hallmarks of animal programmed cell death were found at cellular or molecular level in plant cells in different experimental systems. Additionally the effect of over-expression in plants of animal genes implicated in programmed cell death has been tested, and some plant homologues of these genes have been found. This suggests that, despite some differences, plants and animals could share at least some common components of a core mechanism used to carry out programmed cell death in eukaryotes. In this review, we will concentrate on the last findings that suggest similarity between plant programmed cell death and its better known counterpart in animals. (C) 2000 Editions scientifiques et medicales Elsevier SAS.
Document Type: Review
Language: English
Author Keywords: apoptosis; caspases; DNA ladder; necrosis; plants; programmed cell death; TUNEL
KeyWords Plus: POLY(ADP-RIBOSE) POLYMERASE; HYPERSENSITIVE RESPONSE; ARABIDOPSIS-THALIANA; BARLEY ALEURONE; CYTOCHROME-C; APOPTOSIS; DNA; SENESCENCE; CASPASES; TOBACCO
Reprint Address: Gallois, P (reprint author), Univ Perpignan, Lab Genome & Dev Plantes, CNRS, UMR 5096, 52 Ave Villeneuve, F-66860 Perpignan, France
Addresses:
1. Univ Perpignan, Lab Genome & Dev Plantes, CNRS, UMR 5096, F-66860 Perpignan, France
Publisher: GAUTHIER-VILLARS/EDITIONS ELSEVIER, 23 RUE LINOIS, 75015 PARIS, FRANCE
Subject Category: Plant Sciences
IDS Number: 364CY
ISSN: 0981-9428

Programmed cell death during plant growth and development

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Author(s): Beers EP
Source: CELL DEATH AND DIFFERENTIATION Volume: 4 Issue: 8 Pages: 649-661 Published: DEC 1997
Times Cited: 79 References: 158 Citation MapCitation Map
Abstract: This review describes programmed cell death as it signifies the terminal differentiation of cells in anthers, xylem, the suspensor and senescing leaves and petals. Also described are cell suicide programs initiated by stress (e.g., hypoxia-induced aerenchyma formation) and those that depend on communication between neighboring cells, as observed for incompatible pollen tubes, the suspensor and synergids in some species. Although certain elements of apoptosis are detectable during some plant programmed cell death processes, the participation of autolytic and perhaps autophagic mechanisms of cell killing during aerenchyma formation, tracheary element differentiation, suspensor degeneration and senescence support the conclusion that nonapoptotic programmed cell death pathways are essential to normal plant growth and development. Heterophagic elimination of dead cells, a prominent feature of animal apoptosis, is not evident in plants. Rather autolysis and autophagy appear to govern the elimination of cells during plant cell suicide.
Document Type: Review
Language: English
Author Keywords: programmed cell death; plants; apoptosis; protease
KeyWords Plus: POLLEN-TUBE DEVELOPMENT; LEAF SENESCENCE; ARABIDOPSIS-THALIANA; SEX DETERMINATION; PEA-CHLOROPLASTS; PITH AUTOLYSIS; ZINNIA-ELEGANS; RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE; POLY(ADP-RIBOSE) POLYMERASE; CAENORHABDITIS-ELEGANS
Reprint Address: Beers, EP (reprint author), VIRGINIA POLYTECH INST & STATE UNIV, DEPT HORT, BLACKSBURG, VA 24061 USA
Publisher: STOCKTON PRESS, HOUNDMILLS, BASINGSTOKE, HAMPSHIRE, ENGLAND RG21 6XS
Subject Category: Biochemistry & Molecular Biology; Cell Biology
IDS Number: YJ306
ISSN: 1350-9047

Programmed senescence of plant organs

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Author(s): Hadfield KA, Bennett AB
Source: CELL DEATH AND DIFFERENTIATION Volume: 4 Issue: 8 Pages: 662-670 Published: DEC 1997
Times Cited: 36 References: 139 Citation MapCitation Map
Abstract: The senescence of plant organs associated with reproductive development has been studied extensively during the past century, and it has long been recognized that th is type of death is internally programmed. The regulation of organ senescence as well as its biochemical and genetic determinants has been an historically rich area of research. Certain plant hormones have been implicated as regulators or modulators of organ senescence and many of the biochemical pathways associated with the senescence syndrome have been elucidated. The genetic basis of organ senescence has also been well established by the identification of mutations that impair the senescence program and recently, transgenic plants have been used to critically determine the role of specific enzymes and hormonal signals in mediating programmed senescence of plant organs, Here, we review the current understanding of the processes that regulate leaf, flower and fruit senescence, emphasizing the rate that programmed organ senescence plays in the adaptive fitness of plants.
Document Type: Review
Language: English
Author Keywords: survivorship; fruit ripening; organ senescence
KeyWords Plus: ENCODING 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE; TOMATO FRUIT POLYGALACTURONASE; ETHYLENE-INDUCIBLE EXPRESSION; DELAYED LEAF SENESCENCE; CARNATION FLOWER PETALS; MESSENGER-RNA LEVELS; GENE-EXPRESSION; MOLECULAR-CLONING; PROTEIN-SYNTHESIS; AVOCADO FRUIT
Addresses:
1. UNIV CALIF DAVIS, DEPT VEGETABLE CROPS, MANN LAB, DAVIS, CA 95616 USA
Publisher: STOCKTON PRESS, HOUNDMILLS, BASINGSTOKE, HAMPSHIRE, ENGLAND RG21 6XS
Subject Category: Biochemistry & Molecular Biology; Cell Biology
IDS Number: YJ306
ISSN: 1350-9047

GENE-EXPRESSION DURING LEAF SENESCENCE

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Author(s): SMART CM
Source: NEW PHYTOLOGIST Volume: 126 Issue: 3 Pages: 419-448 Published: MAR 1994
Times Cited: 366 References: 297 Citation MapCitation Map
Abstract: Leaf senescence is a highly-controlled sequence of events comprising the final stage of development. Cells remain viable during the process and new gene expression is required. There is some similarity between senescence in plants and programmed cell death in animals. In this review, different classes of senescence-related genes are defined and progress towards isolating such genes is reported. A range of internal and external factors which appear to cause leaf senescence is considered and various models for the mechanism of senescence initiation are described. The current understanding of senescence at the organelle and molecular levels is presented. Finally, some ideas are mooted as to why senescence occurs and why it should be studied further.
Document Type: Review
Language: English
Author Keywords: LEAF SENESCENCE; PROGRAMMED CELL DEATH; GENES; PLANT GROWTH REGULATORS; ORGANELLES; MOLECULES
KeyWords Plus: NON-YELLOWING MUTANT; SENESCING BARLEY LEAVES; TRITICUM-AESTIVUM L; CYTOSOLIC GLUTAMINE-SYNTHETASE; REJUVENATED SOYBEAN COTYLEDONS; TRANSLATABLE MESSENGER-RNAS; PEPTIDE-HYDROLASE ACTIVITY; DARK-INDUCED SENESCENCE; FESTUCA-PRATENSIS HUDS; PROGRAMMED CELL-DEATH
Reprint Address: SMART, CM (reprint author), AFRC, INST GRASSLAND & ENVIRONM RES, DEPT CELL BIOL, PLAS GOGERDDAN, ABERYSTWYTH SY23 3EB, DYFED WALES
Publisher: CAMBRIDGE UNIV PRESS, 40 WEST 20TH STREET, NEW YORK, NY 10011-4211
Subject Category: Plant Sciences
IDS Number: NG798
ISSN: 0028-646X

Seborrhoeic dermatitis

2009-12-02T02:55:00.000-08:00

Collection of words for searching:
Seborrhoeic dermatitis
biotin, pyridoxine (vitamin B6) and riboflavin (vitamin B2)
yeast, Malassezia furfur

salinity tolerance in cereals

2009-11-26T02:54:00.003-08:00

Rajendran, K., Tester, M. & Roy, S.J. (2009) Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment 32, 237-249

from Naked Scientist:
http://www.thenakedscientists.com/HTML/content/interviews/interview/1239/

Chris - So how did you do that rather clever trick of making that gene only get turned on in those cells around these xylem vessels because that’s the breakthrough step, isn’t it really?

Mark - Yes, it is. There’s different ways you can do this. One is to try to discover little bits of DNA which will activate genes in specific parts of the plant. So that’s one way you can do it a very direct way. In the meantime, we’re using a model plant. It’s called Arabidopsis. It’s a silly little weed, but you can do lots of really nice molecular genetics with it. And what we did was throw into the genome of this little plant, this little weed, a little bit of DNA which allows us to turn on genes. But we threw it on in random in the genome, made thousands of these plants and then looked at the plants to find ones which had the right pattern of expression. So the initial generation of the plants was random and then we looked to find plants which had by fluke, this bit of DNA landing in a part of the genome that would activate that gene only in the inner half of the root.

Chris - Big question though, Mark must be a course, it’s one thing to do this in thale cress, the plant sciences fruit fly. But we don’t eat that. So what about things that we do eat? Could you put this same genetic combination into rice, into barley, wheat, and so on? The kinds of things we do rely on for food staples.

Mark - Absolutely, Chris. We have done this in rice and the results are looking very promising. We look like we’ve worked out how to reduce the sodium concentration. Well we have reduced the sodium concentration in the shoots of rice and we’re in the process of testing the effect of that on yield. The first experiments did improve yield, but we just want to again be fairly conservative rather than just shooting off the results rapidly. But it’s looking very promising. To turn the genes on in wheat and barley, maze, it’s actually quite difficult because these molecular genetic tricks that we’re able to use on Arabidopsis, we just simply can’t do it. We’re technically not able to do it in wheat and barley. So what we’re doing now is we’re having a program to discover the promoters that would help us turn this on, so going back to a very direct but slower way of manipulating an expression in wheat and barley. We actually have transgenic plants in the glasshouse at the moment for wheat and barley and we’re limited by bulking up the seed, and we’re just at that stage at the moment. So keep your fingers crossed for us and hopefully in the year’s time, we’ll be able to say if we’ve done it in the other crops as well.

salinity tolerance in cereals

2009-11-26T02:54:00.001-08:00

Rajendran, K., Tester, M. & Roy, S.J. (2009) Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment 32, 237-249

Fusarium ओक्स्य्स्पोरुम genome

2009-10-14T04:12:00.001-07:00

Genome information:
Name GenBank
Master WGS AAXH00000000
Chromosome 1 CM000589
Chromosome 2 CM000590
Chromosome 3 CM000591
Chromosome 4 CM000592
Chromosome 5 CM000593
Chromosome 6 CM000594
Chromosome 7 CM000595
Chromosome 8 CM000596
Chromosome 9 CM000597
Chromosome 10 CM000598
Chromosome 11 CM000599
Chromosome 12 CM000600
Chromosome 13 CM000601
Chromosome 14 CM000602
Chromosome 15 CM000603

Whole genome sequence fungi list

2009-10-14T04:04:00.000-07:00

Aspergillus clavatus
Aspergillus fumigatus
Aspergillus niger
Candida glabrata
Cryptococcus neoformans
Debaryomyces hansenii
Encephalitozoon cuniculi
Eremothecium gossypii
Gibberella zeae
Kluyveromyces lactis
Magnaporthe grisea
Neurospora crassa
Pichia stipitis
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Ustilago maydis
Yarrowia lipolytica

A MicroRNA Imparts Robustness against Environmental Fluctuation during Development

2009-10-06T16:45:00.001-07:00

Article
A MicroRNA Imparts Robustness against Environmental Fluctuation during Development

Xin Li1, 2, 3, Justin J. Cassidy1, 2, Catherine A. Reinke1, Stephen Fischboeck1 and Richard W. Carthew1, ,

1Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Drive, Northwestern University, Evanston, Illinois 60208, USA

Received 16 May 2008; revised 25 November 2008; accepted 29 January 2009. Published: April 16, 2009. Available online 16 April 2009.

Summary
The microRNA miR-7 is perfectly conserved from annelids to humans, and yet some of the genes that it regulates in Drosophila are not regulated in mammals. We have explored the role of lineage restricted targets, using Drosophila, in order to better understand the evolutionary significance of microRNA-target relationships. From studies of two well characterized developmental regulatory networks, we find that miR-7 functions in several interlocking feedback and feedforward loops, and propose that its role in these networks is to buffer them against perturbation. To directly demonstrate this function for miR-7, we subjected the networks to temperature fluctuation and found that miR-7 is essential for the maintenance of regulatory stability under conditions of environmental flux. We suggest that some conserved microRNAs like miR-7 may enter into novel genetic relationships to buffer developmental programs against variation and impart robustness to diverse regulatory networks.

Fusarium oxysporum

2009-10-06T06:45:00.000-07:00

Eukaryota;
Fungi;
Dikarya;
Ascomycota;
Pezizomycotina;
Sordariomycetes;
Hypocreomycetidae;
Hypocreales;
mitosporic Hypocreales;
Fusarium;
Fusarium oxysporum species complex;
Fusarium oxysporum

Structure creates Function: Network analysis of genes determining vascular wilt disease

2009-10-05T22:12:00.000-07:00

Structure creates Function: Network analysis of genes determining vascular wilt disease: "Lead Supervisor: Dr Louise Thatcher
Associates: Dr Kemal Kazan, Dr Donald Gardiner

Vascular wilt disease caused by the root infecting fungus Fusarium oxysporum affects over 100 plant species, including many economically important crops. This pathogen survives in soil for long periods and can be extremely difficult to eradicate once soils become infested.

High-throughput screening for altered Fusarium resistance on the model host Arabidopsis thaliana has identified many host mutants with increased susceptibility or resistance. This project aims to confirm the Fusarium disease phenotypes of a selected subset of mutants and develop hypotheses on the involvement of mutated genes in resistance or susceptibility mechanisms to Fusarium. This will involve screening second independent mutant lines in the genes of interest, confirming the mutations through PCR analysis, and the use of other pathogen assays, gene expression analysis and/or bioinformatic approaches to develop a network map of genes determining vascular wilt disease outcomes"

http://www.csiro.au/science/Summer-Studentships-Projects--ci_pageNo-2.html#6

Network analysis of genes determining vascular wilt disease

2009-10-05T22:06:00.000-07:00

Network analysis of genes determining vascular wilt disease.

Lead Supervisor: Dr Louise Thatcher
Associates: Dr Kemal Kazan, Dr Donald Gardiner

Vascular wilt disease caused by the root infecting fungus Fusarium oxysporum affects over 100 plant species, including many economically important crops. This pathogen survives in soil for long periods and can be extremely difficult to eradicate once soils become infested.

High-throughput screening for altered Fusarium resistance on the model host Arabidopsis thaliana has identified many host mutants with increased susceptibility or resistance. This project aims to confirm the Fusarium disease phenotypes of a selected subset of mutants and develop hypotheses on the involvement of mutated genes in resistance or susceptibility mechanisms to Fusarium. This will involve screening second independent mutant lines in the genes of interest, confirming the mutations through PCR analysis, and the use of other pathogen assays, gene expression analysis and/or bioinformatic approaches to develop a network map of genes determining vascular wilt disease outcomes

DNA repair proteins

2009-09-10T13:51:00.000-07:00

Redox signaling between DNA repair proteins for efficient lesion detection

Published online before print August 31, 2009, doi: 10.1073/pnas.0908059106
PNAS September 8, 2009 vol. 106 no. 36

1. Amie K. Boala,
2. Joseph C. Genereuxa,
3. Pamela A. Sontza,
4. Jeffrey A. Gralnickb,
5. Dianne K. Newmanc,1 and
6. Jacqueline K. Bartona,1

+ Author Affiliations

1.
aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125;
2.
bDepartment of Microbiology, BioTechnology Institute, University of Minnesota, St. Paul, MN 55108; and
3.
cDepartments of Biology and Earth, Atomospheric and Planetary Science, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139

1.

Contributed by Jacqueline K. Barton, July 21, 2009 (received for review June 25, 2009)

Abstract

Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.

sexual activity on cycle ergometer stress test parameters

2009-09-08T21:28:00.000-07:00

Effect of sexual activity on cycle ergometer stress test parameters, on plasmatic testosterone levels and on concentration capacity - A study in high-level male athletes performed in the laboratory

Author(s): Sztajzel J, Periat M, Marti V, Krall P, Rutishauser W
Source: JOURNAL OF SPORTS MEDICINE AND PHYSICAL FITNESS
Volume: 40
Issue: 3
Pages: 233-239
Published: SEP 2000

Abstract: Background. The purpose of this study was to investigate the effect of sexual activity on cycle ergometer stress test parameters, on plasmatic testosterone levels and on concentration capacity in high-level mate athletes.

Methods. Experimental design. Analysis of two days of testing accomplished in a laboratory setting, comparing a day with to a day without sexual activity (control day). Participants. Fifteen high-level male athletes, consisting of 8 team players, 5 endurance athletes and 2 weight-lifters, participated in the study. Measures. Each subject completed the following on each test day: two maximal graded stress tests on a cycle ergometer and a one-hour exercise stress test coupled to an arithmetic mental concentration test. Blood samples of testosterone were obtained and cardiac activity of each athlete was monitored with a 24-hour ECG tape recording over the two test days.

Results, Significantly higher differences were achieved for posteffort heart rate (HR) values at 5 minutes (p<0.01) and at 10 minutes (p<0.01) during the recovery phase of the morning stress test 2 hours after sexual activity. These differences disappeared during the recovery phase of the afternoon stress test performed approximately 10 hours after sexual intercourse took place.

Conclusions, Our findings show that sexual activity had no detrimental influence on the maximal workload achieved and on the athletes' mental concentration. However, the higher posteffort HR values after the maximal stress test on the morning of sexual intercourse suggest that the recovery capacity of an athlete could be affected if he had sexual intercourse approximately 2 hours before a competition event.

Does Sex the Night Before Competition Decrease Performance?

2009-09-08T21:24:00.001-07:00

Does Sex the Night Before Competition Decrease Performance?

McGlone, Samantha; Shrier, Ian MD, PhD*†
Author Information
*Department of Physiology, McGill University, and †Centre for Clinical Epidemiology and Community Studies, Montreal, Quebec, Canada
Received June 7, 1999; accepted August 9, 2000.
Address correspondence and reprint requests to Ian Shrier, MD, Centre for Clinical Epidemiology and Community Studies, 3755 Cote Sainte Catherine Road, Montreal, Quebec H3T 1E2, Canada.

For many years, football coaches, Olympic athletes, and even Muhammad Ali have advocated sexual abstinence the night before an athletic event. 1 Marty Liquori, one the world's number one-ranked 5,000-meter runner believes that “Sex makes you happy, and happy people don't run a 3:47 mile.”2 Marv Levy, head coach of the Buffalo Bills, insisted that the team be separated from their wives before their appearance in four Super Bowls; a policy that apparently was not successful (four losses out of four Super Bowls). On the other hand, there are also plenty of anecdotal stories of athletes who claim to have benefited from sex the night before an event. Both U.S. track star David Wottle and Canadian downhill skier Karin Lee Gardner attribute their Olympic gold medals in part to their “pre-race preparation.”2 As legendary New York Yankees manager Casey Stengel put it, “It's not the sex that wrecks these guys, it's staying up all night looking for it.” Considering the controversy surrounding the topic, the objective of this editorial is to summarize the literature on whether sex the night before competition affects performance, and to suggest possible future areas for research.

The long-standing myth that athletes should practice abstinence before important competitions may stem from the theory that sexual frustration leads to increased aggression, and that the act of ejaculation draws testosterone from the body. 1 In actual fact, sex could alter performance through either physiological or psychological factors. To answer this question, we searched SportDiscus (1975–1988/1989, key words: Coitus and Sexual Intercourse) and MEDLINE for relevant articles. Of the 31 articles we retrieved, only 3 were scientific studies (all physiological). All of these studies suggested that sex the night before competition does not alter physiological testing results. For instance, 14 married male former athletes were given a maximum-effort grip strength test the morning after coitus, and the same test following at least 6 days of abstinence. 3 The results suggested that strength and endurance of the palmar flexing muscles are not adversely affected by sex the previous night. An unpublished follow-up to this study was conducted by researchers at Colorado State University on 10 fit, married men, ages 18–45 years (cited in ref. 4). In their tests for grip strength, balance, lateral movement, reaction time, aerobic power (stair-climbing exercise), and VO2max (treadmill test), the results did not change with sexual activity. Finally, the results from a 1995 randomized cross-over study suggested that sexual intercourse 12 hours prior to the test had no significant effects on maximal aerobic power, oxygen pulse, or double product. 5

Based on the results of these studies, one might conclude that sexual activity the night before competition would not affect performance. However, each of the above-mentioned studies focused on the physiological effects of precompetition sex, which would only be expected to decrease performance if the activity led to exhaustion. Considering that normal sexual intercourse between married partners expends only 25–50 calories (the energy equivalent of walking up two flights of stairs), 6 it is doubtful that sex the previous night would affect laboratory physiological performance tests.

Remembering that the original hypothesis suggested that performance would only be affected through a change in aggression, researchers really should have measured variables that are affected by aggression (e.g., motivation, alertness, and attitude toward competition). According to the current “inverted U” sport psychology hypothesis, 4 there is an optimal level of alertness/anxiety before a competition, and a poor performance will result from either being too anxious or not alert enough. If athletes are too anxious and restless the night before an event, then sex may be a relaxing distraction. If they are already relaxed or, like some athletes, have little interest in sex the night before a big competition, then a good night's sleep is all they need. This theory predicts that the results will be dependent on individual preferences and routines. The night before an important race is not a good time for drastic changes in routine. Consistency is the key.

Clearly there is a need for more research on the topic of sexual activity and athletic performance. However, any research will have difficulty controlling factors related to such sexual behavior such as the time of day, frequency and duration of sexual activity, behavior of subjects between data collection, diet, fatigue, stress, and individual response to sexual activity. Anshel also poses a question worth considering: “How valid are test results when a natural activity such as coitus becomes a required act occurring within a specific time period?”7 In addition, results may be dependent on the sexual partner. For instance, heart rate and blood pressure responses are different if sex is with a spouse of 10 years, compared to a new partner or in strange surroundings. 8 Therefore, any future research will have to control for interindividual variation of the above-mentioned variables with a randomized design, or at least control for differences at the analysis stage.

Finally, the inverted U hypothesis for alertness/anxiety suggests that the performance of some people will improve with sex the night before competition (i.e., responders) and the performance of others will be hindered (i.e., nonresponders). If true, a randomized controlled trial may not be able to detect any differences. For instance, if the “truth” is that 50% of the population improves with sex the night before a competition and 50% is hindered by sex the night before competition, a randomized controlled trial will show that on average there is no effect. Therefore, the best way to test the hypothesis is with a repeated-measures, cross-over design in which the same athletes are tested several times following abstinence, and several times following sex the night before competition. This would allow one to determine not only if sex the night before competition affects performance in certain individuals, but also if there are indeed “responders” and “nonresponders.”

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REFERENCES

1. Krieger L. Scoring before a big event. Winning 1997; 1:88–89. [Context Link]

2. Bloom M. The sex factor. Runner's World 1994; 11:71–74. [Context Link]

3. Johnson W. Muscular performance following coitus. J Sex Res 1968; 4:247–248. Library Holdings [Context Link]

4. Thornton J. Sexual activity and athletic performance: is there a relationship? Phys Sport Med 1990; 18:148–153. [Context Link]

5. Boone T, Gilmore S. Effects of sexual intercourse on maximal aerobic power, oxygen pulse, and double product in male sedentary subjects. J Sports Med Phys Fitness 1995; 35:214–217. Library Holdings [Context Link]

6. Mirkin G. Sex before competition. Report #6750. Mar. 10, 1996. http://drmirkin.com/archive/6750.html [Context Link]

7. Anshel M. Effects of sexual activity on athletic performance. Phys Sports Med 1981; 9:65–68. Library Holdings [Context Link]

8. Bohlen J, Held J, Sanderson M, et al. Heart rate, rate pressure point, and oxygen uptake during four sexual activities. Arch Intern Med 1984; 144:1745–1748. Bibliographic Links Library Holdings [Context Link]

The effect of losing virginity on academic performance

2009-09-08T21:13:00.000-07:00

Reading, writing, and sex: The effect of losing virginity on academic performance

View full text from the publisher Blackwell Science
Author(s): Sabia JJ (Sabia, Joseph J.)
Source: ECONOMIC INQUIRY
Volume: 45
Issue: 4
Pages: 647-670
Published: OCT 2007

Abstract: Controlling for a wide set of individual- and family-level observables available in the National Longitudinal Study of Adolescent Health, ordinary least squares (OLS) estimates show that sexually active adolescents have grade point averages that are approximately 0.2 points lower than virgins. However, when information on the timing of intercourse decisions is exploited and individual fixed effects are included, the negative effect of sexual intercourse disappears for females, but persists for males. Taken together, the results of this study suggest that while there may be adverse academic spillovers from engaging in intercourse for some adolescents, previous studies' estimates are overstated due to unmeasured heterogeneity.

DNA methylation analysis by प्य्रोसेक़ुएन्किन्ग

2009-09-02T03:40:00.000-07:00

Figure 1 - Enzymatic cascade of the pyrosequencing reaction in the example of a bisulfite-treated template sequence, including a CpG position that is methylated on approximately 50% of all molecules.
From the following article

DNA methylation analysis by pyrosequencing

Jörg Tost & Ivo G Gut

Nature Protocols 2, 2265 - 2275 (2007) Published online: 6 September 2007

doi:10.1038/nprot.2007.314

Importing Mitochondrial Proteins: Machineries and Mechanisms

2009-08-30T03:36:00.000-07:00

Review
Importing Mitochondrial Proteins: Machineries and Mechanisms

Agnieszka Chacinska1,2, Carla M. Koehler3, Dusanka Milenkovic1, 2, Trevor Lithgow4 and Nikolaus Pfanner1, 2,

1Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany

2Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany

3Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA

4Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia

Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis.

sliding β clamp subunit

2009-08-30T03:00:00.000-07:00

Preview
Clamping Down on Transposon Targeting

Mick Chandler

1Laboratoire de Microbiologie et Génétique Moléculaire UMR 5100, CNRS, 31062 Toulouse CEDEX, France

Cell, Volume 138, Issue 4, 21 August 2009, Pages 685-695,
Adam R. Parks, Zaoping Li, Qiaojuan Shi, Roisin M. Owens, Moonsoo M. Jin, Joseph E. Peters

The sliding β clamp subunit of the DNA replication machinery in the bacterium Escherichia coli coordinates multiple functions in the cell beyond genome duplication. In this issue, Parks et al. (2009) find that the β clamp interacts with the transposition protein TnsE to target the Tn7 transposon to discontinuously replicating DNA at the replication fork.

सेल Volume 138, Issue 2, 23 July 2009, Pages 271-285

2009-08-10T04:15:00.000-07:00

Article
CD47 Is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis

Cell, Volume 138, Issue 2, 23 July 2009, Pages 271-285

References and further reading may be available for this article. To view references and further reading you must purchase this article.

Siddhartha Jaiswal1, Corresponding Author Contact Information, E-mail The Corresponding Author, Catriona H.M. Jamieson2, Wendy W. Pang1, Christopher Y. Park1, Mark P. Chao1, Ravindra Majeti1, David Traver3, Nico van Rooijen4 and Irving L. Weissman1

Summary

Macrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

Ergot alkaloid biosynthesis in Aspergillus fumigatus

2009-06-03T21:41:00.000-07:00

Ergot alkaloid biosynthesis in Aspergillus fumigatus - Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase

Author(s): Rigbers O (Rigbers, Ole), Li SM (Li, Shu-Ming)1
Source: JOURNAL OF BIOLOGICAL CHEMISTRY
Volume: 283 Issue: 40 Pages: 26859-26868 Published: OCT 3 2008

Abstract: The putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60, and overexpressed in Escherichia coli. FgaMT comprises 339 amino acids with a molecular mass of about 38.1 kDa. The soluble dimeric His(6)-FgaMT was purified to near homogeneity and characterized biochemically. FgaMT was found to catalyze the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine, resulting in the formation of 4-dimethylallyl-L-abrine, which was identified by NMR and mass spectrometry analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity toward the prenyl moiety at position C-4 of the indole ring. 4-Dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. Km values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mM, respectively. The turnover number was 2.0 s(-1).

Determination of Ergot Alkaloids

2009-06-03T21:40:00.000-07:00

Determination of Ergot Alkaloids: Purity and Stability Assessment of Standards and Optimization of Extraction Conditions for Cereal Samples

Author(s): Krska R (Krska, Rudolf)1,2, Berthiller F (Berthiller, Franz)1, Schuhmacher R (Schuhmacher, Rainer)1, Nielsen KF (Nielsen, Kristian F.)3, Crews C (Crews, Colin)2

Source: JOURNAL OF AOAC INTERNATIONAL Volume: 91 Issue: 6 Pages: 1363-1371 Published: NOV-DEC 2008

Abstract: Results obtained from a purity study on standards of the 6 major ergot alkaloids ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine and their corresponding epimers are discussed. The 6 ergot alkaloids studied have been defined by the European Food Safety Authority as those that are the most common and physiologically active. The purity of the standards was investigated by means of liquid chromatography with diode array detection, electrospray ionization, and time-of-flight mass spectrometry (LC-DAD-ESI-TOF-MS). All of the standards assessed showed purity levels considerably above 98% apart from ergocristinine (94%), ergosine (96%), and ergosinine (95%). Also discussed is the optimization of extraction conditions presented in a recently published method for the quantitation of ergot alkaloids in food samples using solid-phase extraction with primary secondary amine (PSA) before LC/MS/MS. Based on the results obtained from these optimization studies, a mixture of acetonitrile with ammonium carbonate buffer was used as extraction solvent, as recoveries for all analyzed ergot alkaloids were significantly higher than those with the other solvents. Different sample-solvent ratios and extraction times showed just minor influences in extraction efficacy. Finally, the stability of the ergot alkaloids in both raw cereals and cereal-based processed food extracts was studied. According to these studies, extracts should be prepared and analyzed the same day or stored below ambient temperatures. Barley and rye extracts, which were stored at 4 and 15 degrees C after PSA cleanup, proved to be stable overnight. However, storage over a period of 14 days at 4 degrees C resulted in significant epimerization, which was most pronounced in rye and particularly for ergocornine, ergocryptine, and ergocristine.

The Venus of Hohle Fels.

2009-05-20T18:17:00.000-07:00

The Venus of Hohle Fels. (Credit: Photo by H. Jensen; Copyright: Universität Tübingen)

Good News for Night Owls

2009-05-06T06:21:00.001-07:00

Good News for Night Owls
By Elsa Youngsteadt
ScienceNOW Daily News
23 April 2009

Night owls seem to have a cognitive edge over early risers--at least when they're on their natural sleep schedule. That's one upshot of a new brain-imaging study that also gives surprising new insights into how the brain manages the urge to sleep and wake. The results, sleep researchers say, may improve predictions of when people are most at risk for drowsy accidents.
Two factors control our bedtime. The first is hardwired: A master clock in the brain regulates a so-called circadian rhythm, which synchronizes activity patterns to the 24-hour day. Some people's clocks tell them to go to bed at 9 p.m., others' at 3 a.m., (ScienceNOW, 24 June 2003). The second factor--called sleep pressure--depends not on time of day but simply on how long someone has been awake already.

Because sleep pressure accumulates during waking hours, logic suggests that we should be most alert--and hence sharpest--shortly after we get up versus right before we go to bed, regardless of whether we're night owls or larks.

But that's not what Christina Schmidt found. The doctoral student at the University of Liège in Belgium and her collaborators, led by sleep researcher Philippe Peigneux, recruited 16 morning people and 15 night people to take alertness tests in a brain scanner. Subjects had to pay attention to numbers on a computer screen and hit a button whenever the numbers began to change. To control for the effect of the circadian clock, the subjects were allowed to sleep on their own natural schedules and take the test 1.5 hours and 10.5 hours after waking, regardless of the actual time of day.

Both groups performed equally well on the test when they took it 1.5 hours after waking. But after 10.5 hours without sleep, the night owls pulled ahead. Their reaction times improved by about 6% relative to the morning people and to their own earlier performance, the researchers report in tomorrow's issue of Science. This suggests that once they wake up, sleep pressure builds up faster in early birds, says Peigneux, and that this hurts their cognition over time.

It's a result with "real-world consequences," says sleep researcher David Dinges of the University of Pennsylvania School of Medicine in Philadelphia. Current risk analyses use the time of day and hours worked to predict when people are in greatest danger of accidents--such as aviation errors. But now, Dinges says, they may need to take into account that morning people tend to lose their concentration faster. At the very least, according to sleep researcher Amita Sehgal, also at the University of Pennsylvania School of Medicine, this is a new and "intriguing" explanation for larks' and owls' different habits.

But the really provocative result, adds Dinges, came from the brain imaging. The night owls showed greater activity in the master-clock region of their brains--a cluster of cells known as the suprachiasmatic nucleus--than the larks when taking the later test. That suggests that sleep pressure and the circadian clock can influence each other directly--bringing together two systems that, for decades, had been thought to operate separately.

Homeostatic Sleep Pressure and Responses to Sustained Attention in the Suprachiasmatic Area

2009-05-06T06:13:00.001-07:00

Science 24 April 2009:
Vol. 324. no. 5926, pp. 516 - 519
DOI: 10.1126/science.1167337
Prev | Table of Contents | Next
Reports
Homeostatic Sleep Pressure and Responses to Sustained Attention in the Suprachiasmatic Area
Christina Schmidt,1,2,* Fabienne Collette,1,2 Yves Leclercq,1 Virginie Sterpenich,1 Gilles Vandewalle,1 Pierre Berthomier,3 Christian Berthomier,3 Christophe Phillips,1 Gilberte Tinguely,1 Annabelle Darsaud,1 Steffen Gais,1 Manuel Schabus,1 Martin Desseilles,1 Thien Thanh Dang-Vu,1 Eric Salmon,1 Evelyne Balteau,1 Christian Degueldre,1 André Luxen,1 Pierre Maquet,1 Christian Cajochen,4 Philippe Peigneux1,5,*

Throughout the day, cognitive performance is under the combined influence of circadian processes and homeostatic sleep pressure. Some people perform best in the morning, whereas others are more alert in the evening. These chronotypes provide a unique way to study the effects of sleep-wake regulation on the cerebral mechanisms supporting cognition. Using functional magnetic resonance imaging in extreme chronotypes, we found that maintaining attention in the evening was associated with higher activity in evening than morning chronotypes in a region of the locus coeruleus and in a suprachiasmatic area (SCA) including the circadian master clock. Activity in the SCA decreased with increasing homeostatic sleep pressure. This result shows the direct influence of the homeostatic and circadian interaction on the neural activity underpinning human behavior.

2 micron plasmid hitchhikes on mitotic mechanism of Saccharomyces cerevisiae to maintain equal distribution in host

2009-05-05T16:23:00.000-07:00

Published online April 13, 2009
doi:10.1083/jcb.200810130
The Journal of Cell Biology, Vol. 185, No. 2, 251-264
The Rockefeller University Press, 0021-9525 $30.00
© 2009 Cui et al.

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

Hong Cui, Santanu K. Ghosh, and Makkuni Jayaram
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712

Correspondence to Makkuni Jayaram: jayaram@icmb.utexas.edu

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a "partitioning center" represents yet another facet of the benign parasitism of the yeast plasmid.

Biogeochemistry: Less nickel for more oxygen

2009-04-28T15:57:00.001-07:00

News and Views
Nature 458, 714-715 (9 April 2009) | doi:10.1038/458714a; Published online 8 April 2009

Biogeochemistry: Less nickel for more oxygen
Mak A. Saito1

Top of pageAbstractThe availability (or lack) of oceanic trace elements is providing fresh lines of explanation for turning points in Earth's history — the Great Oxidation Event being one such momentous occasion.

About 2.4 billion years ago, the oxygen content of Earth's atmosphere increased in what is called the Great Oxidation Event (GOE). This marked the beginning of the most significant series of chemical changes Earth has ever experienced, setting the stage for oxidative weathering of the continents, successive changes in ocean chemistry, and the eventual rise of multicellular life.

Yet the sequence of events leading up to the GOE is not well understood. Most researchers agree that the evolution of oxygenic photosynthesis within a group called the cyanobacteria was the source of the molecular oxygen that caused the GOE1. But the timing of the rise of these bacteria is uncertain2, 3, and there may have been a period of inertia — due, for example, to chemical reactions with methane that consumed oxygen4 — that prevented a swift increase in atmospheric oxygen. It remains a matter of debate how these two phenomena might have induced the GOE: an early rise of cyanobacteria and slow crumbling of chemical resistance3, 4; or a late rise of cyanobacteria leading to rapid initiation of the GOE5.

On page 750 of this issue6, Konhauser et al. report evidence for an alternative driving mechanism of the GOE, one that would have decreased microbial methane production in the oceans and paved the way for increased oxygen abundances. The authors find significant decreases in the nickel-to-iron ratios in ancient rocks, known as banded iron formations, that provide records of element concentrations in the oceans (Fig. 1). They estimate that a major decrease in the oceanic inventory of nickel must have occurred around 2.7 billion years ago. This, they conclude, led to a cascade of events in which methanogens, with their gluttonous appetite for nickel to feed three nickel-containing metalloenzymes, would have become starved of the element and so have produced much less methane. With the decrease in chemical inertia associated with methane4, the stage was set for cyanobacterial oxygen to accumulate, leading to the GOE. Moreover, although Konhauser et al. don't go into detail, the decline in atmospheric methane, a powerful greenhouse gas, is believed to help account for the initiation of a planetary-scale glaciation known as Snowball Earth that is thought to have begun between 2.3 billion and 2.2 billion years ago4, 5.

Figure 1: Record site.
This is a view of Dales Gorge, northwest Australia, one of the banded iron formations sampled by Konhauser et al.6.

High resolution image and legend (151K)

The idea that significant changes in seawater trace-metal abundance have occurred during Earth's history is becoming popular7, 8. For example, it is thought that iron and cobalt were abundant in ancient oceans, whereas zinc and copper were probably extremely scarce owing to precipitation with sulphides8. When the oceans became oxygenated, it is likely that this scheme was reversed, with iron and cobalt becoming scarce through oxidation and precipitation as oxyhydroxides, and zinc and copper becoming much more abundant upon the oxidation of sulphide to sulphate in sea water. These predictions of broad changes in ocean chemistry are mirrored by the physiological and genomic traits of archaea and bacteria, relative to those of the later-evolving eukaryotes8, 9.

Nickel has largely been left out of this intriguing story. On the evidence of chemical modelling8, it seems that nickel was not as strongly affected by the variations in sulphide and oxygen during Earth's history. But such a conclusion does not take into account the possible involvement of external factors. Konhauser et al. show how such a factor might have come into play, with the cooling of Earth's mantle resulting in decreased eruption of nickel-rich rocks and causing an estimated 50% fall in the oceanic nickel inventory.

Konhauser and colleagues' thinking6 may come as a surprise to those familiar with the chemistry of the modern oceans. Trace metals — as their name suggests — are extraordinarily scarce in sea water. In vast regions of the modern oceans, photosynthesis is limited by low iron availability, with iron concentrations often being less than 0.05 nanomoles per litre10. Yet, of the trace metals required by life, nickel is one of the more abundant in sea water, with surface water concentrations of 1–2 nanomoles per litre11. In this modern context, the idea of a nickel famine seems odd. But the nickel requirements of methanogens are reported6 to be in the hundreds of nanomoles per litre, suggesting that methanogens cannot live in the modern oceans and are perhaps relegated to sedimentary, coastal and freshwater environments, where nickel is more abundant.

By connecting changes in mantle temperature to nickel fluxes and methanogens, Konhauser and colleagues' study is particularly satisfying. Instead of relying on the uncertain timing of the rise of cyanobacteria to explain the GOE, that event can instead be tied to a specific mechanism recorded in the banded iron formations. In addition, this 'nickel famine' mechanism is consistent with evidence12 of 'whiffs of oxygen' that occurred more than 50 million years before the GOE. But I cannot help but wonder whether there is a reason — such as the slow chemical kinetics of nickel ions — why methanogens could not evolve a high-affinity nickel-uptake mechanism similar to those that exist for the uptake of iron, zinc and cobalt13, 14, 15.

Finally, there is another context in which the research of Konhauser et al. is set — the exciting endeavour of trying to understand how the elemental cycles (of nickel, carbon, iron, nitrogen and so on) have 'co-evolved' with microbial life. Many of the changes in element cycling were probably caused by the rise and fall of specific microbial metabolisms, while also strongly affecting the trajectory and composition of life on Earth. Life and the cycling of elements have both been changing throughout Earth's history, often influencing each other profoundly along the way. One of the sobering realizations of studies such as this is that, although natural selection provides a clear, single positive-feedback mechanism for the continuation of life, elemental cycles are instead influenced by an aggregate of mechanisms, including biological evolution, chemical reactions, changes in ocean circulation and geological events. If, as Konhauser et al. suggest, a single geological change can starve a major oceanic microbial community, and thereby change the trajectory of life on Earth, it suggests that there is a fragility to Earth's elemental cycles that we are only beginning to uncover.

Kinetochore geometry defined by cohesion within the centromere

2009-04-28T15:50:00.001-07:00

Article
Nature 458, 852-858 (16 April 2009) | doi:10.1038/nature07876; Received 9 September 2008; Accepted 12 February 2009

Kinetochore geometry defined by cohesion within the centromere
Takeshi Sakuno1,2, Kenji Tada1,3 & Yoshinori Watanabe1,3

Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences,
Promotion of Independence for Young Investigators,
Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Yayoi, Tokyo 113-0032, Japan
Correspondence to: Yoshinori Watanabe1,3 Correspondence and requests for materials should be addressed to Y.W. (Email: ywatanab@iam.u-tokyo.ac.jp).

Top of pageAbstractDuring cell division microtubules capture chromosomes by binding to the kinetochore assembled in the centromeric region of chromosomes. In mitosis sister chromatids are captured by microtubules emanating from both spindle poles, a process called bipolar attachment, whereas in meiosis I sisters are attached to microtubules originating from one spindle pole, called monopolar attachment. For determining chromosome orientation, kinetochore geometry or structure might be an important target of regulation. However, the molecular basis of this regulation has remained elusive. Here we show the link between kinetochore orientation and cohesion within the centromere in fission yeast Schizosaccharomyces pombe by strategies developed to visualize the concealed cohesion within the centromere, and to introduce artificial tethers that can influence kinetochore geometry. Our data imply that cohesion at the core centromere induces the mono-orientation of kinetochores whereas cohesion at the peri-centromeric region promotes bi-orientation. Our study may reveal a general mechanism for the geometric regulation of kinetochores, which collaborates with previously defined tension-dependent reorientation machinery.

The dark side of light at night: physiological, epidemiological, and ecological consequences

2009-04-16T20:37:00.001-07:00

The dark side of light at night: physiological, epidemiological, and ecological consequences

Author(s): Navara KJ (Navara, Kristen J.), Nelson RJ (Nelson, Randy J.)
Source: JOURNAL OF PINEAL RESEARCH Volume: 43 Issue: 3 Pages: 215-224 Published: OCT 2007

Abstract: Organisms must adapt to the temporal characteristics of their surroundings to successfully survive and reproduce. Variation in the daily light cycle, for example, acts through endocrine and neurobiological mechanisms to control several downstream physiological and behavioral processes. Interruptions in normal circadian light cycles and the resulting disruption of normal melatonin rhythms cause widespread disruptive effects involving multiple body systems, the results of which can have serious medical consequences for individuals, as well as large-scale ecological implications for populations. With the invention of electrical lights about a century ago, the temporal organization of the environment has been drastically altered for many species, including humans. In addition to the incidental exposure to light at night through light pollution, humans also engage in increasing amounts of shift-work, resulting in repeated and often long-term circadian disruption. The increasing prevalence of exposure to light at night has significant social, ecological, behavioral, and health consequences that are only now becoming apparent. This review addresses the complicated web of potential behavioral and physiological consequences resulting from exposure to light at night, as well as the large-scale medical and ecological implications that may result.

Light pollution, reproductive function and cancer risk

2009-04-16T20:34:00.002-07:00

Light pollution, reproductive function and cancer risk

Author(s): Anisimov VN
Source: NEUROENDOCRINOLOGY LETTERS Volume: 27 Issue: 1-2 Pages: 35-52 Published: FEB-APR 2006

Abstract: At present, light pollution (exposure to light-at-night) both in the form of occupational exposure during night work and as a personal choice and life style, is experienced by numerous night-active members of our society. Disruption of the circadian rhythms induced by light pollution has been associated with cancer in humans. There are epidemiological evidences of increased breast and colon cancer risk in shift workers. An inhibition of the pineal gland function with exposure to the constant light (LL) regimen promoted carcinogenesis whereas the light deprivation inhibits the carcinogenesis. Treatment with pineal indole hormone melatonin inhibits carcinogenesis in pinealectomized rats or animals kept at the standard light/dark regimen (LD) or at the LL regimen. These observations might lead to use melatonin for cancer prevention in groups of humans at risk of light pollution.

Exposure to light-at-night increases the growth of DMBA-induced mammary adenocarcinomas in rats

2009-04-16T20:34:00.001-07:00

Exposure to light-at-night increases the growth of DMBA-induced mammary adenocarcinomas in rats

Author(s): Cos S, Mediavilla D, Martinez-Campa C, Gonzalez A, Alonso-Gonzalez C, Sanchez-Barcelo EJ
Source: CANCER LETTERS Volume: 235 Issue: 2 Pages: 266-271 Published: APR 28 2006

Abstract: In order to assess whether light exposure at night influences the growth of mammary tumors, as well as the role of melatonin in this process, female rats bearing DMBA-induced mammary adenocarcinomas were exposed to different lighting environments. Animals exposed to light-at-night, especially those under a constant dim light during the darkness phase, showed: (a) significantly higher rates of tumor growth as well as lower survival than controls, (b) higher concentration of serum estradiol, and (c) lower nocturnal excretion of 6-sulfatoxymelatonin, without there being differences between nocturnal and diurnal levels. These results suggest that circadian and endocrine disruption induced by light pollution, could induce the growth of mammary tumors. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Light pollution, melatonin suppression and cancer growth

2009-04-16T20:29:00.000-07:00

Light pollution, melatonin suppression and cancer growth

Author(s): Reiter RJ, Gultekin F, Manchester LC, Tan DX
Source: JOURNAL OF PINEAL RESEARCH Volume: 40 Issue: 4 Pages: 357-358 Published: MAY 2006

Article Text
The naturally occurring daily periods of light and darkness are an important source of internal order as manifested by the circadian rhythmicity exhibited by numerous organismal functions. Alterations of these daily fluctuations, referred to as chronodisruption [1], may be consequential in a number disorders, e.g. 'jet lag' [2], and diseases, e.g. cancer [3–5]. Given that the light:dark cycle is a central factor in circadian regulation, light during the normal dark period (light pollution), is becoming an increasing greater problem as societies become progressively more electrofied. Interruption of the daily dark period may have both subtle and not-so-subtle consequences in terms of human health.

One cycle that is quickly disturbed by light pollution is the pineal and blood melatonin rhythms. The repeated suppression of melatonin by light at night is a condition in which the health consequences may not be at all subtle. This is emphasized by the recent observations of Blask et al. [6] who, after a series of clever experiments, provided a highly plausible mechanism to explain how light suppression of melatonin at night accelerates the metabolic activity and growth of rat hepatoma and human MCF-7 cancer cells. The significance of these findings is reinforced by the earlier epidemiological studies in which the authors claimed that the repeated exposure of women to light at night, e.g. individuals who worked the night shift, had an increased incidence of breast [3,5,7] and colorectal cancer [8].

After transplantation of human MCF-7 cancer cells into nude rats, Blask et al. [6] showed that flooding the tumors with melatonin-deficient blood collected from premenopausal women during the day was inconsequential in terms of inhibiting cancer metabolism and growth. Conversely, melatonin-rich blood collected from the same women at night shut down the uptake and metabolism of the tumor growth promoter, linoleic acid, as well as the proliferation of the cancer cells. The most interesting aspect of the study, however, was that when the female blood donors were exposed to bright light at night to reduce endogenous melatonin levels, their blood was then incapable of inhibiting the cancer cells. Thus, light suppression of physiological melatonin levels negated the ability of the blood to limit tumor linoleic acid metabolism and cellular proliferation.

In the words of Blask, 'physiological nighttime levels of melatonin put tumors to sleep at night' while during the day when melatonin concentrations are normally low there are no limitations on their growth. Blask also is quoted as drawing the analogy that during light exposure at night tumors become 'insomniacs' [9].

There may be factors in addition to reducing linoleic acid uptake and metabolism that may be operative when melatonin, at physiological levels, limits growth of MCF-7 human cancer cells [10]. Leon-Blanco et al. [11] showed that melatonin also inhibits telomerase activity in MCF-7 cells. The activity of this enzyme is typically highly elevated in cancer cells [12] where it maintains the integrity and length of the telomeres of eukaryotic chromosomes. In noncancer cells, the telomeres gradually shorten over time making the chromosomes less stable and vulnerable to damage; this increases the propensity of normal cells to die. In cancer cells, the chromosomes remain durable because of the elevated telomerase activity so the cells are also more sturdy and resistant to death. When nude mice bearing MCF-7 tumors were treated with melatonin in their drinking water, not only was the growth of these tumors reduced but also telomerase activity as well as the levels of the mRNA of its catalytic submit, TERT, were also suppressed in the tumors. Moreover, like Blask et al. [6], Leon-Blanco et al. [11] reported that physiological concentrations of nighttime blood melatonin levels (about 1 nM) shut down telomerase activity of MCF-7 cancer cells. Thus, the ability of physiological levels of melatonin to 'put cancers to sleep at night' [9] extends not only to linoleic acid uptake and metabolism but to telomerase activity as well. Telomerase activity has been targeted as a site for cancer inhibition by the pharmaceutical industry [13].

These findings have additional important implications. Blask et al [6] noted that a 15–25% reduction in nocturnal melatonin levels is sufficient to promote tumor growth. This being the case, the age-related reduction in endogenous melatonin levels (which often exceeds 25%) [14] would be expected to increase the vulnerability of humans to cancer growth. As cancer is an age-related disease, the reduction of melatonin may be significant in mediating accelerated tumor growth in the elderly.

Finally, the amplitude of the nocturnal melatonin rhythm is determined by a variety of factors [15] and there are individuals who have a genetically attenuated nighttime melatonin rise. Given the high fidelity of the melatonin cycle over time, it is possible that these relatively melatonin-depressed humans are more susceptible to aggressive tumor metabolism and growth. These are considerations to ponder for future studies.

Lighting for the human circadian clock: recent research indicates that lighting has become a public health issue

2009-04-16T20:28:00.000-07:00

Author(s): Pauley SM
Source: MEDICAL HYPOTHESES Volume: 63 Issue: 4 Pages: 588-596 Published: 2004

Abstract: The hypothesis that the suppression of melatonin (MLT) by exposure to light at night (LAN) may be one reason for the higher rates of breast and colorectal cancers in the developed world deserves more attention. The literature supports raising this subject for awareness as a growing public health issue. Evidence now exists that indirectly links exposures to LAN to human breast and colorectal cancers in shift workers. The hypothesis begs an even larger question: has medical science overlooked the suppression of MLT by LAN as a contributor to the overall incidence of cancer?
The indirect linkage of breast cancer to LAN is further supported by laboratory rat experiments by David E. Blask and colleagues. Experiments involved the implanting of human MCF-7 breast cancer cell xenografts into the groins of rats and measurements were made of cancer cell growth rates, the uptake of linoleic acid (LA), and MLT levels. One group of implanted rats were placed in light-dark (12L:12D) and a second group in tight-light (12L:12L) environments. Constant light suppressed MLT, increased cancer cell growth rates, and increased LA uptake into cancer cells. The opposite was seen in the light-dark group. The proposed mechanism is the suppression of nocturnal MLT by exposure to LAN and subsequent tack of protection by MLT on cancer cell receptor sites which allows the uptake of LA which in turn enhances the growth of cancer cells.

MLT is a protective, oncostatic hormone and strong antioxidant having evolved in all plants and animals over the millennia. In vertebrates, MLT is normally produced by the pineal gland during the early morning hours of darkness, even in nocturnal animals, and is suppressed by exposure to LAN.

Daily entrainment of the human circadian clock is important for good human health. These studies suggest that the proper use and color of indoor and outdoor Lighting is important to the health of both humans and ecosystems. Lighting fixtures should be designed to minimize interference with normal circadian rhythms in plants and animals.

New discoveries on blue-light-sensitive retinal ganglion cell light receptors that control the circadian clock and how those receptors relate to today's modern high intensity discharge (HID) lamps are discussed. There is a brief discussion of circadian rhythms and light pollution. With the precautionary principle in mind, practical suggestions are offered for better indoor and outdoor lighting practices designed to safeguard human health. (C) 2004 Elsevier Ltd. All rights reserved.

hairpin-dependent pausing

2009-03-25T17:27:00.000-07:00

Possible effects of antisense oligonucleotides on hairpin-dependent pausing. (Top) The mechanism of hairpin-dependent transcriptional pausing. The four components of thehis pause signal are labeled on the paused TC, which is formed in competition with bypass of the site and then slowly escapes back into the elongation pathway. How the pause hairpin inhibits nucleotide addition is unknown, but it presumably disrupts reactive alignment of the RNA 3′OH and incoming NTP (depicted here by separation of the 3′ OH and NTP-binding subsites, i and i + 1; see Fig. 7). (Bottom) In the direct model of hairpin-dependent pausing, a specific interaction between the RNA hairpin and its binding site on RNAP disrupts nucleotide addition in the active site of RNAP (Chan et al. 1997; Wang and Landick 1997). In the indirect model, the hairpin merely defines a particular length of 3′-proximal, single-stranded RNA transcript and thus could both disrupt RNA–RNAP interactions required for elongation or TC stability and prevent backtracking of RNAP along the DNA template (Komissarova and Kashlev 1997b; Nudler et al. 1997). Annealing of antisense oligonucleotides to the nascent RNA would be able to recapitulate indirect effects of hairpins, but not direct effects.

Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release
Irina Artsimovitch and Robert Landick1
Genes & Dev. 1998. 12: 3110-3122

Rust fungi of New Zealand - An introduction, and list of recorded species

2008-11-28T08:47:00.000-08:00

Rust fungi of New Zealand - An introduction, and list of recorded species
more options
Author(s): McKenzie EHC
Source: NEW ZEALAND JOURNAL OF BOTANY Volume: 36 Issue: 2 Pages: 233-271 Published: JUN 1998
Times Cited: 9 References: 128 Citation Map
Abstract: An overview of the rust fungi (Basidiomycota, Teliomycetes, Uredinales) is presented as an introduction towards a new rust mycoflora for New Zealand. All species recorded from New Zealand are listed, together with details on their host plants, a reference to the first New Zealand record of each unique rust/host combination, and a separate alphabetical list of host plants and the rust fungi which parasitise them. New Zealand has a depauperate rust flora consisting of 234 recorded species, of which 54% are native. Of 22 genera recorded from New Zealand only five genera (Hamaspora, Kuehneola, Phragmidium, Puccinia, Uromyces) and three form genera (Aecidium, Caeoma, Uredo) contain native species. Only five genera (Phragmidium, Melampsora, Puccinia, Uromyces, Uromycladium) and two form genera (Aecidium, Uredo) are represented by more than two species. Melampsora contains mainly adventive species; approximately half of the Phragmidium, Puccinia, and Uromyces species and all the Uromycladium species are adventive. Some 95% of the Uredo species and all the Aecidium species are native. Only eight native rusts have spread to exotic hosts; Uredo puawhananga commonly infects some exotic, cultivated Clematis species, while Puccinia lagenophorae is sometimes troublesome on cultivars of Bellis perennis. None of the 17 exotic rusts infecting native plants is of economic or conservation concern.The widespread rusts in New Zealand are often adventive species. Only 5% of adventive rusts are confined to the South Island, but 30% are confined to the North Island. This inequity probably reflects the warmer conditions in the north, and the fact that adventive species are often of tropical origin. Of the native species, 34% occur only in the South Island and just 14% are restricted to the North Island. Since 1945, on average, more than one new adventive rust has been found per year. Most of them are of northern temperate origin, but often considered to be introduced from Australia by trans-Tasman airflows.
Document Type: Review
Language: English
Author Keywords: Uredinales; New Zealand; rusts; fungi; checklist; host list
KeyWords Plus: PLANT-DISEASE RECORDS; CHATHAM-ISLANDS; PATHOGENS; AUSTRALIA
Reprint Address: McKenzie, EHC (reprint author), Landcare Res, Herbarium PDD, Private Bag 92170, Auckland, New Zealand
Addresses:
1. Landcare Res, Herbarium PDD, Auckland, New Zealand
Publisher: SIR PUBLISHING, PO BOX 399, WELLINGTON, NEW ZEALAND
Subject Category: Plant Sciences
IDS Number: 100MK
ISSN: 0028-825X

पुच्सिनिलेस ट्री

2008-11-26T17:21:00.001-08:00

http://www.sciencedirect.com.ezp02.library.qut.edu.au/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B7XMR-4N7XPJ0-1&_image=fig3&_ba=3&_user=62921&_coverDate=05%2F31%2F2007&_alid=831657505&_rdoc=2&_fmt=full&_orig=search&_cdi=29677&_st=13&_acct=C000005418&_version=1&_urlVersion=0&_userid=62921&md5=0a06df9b20bc70321ef935cb7a6ebd20

Two Dozen Science Podcasts

2008-11-24T13:31:00.000-08:00

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The role of tip-localized mitochondria in hyphal growth

2008-11-23T21:11:00.000-08:00

Copyright © 2005 Elsevier Inc. All rights reserved.

The role of tip-localized mitochondria in hyphal growth

Natalia N. Levina and Roger R. Lew,

Department of Biology, York University, 4700 Keele Street, Toronto, Ont., Canada M3J 1P3

Received 25 May 2005; accepted 23 June 2005. Available online 7 February 2006.

Abstract
Hyphal tip-growing organisms have a high density of tip-localized mitochondria which maintain a membrane potential based on Rhodamine 123 fluorescence, but do not produce ATP based on the absence of significant oxygen consumption. Two possible roles of these mitochondria in tip growth were examined: Calcium sequestration and biogenesis, because tip-high cytoplasmic calcium gradients are a common feature of tip-growing organisms, and the volume expansion as the tip extends would require a continuous supply of additional mitochondria. Co-localization of calcium-sensitive fluorescent dye and mitochondria-specific fluorescent dyes showed that the tip-localized mitochondria do contain calcium, and therefore, may function in calcium clearance from the cytoplasm. Short-term inhibition of DNA synthesis or mitochondrial protein synthesis did not affect either tip growth, or mitochondrial shape or distribution. Therefore, mitochondrial biogenesis may not occur from the tip-localized mitochondria in hyphal organisms.

Keywords: Tip growth; Calcium; Mitochondria; Hyphal growth; Biogenesis; Neurospora crassa; Saprolegnia ferax

Article Outline
1. Introduction
2. Materials and methods
2.1. Strains and growth conditions
2.2. Preparation for microscopy
2.3. Treatment with fluorescent dyes
2.4. Treatment with inhibitors
2.5. Microscopy and objectives
2.6. Image processing
2.7. Mitochondria isolation
2.8. Fluorometric quantitation of Ca2+ dependence of chlortetracycline fluorescence
3. Results
3.1. Tip-localized mitochondrial energization
3.2. Ca2+ sequestration in tip-localized mitochondria
3.3. Tip-localized mitochondria do not undergo biogenesis
4. Discussion
Acknowledgements
References
1. Introduction
The dominant growth form in fungal organisms is a mycelial structure in which hyphal extension is used to invade new territory. Normally under high hydrostatic pressure (Lew et al., 2004), hyphae undergo continuous expansion solely at the tip, creating a tubular extension in a dynamic process called tip growth. Tip growth occurs in many organisms that have cell walls, often in specialized cells such as pollen tubes, root hairs, and rhizoids. Neuronal growth cones exhibit a similar tip extension, but using an amoeboid mechanism. In all the tip-growing cells examined to date, there appears to be a consistent role for calcium, which is found at a high concentration at growing tips (reviewed by Holdaway-Clarke and Hepler, 2003 and Torralba and Heath, 2000).

Tip-growing cells have a polarized cytological architecture. Vesicles often fill the apex, presumably to supply membrane, and cell wall precursors for the expanding tip. The cytoskeleton has been implicated as an organizing element that maintains the tip-localized vesicles during tip extension (Geitmann and Emons, 2000). Amongst tip-growing organisms, the cytology of fungi has been mapped extensively. In hyphal tips of the ascomycete Neurospora crassa, wall vesicles, which fuse with the membrane at the expanding tip are located in a steep gradient 0–5 μm behind the tip (Collinge and Trinci, 1974). This region is also the site of cell wall synthesis based on the incorporation of radioactive wall precursors (Gooday, 1971). A cytoplasmic Ca2+ gradient extends in a less steep gradient from 0 to 15 μm behind the tip, probably due to diffusion of Ca2+ released at the apex during growth (Silverman-Gavrila and Lew, 2003). The Ca2+ gradient is required for hyphal growth (Silverman-Gavrila and Lew, 2000), it is generated by IP3-activated Ca2+ release (Silverman-Gavrila and Lew, 2001 and Silverman-Gavrila and Lew, 2002) from Ca2+-containing vesicles (Torralba et al., 2001). Putative components of vesicle docking mediators have also been located at the apex (Gupta and Heath, 2000).

Mitochondria are present at a high density in some, but not all, tip-growing cells. During root hair initiation, mitochondria are spatially associated with the initiation bulge (Ciamporova et al., 2003). However, there is no indication of a unique tip-localized mitochondrial population behind the vesicle-filled apex of the root hair (Galway, 2000). In pollen tubes, numerous mitochondria are observed in the sub-apical zone behind the vesicle-filled apex (Pierson et al., 1990 and Uwate and Lin, 1980), which are elongate, compared to spherical in the vacuolated zone (Cresti et al., 1977). The establishment of polarity during neuronal growth is closely associated with mitochondrial location (Mattson, 1999), including high mitochondrial densities at the neuronal growth cone, which may function in energy supply (Chada and Hollenbeck, 2004 and Morris and Hollenbeck, 1993) and calcium clearing (Rumpal and Lnenicka, 2003).

Hyphal organisms have a high density of tip-localized mitochondria. In the oomycete Saprolegnia ferax, mitochondria first appear about 5 μm behind the tip, in the central cytoplasm, shifting to peripheral location about 15 μm behind the tip (Heath and Kaminskyj, 1989). The volume fraction of cytoplasm occupied by mitochondria is highest 5–15 μm behind the tip, which was also observed in Neurospora crassa (Lew, 1999). Zalokar (1959) mapped the distribution of mitochondria and mitochondrial biochemical activity in Neurospora, and found that, while mitochondria are located throughout the hyphal apical region (0–150 μm), cytochrome oxidase and succinic dehydrogenase, which were identified histochemically, are first observed 50 μm behind the tip, and increase to a maximal level 100–150 μm behind the tip. Of necessity, Zalokar’s measurements required fixation, which may alter the natural distribution of mitochondria. With a vibrating oxygen electrode, respiratory activity along growing hyphae was measured with 1–2 μm spatial resolution, and was first observed 15 μm behind the tip (Fig. 1C) (Lew and Levina, 2004), behind the high density of tip-localized mitochondria, measured by quantitation of electron micrographs (Fig. 1C) (Lew, 1999) or mitochondria-specific fluorescent dyes in growing hyphae (Figs. 1A and B). This corroborates Zalokar’s observations, and leads to the research question: What are the roles of the tip-localized mitochondria during hyphal growth in hyphal organisms?

Full-size image (66K)

Fig. 1. Tip-localized mitochondria and respiratory activity along growing hyphae. (A) MitoFluor Red fluorescence imaging of mitochondria in a growing hyphae and quantitative fluorescence intensity transects (see Section 2.6). (B) Rhodamine 123 fluorescence imaging of mitochondria in a growing hyphae and quantitative fluorescence intensity transects. (C) Mitochondrial densities from electron micrographs (squares) and oxygen influx measurements (circles) to show that the tip-localized mitochondria do not respire (data are re-drawn from Lew, 1999 and Lew and Levina, 2004).

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Since the tip-localized mitochondria do not supply ATP during hyphal extension, we explored two alternative functions: Ca2+ clearing and mitochondrial biogenesis. Ca2+ clearance is now accepted as a physiological role of mitochondria in animal cells (Gunter et al., 2004 and Nicholls and Chalmers, 2004). N. crassa has been used extensively in studies of biogenesis (Neupert, 1997), which have focused primarily on the mechanisms responsible for protein import into mitochondria. Only limited research has been done on the location of mitochondrial biogenesis. Luck (1963) used [3H]choline to monitor phospholipid incorporation into mitochondria in a choline-requiring mutant of N. crassa. The distribution of [3H]choline followed a Poisson distribution, indicative of incorporation throughout the mycelium. Tip-localized biogenesis may also occur, since the supply of mitochondria must keep pace with the hyphal volume expansion during tip growth; or, the tip-localized mitochondrial population may be maintained by molecular motors and the cytoskeleton (Westermann and Prokisch, 2002). We explored both alternative mitochondrial roles, Ca2+ clearing and biogenesis, using fluorescence microscopy of growing hyphae.

2. Materials and methods
2.1. Strains and growth conditions
Wild-type (74-OR23-1A) N. crassa was obtained from the Fungal Genetics Stock Center (FGSC 987; School of Biological Sciences, University of Missouri, Kansas City, Missouri, USA) (McCluskey, 2003) and maintained on 2% (w/v) agar slants containing Vogel’s minimal medium (Vogel, 1956) plus 1.5% (w/v) sucrose.

2.2. Preparation for microscopy
Conidia from the slants were sown on Petri dishes containing 1.5% agar and Vogel’s minimal medium (with 1.5% sucrose) or OM (organic medium (w/v): 1% glucose, 0.1% peptone, 0.01% yeast extract, 0.1% KH2PO4, and 0.03% MgSO4·7H2O). After overnight incubation at 28 °C, the cultures were flooded with OM. In preliminary experiments imaging chlortetracycline fluorescence, when a simple salt solution was used (BS: 10 mM Mes, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 133 mM sucrose, pH adjusted to 5.8 with KOH) the dye fluorescence tended to be diffuse, although tip-localized, possibly due to inefficient dye loading. OM was chosen as the medium of choice because of the better imaging clarity and well-defined structures observed in the hyphae loaded with chlortetracycline.

2.3. Treatment with fluorescent dyes
To load cells with chlortetracycline, 500 μl of OM with 50 μM chlortetracycline was added on the surface of the culture. After chlortetracycline addition, cells stopped growing for 10–15 min, some cells formed multiple tips, then continued to grow with normal hyphal morphology. After the cells had recovered for at least 2 h, chlortetracycline fluorescence was imaged on the confocal microscope using an Argon laser (458 nm excitation line). After scanning, hyphal growth slowed and mitochondrial morphology was altered as detected with Rhodamine 123 (Molecular Probes R-302, Abs/Em 507/529, final concentration 2.5 μM from a 2000× stock in methanol) or MitoFluor Red 589 (Molecular Probes M-22424, Abs/Em 588/622, final concentration 500 nM) fluorescence. Thus repeated scanning of the cells damaged them, so that continuous observation of one cell was difficult. Instead, different cells from the same culture were examined before and after various treatments. The same technique was used with other fluorescent dyes for single or dual dye imaging. After growth recovery of chlortetracycline-treated cells, 1 ml of OM with MitoFluor Red (500 nM final concentration) was added to the plate. Within 15–20 min after addition of the solution, cells resumed normal growth. Rhodamine 123 was used in some experiments to determine whether the mitochondria maintain a membrane potential (Scaduto and Grotyohann, 1999) (Fig. 1B; Figs. 2C–F), by adding the dye (2.5 μM final concentration) to the plate from a 2000× stock in methanol as noted above.

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Fig. 2. Mitochondria potential. Mitochondria distribution was monitored with MitoFluor Red (A, fluorescence; B, brightfield images). The mitochondria potential was monitored with Rhodamine 123 fluorescence (C and E, fluorescence; D and F, brightfield images). Images were taken at the times shown, inhibitors were added at time 0 s. Inhibition of growth by cyanide addition is observed at +30 s. Cyanide inhibition of growth had no immediate effect on mitochondria distribution (A and B). Rhodamine 123 fluorescence declined slightly (C and D). Direct depolarization of the mitochondrial potential with μm valinomycin caused complete dissipation of Rhodamine 123 fluorescence (E and F) and mitochondrial shape change (arrow). Bars = 10 μm. Under normal conditions, tip-localized mitochondria do maintain a potential, an important prerequisite for Ca2+ sequestration.

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2.4. Treatment with inhibitors
Hydroxyurea (Sigma) was added at a final concentration of 20 mM. Hydroxyurea is an inhibitor of ribonucleotide reductase and therefore inhibits both nuclear DNA and organellar DNA synthesis (Heinhorst et al., 1985), and would cause depletion of mitochondrial enzymes, such as cytochrome c oxidase, a respiratory chain enzyme encoded from both mitochondrial and nuclear DNA, and therefore should inhibit mitochondrial biogenesis. Hydroxyurea at 10–20 mM is sufficient to induce cell cycle arrest in filamentous fungi (Garcia-Muse et al., 2003); at 30 mM (the lowest concentration examined), Srivastava et al. (1988) reported that hydroxyurea immediately inhibits DNA synthesis in N. crassa. Chloramphenicol (Sigma) was added from a 100 mg/ml ethanol stock solution at a final concentration of 2 mg/ml. Ethanol, at the same final concentration (2% v/v), was used in growth measurement controls. Chloramphenicol is an antibiotic inhibiting mitochondrial protein synthesis, e.g., cytochrome c oxidase and malate dehydrogenase (Howell et al., 1971). Concentrations of 1–4 mg/ml are reported to inhibit mitochondrial protein synthesis in vitro (Sebald et al., 1968) and in vivo (Howell et al., 1971). We used chloramphenicol to inhibit the replication of mitochondria in hyphae. Cyanide (NaCN) was added at a final concentration of 1 mM. Nocodazole (Sigma) was added from 33 mM stock in DMSO to the final concentration 10 μM. For all inhibitor treatments, we measured their short-term effect (20 min) on apical growth rates and tip-localized mitochondrial morphology and distribution by fluorescent labeling with MitoFluor Red.

2.5. Microscopy and objectives
Observations were made on Olympus Fluoview 300 confocal system with Fluoview software. For most experiments, either 40, 60, or 63× water immersion objectives (all infinity tube length) were used. Multi-argon laser excitation line 488 nm and He–Ne laser excitation line 543 nm were used for visualization of cells simultaneously stained with chlortetracycline and MitoFluor Red, with FITC and Texas Red filter cubes and scanned in a linear sequencing mode. Control experiments with single labeled cells demonstrated that there was no fluorescence ‘leakage’ of chlortetracycline fluorescence through the Texas Red filter cube, nor ‘leakage’ of MitoFluor Red fluorescence through the FITC filter cube.

2.6. Image processing
Image processing and analysis were performed using the public domain ImageJ program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij/). When performed, image processing was limited to linear contrast stretch. Analysis included growth rate measurements and RGB merge of chlortetracycline (green) and mitochondria-specific fluorescent dyes (red) to identify regions of co-localization (yellow). Care was taken to assure that the fluorescent images exhibited well-defined structure, not just a diffuse fluorescence that would cause an erroneous identification of co-localized regions of fluorescence. To measure the fluorescence intensity of mitochondria labeled with MitoFluor Red or Rhodamine 123, transects (4 μm wide) were abutted to the hyphal tip in the center of hyphae, which were selected for medial focus extending from the tip to 30–40 μm behind the tip. The intensities for each column of pixels were summed to obtain total fluorescence intensity in arbitrary units.

2.7. Mitochondria isolation
Conidia were inoculated into liquid Vogel’s minimal medium (106 cells/ml) and grown for 6 h at 28 °C in shaker flasks (250 ml) at 100 rpm until almost 90% of conidia germinated and germ tubes were up to 200 μm length.

Germlings were harvested by centrifugation for 10 min at 1500g and resuspended in approximately 10 ml of homogenization medium (HM) (1:1 v/v) (0.25 M sucrose, 10 mM Na2EDTA, 5 mM MgSO4, 25 mM MES, 2.5 mM dithiothreitol, and 1% BSA, pH 7.0 (KOH)). Pre-chilled glass beads (1:1, acid-washed, 150–212 μm (Sigma G-1145), 106 μm Sigma, G-4649) were added in an amount equal to the volume of the germlings suspension (approximately 10 g total). Germlings were homogenized by grinding with a pestle in a pre-chilled porcelain mortar. The progress of homogenization and appearance of broken cells was monitored under a microscope with a X10 phase-objective, to determine when most cells had been disrupted. Then, beads were washed with additional HM; the final suspension was approximately 35 ml.

The homogenate was centrifuged for 10 min at 100g to pellet beads and cell material. Supernatant was then centrifuged for 30 min at 14,700g. The mitochondrial pellet had a characteristic rust color. Purity was confirmed by microscopic examination using a dark-field condenser. Mitochondria were resuspended with a fine camel hair brush in suspension medium (SM) (0.25 M sucrose, 25 mM MOPS, 50 mM KCl, and 5 mM EGTA, pH 7.2 with KOH); the final protein concentration varied between 7 and 16 mg/ml (in four preparations).

2.8. Fluorometric quantitation of Ca2+ dependence of chlortetracycline fluorescence
The Ca2+ dependence of chlortetracycline fluorescence (final concentration 50 μM) of mitochondria suspended in SM (protein concentration 1.0–1.5 mg/ml) was measured with a Cary Eclipse Fluorescence spectrophotometer (Varian, Canada). The excitation wavelength was 380 nm (5 nm slit), emission was at 540 nm (5 nm slit). Free Ca2+ concentration was calculated based on an iterative algorithm and binding constants for EGTA according to Goldstein (1978).

3. Results
To visualize mitochondria in N. crassa we used fluorescent mitochondrial dyes, Rhodamine 123 and MitoFluor Red. Both dyes labeled cable-like structures, which were highly dynamic and moved forward with the growing tip. The density of mitochondria labeled with fluorescent dyes was higher in the growing tips. When growth was temporarily slowed or stopped as a result of solution change, the tip-high gradient in mitochondrial density was not disrupted, while permanently non-growing cells (not treated with inhibitors) had either diffuse fluorescence still brighter at the tip, or round-shaped structures (data not shown).

3.1. Tip-localized mitochondrial energization
The high density of mitochondria at the hyphal tip is well-established based upon imaging of growing cells and electron microscopy; but oxygen influx measurements indicate that the tip-localized mitochondria do not respire (Fig. 1C). Rhodamine 123 fluorescence is reported to depend upon the presence of a membrane potential in isolated mitochondria (Scaduto and Grotyohann, 1999), therefore, we used Rhodamine 123 to confirm that tip-localized mitochondria maintain a potential. Tip-localized mitochondria fluoresced strongly, while mitochondria in hyphal compartments behind the colony edge were only weakly fluorescent (data not shown). Cyanide was used to de-energize the mitochondria, it caused both growth inhibition and a slight decline of Rhodamine 123 fluorescence (Figs.2C and D), but not MitoFluor Red fluorescence (Figs. 2A and B). Direct dissipation of the mitochondrial potential with μm valinomycin caused disappearance of Rhodamine 123 fluorescence (Figs. 2E and F) and growth to slow to about 10% of the normal rate. In 14/21 experiments, before fluorescence disappeared, the vermiform mitochondria changed shape to disk-like structures (Figs. 2E and F). The appearance of disk-like structures appeared to be related to a slower time for valinomycin to diffuse to the hyphae and inhibit growth. Therefore, the tip-localized mitochondria have a membrane potential, but are not synthesizing ATP at a rate sufficient to cause oxygen influx, while mitochondria behind the tip do synthesize ATP at a rate high enough to require oxygen uptake. Because the tip-localized mitochondria are energized, they may function as a Ca2+-sequestering organelle at the tip. We tested this possibility by using chlortetracycline.

3.2. Ca2+ sequestration in tip-localized mitochondria
To ensure that chlortetracycline can be used to image mitochondrial calcium, we assayed for Ca2+-dependent chlortetracycline fluorescence in vitro (Fig. 3). Chlortetracycline fluorescence begins to increase at about 0.75 mM free [Ca2+], and appears to reach a maximum at about 10 mM. Therefore, in vivo, we expect chlortetracycline to ‘report’ on mitochondrial calcium, if the mitochondria contain high levels of calcium.

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Fig. 3. Ca2+ dependence of chlortetracycline fluorescence. Unenergized mitochondria (1 mg/ml) were incubated with 50 μM chlortetracycline and varying free [Ca2+] concentrations as shown. Experiments from three mitochondria isolations are shown by different symbols (circles, left scale; triangles and squares, right scale; one experiment (squares) is the average of two assays from the same mitochondria isolation). Note that chlortetracycline fluorescence begins increasing at about 0.75 mM, and approaches maximal levels at about 10 mM.

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Dual dye imaging was used to determine the localization of chlortetracycline and mitochondrial dye fluorescence in growing N. crassa hyphal tips (Figs. 4A–H). The fluorescence of both dyes was higher in the hyphal tip, and was associated with elongate structures. When the chlortetracycline and MitoFluor Red fluorescence images were merged, for the majority of the fluorescent structures, there was co-localization, indicating that mitochondria do contain high levels of Ca2+. There were also chlortetracycline-fluorescing structures which were not co-localized with mitochondria, indicating at least two calcium-storing organelles in the growing tips. Another hyphal organism, the oomycete Saprolegnia ferax, is reported to have a high density of tip-localized mitochondria (Lew, 1999), and chlortetracycline fluorescing structures identified as mitochondria (Yuan and Heath, 1991); we observed co-localization of chlortetracycline and MitoFluor Red fluorescence (Figs. 4I–L).

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Fig. 4. Partial co-localization of Ca2+ stores and mitochondria at the growing hyphal apex and behind the tip in Neurospora crassa (A–H) and Saprolegnia ferax (I–L). Dual imaging of chlortetracycline-fluorescing Ca2+ stores (A, E, and I) and MitoFluor Red imaging of tip-localized mitochondria (B, F, and J) were merged using green and red pseudocoloring to reveal partial co-localization of the two structures (C, G, and K). Bright field images of the hyphae are shown in (D), (H), and (I). For Neurospora crassa (A–H), the tip-localized mitochondria and calcium stores are shown in (A)–(D). the same hyphae was then scanned just behind the tip (the branch structure in D and H serves as an internal marker of location) in (E)–(H). Images in (A), (E), (F), (I), and (J) were linear contrast stretched to maximize the dynamic range prior to green/red merging in (C), (G), and (K). Bars = 10 μm.

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To confirm that the mitochondrial membrane potential is required for Ca2+ sequestration, we treated the growing tips with valinomycin to dissipate the potential (cf. Figs. 2E and F) and examined the effect on co-localization (Fig. 5). Prior to complete inhibition of growth, mitochondria changed shape, from vermiform to disk-like structures. The shape changes were probably due to the dissipation of the mitochondrial potential (Figs. 2E and F). Eventually, the MitoFluor Red fluorescence became diffuse. However, in some experiments, chlortetracycline still labeled disk-like structures even though the MitoFluor Red fluorescence had become diffuse. Thus, MitoFluor Red localization in mitochondria may depend upon the mitochondrial potential. The chlortetracycline fluorescence observed after dissipating the mitochondrial potential with valinomycin indicates that much of the mitochondrial Ca2+ is unavailable for release.

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Fig. 5. Ca2+ retention in tip-localized mitochondria after depolarization. Valinomycin was used to dissipate the mitochondrial potential (Figs. 2 D and F). It caused mitochondrial shape to change from vermiform to round, most noticeable in the third column (arrow), and growth slowed considerably. Co-localization of mitochondria and chlortetracycline fluorescence was observed after valinomycin treatment. However, MitoFluor Red fluorescence became diffuse, suggesting its localization in mitochondria is dependent on the potential. In other experiments, chlortetracycline continued to label disk-like structures after valinomycin treatment, indicating that much of the mitochondrial Ca2+ is not free to diffuse from the mitochondria. The images were taken at the times shown, valinomycin was added at time 0 s. RGB merging of chlortetracycline fluorescence (A, green) and MitoFluor Red fluorescence (B, red) is shown in (C). Brightfield images are shown in (D). Bar = 10 μm.

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3.3. Tip-localized mitochondria do not undergo biogenesis
Given the high density of mitochondria at the hyphal tip, it is possible that the tip is the site of localized mitochondrial biogenesis. Attempts to quantify mitochondrial DNA (assuming actively dividing mitochondria would contain higher DNA quantities) failed. The vital DNA-specific dye SYTO13 (Molecular Probes) selectively stained mitochondria based upon co-localization with MitoFluor Red. SYTO13 fluorescent-labeling of mitochondria disappeared after inhibition with cyanide, when it stained multiple 2–3 μm round structures behind the tip (presumed to be nuclei). This occurred concomitant with morphological changes to mitochondria, which became compacted and condensed into the apical region (data not shown). Therefore, we were unable to quantitatively assess whether tip-localized mitochondria in growing hyphae contained higher levels of DNA, an indicator of mitochondrial biogenesis.

The alternative strategy was to inhibit mitochondrial biogenesis by inhibiting either DNA synthesis, or protein synthesis. With a growth rate of 20 μm min−1, volume 0–20 μm behind the tip doubles every minute, so tip-localized mitochondrial biogenesis should be rapid. Hydroxyurea treatment had no effect upon growth rates; mitochondrial shape and distribution were also unaffected (Fig. 6). Inhibition of mitochondrial protein synthesis using chloramphenicol caused an occasional transient inhibition of growth, also observed with control additions of ethanol or mock solution changes, and no effect on mitochondrial shape and distribution (Fig. 7).

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Fig. 6. Mitochondrial biogenesis: Hydroxyurea treatment. Hydroxyurea was applied at time 0. Images of hyphae, MitoFluor Red fluorescence and bright field images, are shown −6, 7, and 25 min after treatment. Open symbols show control treatments with OM alone, closed symbols show hydroxyurea treatments. Hydroxyurea had no effect upon mitochondrial shape and distribution, or growth rate. Each image shows a different hypha. Bar = 10 μm.

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Fig. 7. Mitochondrial biogenesis: Chloramphenicol treatment. Chloramphenicol was applied at time 0. Images of hypha, MitoFluor Red fluorescence and bright field images, are shown −6, 5 min, and 20 min after treatment. Open symbols show control treatments with OM plus ethanol. Closed symbols show chloramphenicol treatments; squares, measurements taken during fluorescence imaging on the confocal microscope; circles, growth measurements taken on a Zeiss Axioskop microscope. Chloramphenicol had no effect upon mitochondrial shape and distribution, transient inhibition of growth rate was occasionally observed, but also observed in control treatments, probably due to ethanol. Each image shows a different hypha. Bar = 10 μm.

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To assess whether the cytoskeleton maintains the tip-high distribution of mitochondria, growing cells were perfused with OM containing 10 μM nocodazole, an inhibitor causing microtubule depolymerization (Steinberg and Schliwa, 1993). After treatment, hyphal growth slowed from 12 down to 8 ± 1.3 μm min−1 after 3–5 min, typically hyphae emerged from the swollen tips formed immediately after nocodazole application. After 15–20 min, the hyphae continued growing but very slowly (2–3 μm min−1), with a narrow morphology. Nocodazole also affected the distribution of MitoFluor Red-labeled mitochondria. The mitochondria stopped moving along with the extending tip and clustered in a fixed position behind the tip. The slow growing tips initially did not have any fluorescent mitochondrial structures, but after 20–30 min single vermiform structures were occasionally visible in the tips (Fig. 8).

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Fig. 8. Mitochondrial distribution: The role of microtubules. Nocodazole was applied at time 0. Images of hypha, MitoFluor Red fluorescence and bright field images, are shown −1, 5, 11, and 24 min after treatment. Open symbols show control treatments with OM. Closed symbols show nocodazole treatments. Disruption of microtubules with nocodazole affects mitochondria distribution causing them to be distributed basal to the tip. Growth is not inhibited completely, but slows considerably. Each image shows a different hypha. Bar = 10 μm.

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4. Discussion
Tip-localized mitochondria are observed in some (fungi, oomycetes, and amoeboidal growth of neuronal growth cones), but not all tip-growing cells (root hairs and pollen). Thus they cannot be considered an obligatory cytological feature of tip growth. In hyphal organisms, tip-high mitochondria densities are observed in Saprolegnia ferax, based upon quantitation with electron microscopy. In N. crassa, the tip-high gradient observed with electron microscopy was confirmed in growing cells using a variety of mitochondrial-specific dyes. The two we used, MitoFluor Red and Rhodamine 123, are both reported to accumulate in mitochondria (Haugland, 2002). MitoFluor Red is reported to accumulate in mitochondria regardless of the membrane potential, while Rhodamine 123 is cationic, and accumulates in mitochondria maintaining a potential. There is a tip-high density of mitochondria whether hyphae were growing in a minimal salt solution plus sucrose (BS) or a nutrient-replete solution (OM). Growth rates are the same in either solution (Lew, 1999), which is expected since both solutions supply glucose to fuel growth, either via extracellular invertase (BS) or directly (OM). The principal difference is that OM will supply amino acids, the likely cause of higher H+ influx observed in hyphae growing in OM compared to BS, probably due to H+/amino acid symport activity (Lew, 1999). In N. crassa growing in BS, oxygen flux measurements showed that the tip-localized mitochondria do not consume oxygen, pointing to a unique role of tip-localized mitochondria, separate from ATP production. We have not performed the oxygen flux measurements on hyphae growing in OM, but expect the same result. We did observe that tip-localized mitochondria exhibit higher Rhodamine 123 fluorescence than mitochondria in hyphal trunks behind the growing edge when growing in OM (data not shown), consistent with a difference in respiratory function. To examine one possible role of tip-localized mitochondria, Ca2+ clearing, we used chlortetracycline.

The partial co-localization of chlortetracycline fluorescence and mitochondrial-specific dyes suggested that chlortetracycline is ’reporting’ high Ca2+ levels in mitochondria. Although the [Ca2+] dependence of chlortetracycline fluorescence has been reported for microsomal membranes (Lew et al., 1986), the lipid composition of mitochondria is very different, with significant levels of cardiolipin, an acidic phospholipid that can bind Ca2+. Therefore, we examined the [Ca2+] dependence of chlortetracycline fluorescence in isolated unenergized mitochondria. Free [Ca2+] of at least 500 μM was required for chlortetracycline fluorescence to occur, fluorescence intensity approached maximal levels at about 10 mM. Mitochondria in State four (not synthesizing ATP) have a potential of about −170 mV (Mitchell and Moyle, 1969), sufficient to accumulate [Ca2+] as high as 140 mM when cytoplasmic [Ca2+] is 200 nM. The sensitivity of Rhodamine 123 fluorescence to the mitochondrial membrane potential is well-established in vitro (Scaduto and Grotyohann, 1999). In vivo, Rhodamine 123 fluorescence indicates that the tip-localized mitochondria do have a membrane potential, consistent with in vitro results. Therefore, the mitochondria are competent to accumulate Ca2+. However, recent measurements of mitochondrial free [Ca2+] are about 3–4 μM (reviewed by Nicholls and Chalmers, 2004), contradicting the chlortetracycline quantitation, which suggests that at least 750 μM free [Ca2+] is required for fluorescence to increase. Chlortetracycline may be ’reporting’ on the total mitochondrial Ca2+ store, and cannot be considered a direct quantitative reporter of mitochondrial free [Ca2+]. This is consistent with the chlortetracycline fluorescence observed after the mitochondrial potential is dissipated by valinomycin, suggesting that much of the mitochondrial Ca2+ is not ’free’ but instead unavailable for release after it is sequestered. Co-localization of chlortetracycline and MitoFluor Red was observed in another hyphal organism, the phylogenetically distant oomycete Saprolegnia ferax, confirming a previous report (Yuan and Heath, 1991). Both organisms exhibit similar hyphal morphology and growth rates, as well as a high density of tip-localized mitochondria (Lew, 1999), also observed in the related oomycete Pythium ultimum (Grove et al., 1970). We can conclude that tip-localized mitochondria do play a role in Ca2+ sequestration during tip growth of hyphal organisms. The converse, that tip-localized mitochondria can function as a redundant source of Ca2+ to induce the vesicle fusion required for hyphal expansion cannot be discounted.

With a growth rate of about 20 μm min−1, hyphal extension involves a large and continuous increase in cellular volume at the hyphal tip. Mitochondrial density at the tip is about 30% of cell volume, basally they comprise about 15% of cell volume (Lew, 1999). It is easy to infer that tip-localized mitochondrial densities are so high because mitochondrial biogenesis at the tip supplies mitochondria for the basal regions behind the tip as it grows. Attempts to demonstrate this directly, by quantifying DNA per mitochondria, failed because we were unable to visualize mitochondrial DNA during hyphal growth. Indirect attempts relied upon the known inhibitors of DNA synthesis (hydroxyurea) and organellar protein synthesis (chloramphenicol). If tip-localized mitochondria are undergoing biogenesis, we expected to see rapid effects on growth and mitochondria, within the time frame of volume-doubling of the tip region. The tip-localized mitochondria occur in a zone from 0–20 μm. During growth at about 20 μm min−1, volume-doubling of this region would occur every minute or so. If mitochondrial biogenesis is occurring in the tip region, the tip-high mitochondrial density should decline by about 50% per minute when biogenesis is inhibited. Inhibition of DNA synthesis or organellar protein synthesis had no effect on growth or mitochondrial morphology within 20 min. Therefore, we conclude that tip-localized mitochondria are not undergoing significant biogenesis. Luck (1963) had already documented that mitochondrial biogenesis occurs throughout the mycelium of N. crassa. Our results exclude tip-localized mitochondria biogenesis as an additional source of mitochondria during cellular growth.

The tip-high mitochondria distribution must be maintained by the cytoskeleton, both microtubules (Fuchs et al., 2002) and molecular motors (Fuchs and Westermann, 2005 and Westermann and Prokisch, 2002). Disruption of the molecular motors that move along microtubules decreases growth rate and affects morphogenesis (Seiler et al., 1999). We used disruption of microtubules to corroborate the role of the cytoskeleton in maintaining mitochondria distribution. Mitochondria moved away from the hyphal tip, but growth continued, for at least 60 min at a lower rate. This indicates that tip-high mitochondria distributions are the norm during growth, but are not essential for growth. The decline in growth rate may be because tip-localized mitochondria contribute significantly to growth. However, disruption of the microtubules will affect many other processes that may contribute to maximize growth rates.

To summarize, hyphal organisms maintain a high density of tip-localized mitochondria during hyphal extension. These mitochondria appear to be unique, in that they do not function in ATP production, but do play a role in Ca2+ sequestration and must play a role in maintaining the tip-high Ca2+ gradient required for tip growth (Silverman-Gavrila and Lew, 2003). Their role cannot be considered obligatory, since even after disruption of the distribution of tip-localized mitochondria by depolymerizing the microtubules, growth can still continue.

Acknowledgments
We are grateful for the technical assistance of Ms. Karen Rethoret, and critical comments of Dr. I. Brent Heath.

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Funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.

Corresponding author. Fax: +1 416 736 5698.

Expressed sequence tag analysis of the soybean rust pathogen Phakopsora pachyrhizi

2008-11-23T21:09:00.000-08:00

Published by Elsevier Inc.

Expressed sequence tag analysis of the soybean rust pathogen Phakopsora pachyrhizi

Martha Lucia Posada-Buitrago1 and Reid D. Frederick,

USDA-Agricultural Research Service, Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, MD 21702, USA

Received 5 November 2004; accepted 10 June 2005. Available online 15 November 2005.

Abstract
Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi Sydow. A unidirectional cDNA library was constructed using mRNA isolated from germinating P. pachyrhizi urediniospores to identify genes expressed at this physiological stage. Single pass sequence analysis of 908 clones revealed 488 unique expressed sequence tags (ESTs, unigenes) of which 107 appeared as multiple copies. BLASTX analysis identified 189 unigenes with significant similarities (Evalue < 10−5) to sequences deposited in the NCBI non-redundant protein database. A search against the NCBI dbEST using the BLASTN algorithm revealed 32 ESTs with high or moderate similarities to plant and fungal sequences. Using the Expressed Gene Anatomy Classification, 31.7% of these ESTs were involved in primary metabolism, 14.3% in gene/protein expression, 7.4% in cell structure and growth, 6.9% in cell division, 4.8% in cell signaling/cell communication, and 4.8% in cell/organism defense. Approximately 29.6% of the identities were to hypothetical proteins and proteins with unknown function.

Keywords: Phakopsora pachyrhizi; Genome analysis; cDNA sequencing; Expressed sequence tags; Gene expression

Article Outline
1. Introduction
2. Materials and methods
2.1. Fungal isolate and growth conditions
2.2. cDNA library construction
2.3. DNA sequencing
2.4. Data handling
3. Results
3.1. EST analysis
3.2. Gene families
4. Discussion
Acknowledgements
References
1. Introduction
Soybean rust causes significant yield loss to soybean crops in Asia, Africa, Australia, and nearly all tropical countries in the Eastern Hemisphere where soybeans are grown have reported its occurrence (AVRDC, 1987 and AVRDC, 1992). Recent findings of soybean rust in Hawaii in 1994 (Killgore and Heu, 1994), Zimbabwe in 1998 (Levy, 2003), Nigeria in 1999 (Akinsanmi and Ladipo, 2001), South Africa in 2001 (Pretorius et al., 2001), Paraguay in 2001 (Morel, 2001), Brazil and Argentina in 2002 (Rossi, 2003 and Yorinori et al., 2002), and Bolivia in 2003 (Yorinori et al., 2005) demonstrate that Phakopsora pachyrhizi is spreading to new geographic regions. Rust is considered to be a major threat to soybean production in the United States (Sinclair, 1989), especially with the identification of P. pachyrhizi in Louisiana in November 2004 (Schneider et al., 2005). In Brazil, this disease was estimated to cost growers approximately $1.2 billion (USD) in 2003 alone: $500 million in direct yield losses to the disease and $700 million resulting from inappropriate use of fungicides (Yorinori et al., 2005). If P. pachyrhizi becomes established in the continental USd, serious yield losses are likely to occur. It has been estimated that yield losses could exceed 10% in most of the United States with up to 50% yield loss in the Mississippi Delta and southeastern states (Yang et al., 1991).

Four single resistances genes, Rpp1–4 (for resistance to P. pachyrhizi), have been described that impart resistance to some isolates of P. pachyrhizi (Bromfield and Hartwig, 1980, Hartwig, 1986, Hartwig and Bromfield, 1983 and McLean and Byth, 1980). However, no soybean lines have been found with broad-spectrum resistance to all isolates of P. pachyrhizi, and all of the commercial soybean cultivars currently grown in the US are susceptible to soybean rust. In countries where rust has become problematic to commercial production, control strategies have relied on the use of fungicides; however, most growers in the US currently do not apply fungicides to soybeans. The increased costs associated with multiple applications of fungicides might be prohibitive for some growers in the US, and there are concerns about the potential negative effects to the environment if fungicides are applied to such large production acreage.

Soybean rust is caused by two closely related species of fungi, P. pachyrhizi Sydow and P. meibomiae (Arthur) Arthur, which are differentiated based upon morphological characteristics of the telia (Ono et al., 1992). Sequence analysis of the internal transcribed spacer region of the ribosomal RNA genes revealed approximately 80% similarity between these two Phakopsora species; however, only a few nucleotide differences were observed among isolates of P. pachyrhizi or P. meibomiae (Frederick et al., 2002). Unlike most other rust pathogens, both Phakopsora species infect and produce disease symptoms on a wide range of host plants. P. pachyrhizi naturally infects 31 species in 17 genera of Leguminosae, and it has been found to infect 60 species in other genera under controlled conditions (Rytter et al., 1984 and Sinclair and Hartman, 1996). Similarly, P. meibomiae infects 42 species in 19 genera of Leguminosae, and it can infect 18 species in another 12 genera following artificial inoculation (Sinclair and Hartman, 1996). On soybeans, P. pachyrhizi is the more aggressive pathogen and causes considerably more yield loss compared to P. meibomiae.

Phakopsora pachyrhizi produces three types of spores. The urediniospore is the most common spore type and is found throughout the growing season on soybeans and other legume hosts. Urediniospores are produced in large quantities, easily wind disseminated, and multiple spore cycles occur throughout the growing season. Telia and teliospores have been observed on infected plants late in the season in Asia as well as in greenhouse studies (Bromfield, 1984 and Yeh et al., 1981). Teliospore germination and the subsequent production of basidiospores have been reported, but only under laboratory conditions (Saksirirat and Hoppe, 1991). As no alternate host has been identified, there has been no further characterization of the life cycle.

Most of the published research on soybean rust has focused on monitoring disease development, evaluating yield losses, modeling epidemics, host range studies, developing risk assessment models, and screening for sources of resistance. In addition, there have been several reports on the basic biology of the fungus, including histological studies using susceptible lines and those containing single resistance genes (Bonde et al., 1976, Hartwig and Bromfield, 1983 and Sinclair and Hartman, 1996). The infection process employed by P. pachyrhizi consists of several distinct steps: attachment of the spore to the host surface, spore germination, formation of the appressorium, penetration through the cuticle, and invasive growth within the host plant (Bonde et al., 1976). Understanding these processes at both the biochemical and molecular levels is essential for developing new methods of disease management.

Here, we report the first assessment of gene expression at a critical stage of the P. pachyrhizi life cycle: urediniospore germination. This study identifies transcripts present in germinating urediniospores and provides insight into the biochemical processes that occur at this developmental stage. Some of the genes expressed display a high degree of similarity to genes described in other fungi and plants, but the majority corresponded to unclassified genes or genes of unknown function. A preliminary report of this work has been given (Posada and Frederick, 2002).

2. Materials and methods
2.1. Fungal isolate and growth conditions
The P. pachyrhizi isolate Taiwan 72-1 (TW 72-1) used in this study was maintained at the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) Plant Pathogen Biosafety Level 3 Containment Facility at Ft. Detrick, MD (Melching et al., 1983) under the appropriate USDA Animal and Plant Health Inspection Service (APHIS) permit. TW 72-1 was propagated by spray inoculation onto soybean plants, and urediniospores were harvested from infected leaves 10–14 days following inoculation and at subsequent intervals using a mechanical harvester (Cherry and Peet, 1966). Urediniospores were maintained under liquid nitrogen. Frozen urediniospores were heat shocked at 42 °C for 5 min, and 300 mg of spores was germinated in 300 ml distilled water in a sterile 13 in. × 9 in. Pyrex baking dish for 16 h at room temperature. The fungal tissue was collected using a spatula, frozen in liquid nitrogen, and used for RNA extractions.

2.2. cDNA library construction
Total RNA was isolated from germinating spores of P. pachyrhizi isolate TW 72-1 using the ToTally RNA kit (Ambion, Austin, TX, USA), and the poly(A)+ mRNA was purified using an OLIGOTEX mRNA purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. A unidirectional cDNA library was constructed in the plasmid pSPORT1 using the Superscript Plasmid System for cDNA synthesis and Cloning (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The titer of the library was approximately 20,000 colonies, and 5000 individual colonies were transferred to 96-well microtiter plates containing Luria broth with 15% (v/v) sterile glycerol. The plates were archived by storing in a freezer at −80 °C, and 908 clones were sent for sequencing.

2.3. DNA sequencing
Prior to sequencing, all colonies were checked for the presence of an insert by colony-PCR using the SP6 and T7 primers. The PCR products were separated by electrophoresis using 1.5% agarose gels. DNA was prepared for sequencing reactions using a Qiagen BioRobot 9600 and a Beckman Biomek 2000. Purified plasmid DNA was sequenced from the 5′ end with the M13 reverse primer using an Applied Biosystems (ABI) PRISM big dye terminator kit (Perkin-Elmer) and an ABI Applied Biosystems 3700 DNA analyzer at the USDA Agricultural Research Service, Eastern Regional Research Center, Nucleic Acids Facility (ARS-ERRC-NAF) in Wyndmoor, PA.

2.4. Data handling
Raw sequence data were retrieved electronically from the USDA-ARS-ERRC-NAF using the file transfer protocol (ftp) for subsequent processing and analysis. The sequence data were imported into the computer software package Chromas 2.13 (Technelysium Pty, Helensvale, Australia) and manually trimmed of vector sequences. Ambiguous base calls were corrected by manually inspecting the sequence electropherograms, and the edited sequences were used in similarity searches.

Each cDNA sequence was queried against the current non-redundant (nr) protein database at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) using the BLASTX algorithm and the NCBI EST database using the BLASTN algorithm (Altschul et al., 1997). In both cases, the default BLAST parameters were used. The redundancy of the 908 cDNA sequences was determined by comparing all sequences with one another using the program FastA (Wisconsin Package, Genetic Computer Group, Madison, WI, USA).

3. Results
3.1. EST analysis
The cDNA clones were checked by PCR, and 99% were found to contain inserts ranging in size from 350 to 3000 bp. A total of 908 clones were sequenced from the 5′ end of the cDNA inserts. The single pass sequencing runs generated an average of 650 nucleotides of readable sequences after manual editing.

All ESTs were assembled into a database and compared using the FastA program (Wisconsin Package, Genetic Computer Group, Madison, WI, USA) to identify redundant clones. A total of 488 unique ESTs were identified of which 381 appeared only once and 107 were represented by multiple clones at frequencies ranging from 2 to 142. The frequency of redundant ESTs is shown in Fig. 1. The sequences of the P. pachyrhizi EST clones were submitted to NCBI as dbEST IDs 28583523–28584357 and GenBank Accession Nos. DN739461–DN740295.

Full-size image (3K)

Fig. 1. The frequency of occurrence of EST clones derived from germinating P. pachyrhizi urediniospores. The number of EST clones is shown above each of the number of occurrences.

View Within Article

The BLASTX algorithm (Altschul et al., 1997) was used to translate each edited EST into the six possible reading frames for comparison with data in the current nr protein database at the NCBI. A total of 431 ESTs displayed significant similarity to sequences in the NCBI database, while 477 ESTs did not exhibit significant similarity to the database entries. ESTs with similarity scores of Evalue < 10−5 were grouped according to their putative function (Table 1), according to the Expressed Gene Anatomy Database (EGAD) categories developed by The Institute for Genomic Research (TIGR, Rockville, MD, USA).

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Table 1.

EST clones displaying similarity (BLASTX, Evalue < 1E−05) to proteins in the non-redundant protein NCBI database, grouped into functional categories according to expressed gene anatomy database

Clone Accession No. Description Species Evalue No. of clones Organism
1. Cell division
1.1. DNA synthesis/replication
Pp0906 NP_595357 Checkpoint rad 3 Schizosaccharomyces pombe 9.00E−88 1 Yeast
Pp1817 T41457 DNA repair protein rad 18 S. pombe 4.00E−17 1 Yeast
Pp0244 NP_593482 Exonuclease II S. pombe 2.5E−44 1 Yeast
Pp2018 CAB91747 Related to syntaxin 12 Neurospora crassa 4.00E−14 1 Filamentous fungus

1.2. Apoptosis
Pp0322 AF316601 Metacaspase S. pombe 9.00E−50 1 Yeast

1.3. Cell cycle
Pp1417 AAA34617 G1 cyclin S. cerevisiae 8.00E−12 1 Yeast
Pp1017 AJ272133 Cyclin A. nidulans 7.00E−06 3 Filamentous fungus

1.4. Chromosome structure
Pp0437 P62792 Histone H4 Phanerochaete chrysosporium 3.70E−42 1 Filamentous fungus
Pp0729 P62792 Histone H4 P. chrysosporium 3.70E−45 1 Filamentous fungus
Pp1936 AAA35311 Histone H2A-α S. pombe 6.00E−33 3 Yeast
Pp1709 PN0142 Histone H2B N. crassa 5.00E−39 2 Filamentous fungus
Pp1628 A35072 Non-histone chromosomal protein NHP6A S. cerevisiae 4.00E−19 1 Yeast
Pp1812 S78076 Non-histone chromosomal protein NHP6B S. cerevisiae 2.00E−23 1 Yeast

2. Cell signaling/cell communication
2.1. Cell adhesion
Pp0813 Q28983 Zonadhesin Sus scrofa 1.00E−09 2 Mammal

2.3. Effectors/modulators
Pp0839 NP_593464 Calmodulin kinase I homolog S. pombe 5.00E−34 2 Yeast
Pp2023 AAA21544 Casein kinase-1 S. pombe 7.00E−48 1 Yeast
Pp0948 T18359 Nik-1 protein (histidine kinase) N. crassa 1.00E−39 1 Filamentous fungus
Pp0229 T45137 Phosphoprotein phosphatase catalytic chain 2B S. pombe 2.00E−06 1 Yeast
Pp1003 D84555 Probable protein kinase Arabidopsis thaliana 5.00E−27 1 Plant
Pp0424 T11657 RhoGDP dissociation inhibitor S. pombe 3.00E−30 1 Yeast
Pp1001 NP_596024 RhoGAP GTPase activating protein S. pombe 5.00E−12 1 Yeast
Pp1337 NP_594429 Probable phosphatidylinositol-4-phosphate kinase S. pombe 1.00E−51 1 Yeast

3. Cell structure and growth
3.1. Cytoskeletal
Pp1318 CAC17476 α-Tubulin Ustilago maydis 3.10E−87 1 Filamentous fungus
Pp1432 CAC83953 β-Tubulin Uromyces viciae-fabae 3.00E−98 1 Filamentous fungus
Pp1440 Q90631 Kinectin Gallus gallus 5.00E−07 1 Bird
Pp0920 AB018696 RanBPM Xenopus laevis 6.00E−05 1 Amphibian
Pp0414 U92845 Kinesin motor protein U. maydis 2.00E−37 1 Filamentous fungus

3.2. Growth and sporulation
Pp0432 XP_330886 Conidiation-specific protein 6 N. crassa 8.60E−16 7 Filamentous fungus
Pp0926 AAA33573 Conidiation protein N. crassa 1.00E−06 1 Filamentous fungus
Pp0122 CAD10036 Deacetylase Filobasidiella neoformans 5.00E−39 3 Filamentous fungus
Pp1605 A59290 Csm1 (class V chitin synthase with a myosin motor-like domain) Magnaporthe grisea 3.00E−07 1 Filamentous fungus
Pp1209 AAO49384 Class V chitin synthase Fusarium oxysporum 5.70E−88 1 Filamentous fungus

3.3. Others
Pp0941 EAA57250 Hypothetical protein MG08219.4 M. grisea 1.00E−08 2 Filamentous fungus
Pp0223 BAB13330 N-Acetylglucosaminidase Emericella nidulans 4.00E−25 1 Filamentous fungus
Pp1112 NP_014463 Sortilin homolog S. cerevisiae 1.00E−52 1 Yeast
Pp0811 NP_595238 Putative vacuolar protein; β-catenin family S. pombe 2.00E−27 1 Yeast

4. Cell/organism defense
4.1. Apoptosis
Pp1737 I49285 Defender against death protein 1 Mus musculus 1.00E−26 1 Mammal

4.2. Stress response
Pp0611 CAC20378 14-3-3-like protein Hypocrea jecorina 7.00E−92 1 Filamentous fungus
Pp0528 AAK15159 Heat-induced catalase Pleurotus sajor-caju 2.00E−82 4 Filamentous fungus
Pp1848 1908431A Heat-shock protein A. thaliana 1.00E−62 1 Plant
Pp1303 NP_596091 hsp16 (heat-shock protein 16) S. pombe 1.00E−19 1 Yeast
Pp1121 AAN75572 Copper chaperone TahA Trametes versicolor Trametes versicolor 3.00E−10 1 Filamentous fungus
Pp1929 CAD21425 Related to stress response protein rds1p N. crassa 7.00E−34 3 Filamentous fungus
Pp2004 BAA77283 DyP (peroxidase) Galactomyces geotrichum 3.00E−07 1 Filamentous fungus
Pp1616 T49477 Phenol hydroxylase related protein N. crassa 2.00E−16 1 Filamentous fungus

5. Gene/protein expression
5.1. RNA synthesis
5.1.1. RNA polymerases
Pp0946 P29035 Probable RNA-directed RNA polymerase (2Aprotein) (RNA replicase) Tomato aspermy virus 3.00E−08 1 Virus
Pp2029 NP_049325 Replicase Pea early browning virus 1.00E−07 1 Virus

5.1.2. RNA processing (e.g., spliceosomal, helicases)
Pp0519 O42861 Probable helicase S. pombe 4.00E−22 1 Yeast
Pp1810 S22646 Splicing factor U2AF homolog M. musculus 9.00E−42 1 Mammal
Pp1327 AAF37551 RNA-binding motif protein 8 Homo sapiens 4.00E−25 1 Mammal

5.1.3. Transcription factors
Pp1348 NP_010680 Transcription factor; Spt3p S. cerevisiae 1.00E−25 1 Yeast
Pp1504 AAA79367 TATA-binding protein Pneumocystis carinii 2.00E−95 1 Filamentous fungus
Pp0237 NP_011561 Transcription factor Tfc4p S. cerevisiae 3.40E−17 1 Yeast
Pp2041 Q00659 Sulfur metabolite repression control protein E. nidulans 1.00E−16 1 Filamentous fungus

5.2. Protein synthesis
5.2.1. Post-translational modification/targeting
Pp1724 S34655 Polyubiquitin 5 P. chrysosporium 9.00E−91 2 Filamentous fungus
Pp0936 T06053 Probable ubiquitin-dependent proteolytic protein A. thaliana 2.00E−35 1 Plant

5.2.2. Post-translational modification/trafficking
Pp1547 T39383 t-Complex protein 1, α-subunit S. pombe 1.00E−44 1 Yeast
Pp0719 NP_596649 Putative cytochrome C oxidase copper chaperone protein S. pombe 1.00E−12 2 Yeast
Pp0105 2113205A DNA J-like protein S. pombe 3.00E−19 1 Yeast

5.2.3. Protein turnover
Pp0126 CAA09863 Putative tripeptidyl peptidase I M. musculus 3.00E−06 1 Mammal
Pp1331 BAC56232 Tripeptidyl peptidase A A. oryzae 3.70E−39 2 Filamentous fungus
Pp1031 CAC39600 Prolidase A. nidulans 2.00E−38 1 Filamentous fungus

5.2.4. Ribosomal proteins
Pp1147 XP_326286 40S ribosomal protein S22 (S15A) (YS24) N. crassa 3.70E−74 1 Filamentous fungus
Pp1420 P05736 60S ribosomal protein L2 (YL6) (L5) (RP8) S. cerevisiae 2.00E−68 1 Yeast
Pp2011 T40111 14p-like ribosomal protein S. pombe 2.00E−12 1 Yeast

5.2.5. tRNA synthesis/metabolism
Pp1213 P46655 Cytosolic glutamyl-tRNA synthetase S. cerevisiae 6.00E−64 1 Yeast

5.2.6. Translation factors
Pp2028 S43861 Translation elongation factor eEF-1 α-chain Podospora anserina 1.00E−104 1 Filamentous fungus
Pp0835 NP_595367 eIF3 p48 subunit eIF3/signalosome component S. pombe 2.60E−43 1 Yeast
Pp0206 T48731 Probable translation initiation factor N. crassa 2.00E−100 1 Filamentous fungus
Pp0317 NP_015366 Tif5p S. pombe 1.00E−34 1 Yeast
Pp1107 P32186 Elongation factor eEF-1 α-chain Puccinia graminis 6.20E−82 1 Filamentous fungus
Pp2027 NP_502791 ADP-ribosylation factor-like protein (21.3 kDa) (4P563) Caenorhabditis elegans 7.40E−15 2 Nematode

6. Metabolism
6.1. Amino acid
Pp0134 M10139 3-Dehydroshikimate dehydratase N. crassa 9.00E−31 2 Filamentous fungus
Pp0425 AAN31488 DAHP synthase Phytophthora infestans 1.00E−74 1 Oomycete
Pp1503 NP_289154 DAHP synthetase, tyrosine repressible Escherichia coli 4.00E−40 1 Bacteria
Pp1336 NP_009808 DAHP synthase (is feedback-inhibited by tyrosine) S. cerevisiae 1.00E−37 1 Yeast
Pp1502 NP_012612 Tryptophan 2,3-dioxygenase S. cerevisiae 2.00E−12 1 Yeast
Pp0744 NP_592942 Phospho-2-dehydro-3-deoxyheptonate aldolase S. pombe 7.70E−29 1 Yeast
Pp1343 O94225 Homocitrate synthase, mitochondrial precursor Penicillium chrysogenum 3.00E−116 1 Filamentous fungus
Pp1727 T39244 Probable phospho-2-dehydro-3-deoxyheptonate aldolase S. pombe 3.00E−25 4 Yeast
Pp0806 AAO27751 Monooxygenase Fusarium sporotrichioides 1.00E−24 1 Filamentous fungus

6.2. Cofactor
Pp1931 CAB85691 Riboflavin aldehyde-forming enzyme Agaricus bisporus 2.00E−11 2 Filamentous fungus

6.3. Energy/TCA cycle
Pp1004 P34728 ADP-ribosylation factor F. neoformans 5.00E−108 1 Filamentous fungus
Pp2036 AAK18073 Aldehyde dehydrogenase ALDH15 E. nidulans 3.00E−38 1 Filamentous fungus
Pp1816 BAA09832 Isobutene-forming enzyme and benzoate 4-hydroxylase Rhodotorula minuta 8.00E−37 1 Yeast
Pp0727 CAA67613 Mitochondrial carrier protein S. cerevisiae 2.00E−13 1 Yeast
Pp1722 NP_035016 NADH dehydrogenase (ubiquinone) 1 α subcomplex 4 M. musculus 5.00E−08 2 Mammal
Pp1517 NP_594397 Putative isocitrate dehydrogenase (NADP+) S. pombe 2.00E−15 1 Yeast
Pp2040 T50403 Probable succinate dehydrogenase membrane anchor subunit precursor S. pombe 3.00E−24 1 Yeast
Pp1317 AAN74818 Fum15p Gibberella moniliformis 1.30E−16 1 Filamentous fungus
Pp2033 NP_172773 Putative cytochrome P450 monooxygenase A. thaliana 2.00E−16 1 Plant
Pp0837 NP_593578 Putative mitochondrial carrier S. pombe 2.90E−11 1 Yeast
Pp0127 CAC81058 Mitochondrial F1 ATP synthase β-subunit A. thaliana 1.30E−16 1 Plant
Pp0236 CAC81058 Mitochondrial F1 ATP synthase β-subunit A. thaliana 9.90E−16 1 Plant

6.4. Lipid
Pp1703 AAK26619 Acetyl-CoA acetyl transferase Laccaria bicolor 2.00E−39 1 Filamentous fungus
Pp0546 AAK26620 Acetyl-CoA acetyltransferase L. bicolor 7.00E−23 1 Filamentous fungus
Pp1511 CAB55552 Fox2 protein Glomus mosseae 2.00E−28 1 Filamentous fungus
Pp0337 XP_325309 Glycerol-3-phosphate dehydrogenase precursor related protein N. crassa 1,4E−92 1 Filamentous fungus
Pp0913 AAK63186 Probable acyl-CoA dehydrogenase G. intraradices 1.00E−37 2 Filamentous fungus
Pp1714 AAK63186 Probable acyl-CoA dehydrogenase G. intraradices 7.00E−33 3 Filamentous fungus
Pp0121 T40135 Probable involvement in ergosterol synthesis S. pombe 3.00E−33 1 Yeast
Pp1910 AAQ72469 SCS7p (oxidoreductase) Pichia pastoris 3.00E−09 3 Yeast
Pp0804 AAF27123 Putative glycerolkinase A. thaliana 4.00E−33 2 Plant

6.5. Sugar/glycolysis
Pp1827 AAA34858 6-Phosphofructo-2-kinase S. cerevisiae 4.00E−21 2 Yeast
Pp1140 Q24319 Dolichyl-diphosphooligosaccharide–protein glycosyltransferase Drosophila melanogaster 1.00E−12 1 Insect
Pp1235 AAB22823 Fructose-2,6-biphosphatase S. cerevisiae 2.70E−20 1 Yeast
Pp1735 CAC48025 Mutanase (α-1,3 glucanase) E. nidulans 3.00E−23 1 Filamentous fungus
Pp1048 CAC48025 Mutanase (α-1,3 glucanase) E. nidulans 2.00E−14 2 Filamentous fungus
Pp1033 XP_323561 Neutral trehalase N. crassa 1.00E−83 1 Filamentous fungus
Pp0133 CAA20128 Phosphomannomutase (predicted) S. pombe 6.00E−43 1 Yeast
Pp1501 ZP_00110197 COG0235: Ribulose-5-phosphate 4-epimerase, related epimerases and aldolases Nostoc punctiforme 1.00E−40 1 Bacteria
Pp1306 AAC17104 Endo-1,3(4)-β-glucanase Phaffia rhodozyma 1.00E−30 1 Filamentous fungus

6.6. Transport
Pp0917 CAD21006 ABC transporter (ATP-binding cassette transporter) F. neoformans 3.00E−68 1 Filamentous fungus
Pp1541 AAC08353 Calcium/proton exchanger N. crassa 6.00E−10 1 Filamentous fungus
Pp1713 T40789 Clathrin light chain S. pombe 1.00E−17 2 Yeast
Pp1340 CAA05841 Plasma membrane (H+) ATPase U. viciae-fabae 1.00E−105 1 Filamentous fungus
Pp1536 T38039 Probable nuclear transport factor 2 S. pombe 1.00E−26 1 Yeast
Pp1524 NP_594553 Putative membrane protein required for ER-Golgi transport S. pombe 4.00E−10 1 Yeast

6.7. Nucleotide
Pp2031 BAD00051 Ribonuclease T2 A. bisporus 2.00E−29 1 Filamentous fungus
Pp1845 XP_322797 Ribonucleoside-diphosphate reductase large chain N. crassa 1.00E−90 1 Filamentous fungus
Pp0547 AAN73281 UPL-1 Giardia intestinalis 4.10E−06 1 Protozoa

6.8. Protein modification
Pp0618 BAB56108 Carboxypeptidase Aspergillus nidulans 4.00E−21 1 Filamentous fungus
Pp1631 NP_253798 Lactoylglutathione lyase Pseudomonas aeruginosa 3.00E−13 1 Bacteria
Pp0643 AAA20876 Pepsinogen Aspergillus niger 1.00E−77 3 Filamentous fungus
Pp1341 CAC28786 Related to UDP-acetylglucosamine-peptide N-glucosaminyltransferase N. crassa 7.00E−44 1 Filamentous fungus
Pp1032 NP_035322 Proteasome activator subunit 3 M. musculus 1.00E−11 3 Mammal
Pp0747 AAG05190 ATP-dependent Clp protease proteolytic subunit P. aeruginosa 5.00E−22 2 Bacteria
Pp0915 AAB19394 Aspartate aminotransferase S. cerevisiae 2.00E−47 1 Yeast

6.9. Other metabolism
Pp1221 CAD79489 Glyoxal oxidase 2 Ustilago maydis 8.20E−28 1 Filamentous fungus
Pp0235 CAD79489 Glyoxal oxidase 2 Ustilago maydis 2.00E−34 1 Filamentous fungus
Pp1324 AAF02494 Alcohol oxidase 1 Pichia methanolica 1.00E−23 1 Yeast
Pp0218 T46646 Pyridoxine (Vitamin B6) biosynthesis protein pdx1 Cercospora nicotianae 5.00E−26 1 Filamentous fungus

7. Transposon
Pp0944 NP_921277 Transposase Tn10 Oryza sativa 7.00E−77 1 Plant

8. Unclassified
Pp0116 T30954 Hypothetical protein C44E4.6 C. elegans 6.00E−15 1 Nematode
Pp0115 AAA65309 pB602L African swine fever virus 1.00E−07 2 Virus
Pp1326 NP_780783 Hypothetical protein CTC00065 Clostridium tetani 5.10E−14 1 Bacteria
Pp0529 NP_754280 Transthyretin-like protein precursor E. coli 2.00E−17 1 Bacteria
Pp0819 NP_267806 Hypothetical protein L98109 Lactococcus lactis 7.00E−07 1 Bacteria
Pp0308 CAB85694 Hypothetical protein A. bisporus 7.60E−15 12 Filamentous fungus
Pp0103 AAK25792 Putative Egh16H1 precursor isoform A B. graminis f. sp. hordei 7.00E−12 4 Filamentous fungus
Pp0104 JC4750 gEgh 16 protein B. graminis f. sp. hordei 2.00E−32 36 Filamentous fungus
Pp0417 JC4750 gEgh 16 protein B. graminis f. sp. hordei 3.00E−34 142 Filamentous fungus
Pp0730 AAK25793 Putative Egh16H1 precursor isoform B B. graminis f. sp. hordei 5.00E−11 1 Filamentous fungus
Pp1039 JC4750 gEgh 16 protein B. graminis f. sp. hordei 3.00E−26 1 Filamentous fungus
Pp1043 JC4750 gEgh 16 protein B. graminis f. sp. hordei 3.00E−32 1 Filamentous fungus
Pp0326 CAD10781 Pentahydrophobin Claviceps purpurea 7.90E−06 1 Filamentous fungus
Pp0927 NP_758766 Hypothetical protein Erwinia amylovora 2.00E−24 1 Filamentous fungus
Pp1044 AAK52794 MAS3 protein M. grisea 9.00E−09 1 Filamentous fungus
Pp1429 AF264035 MAS1 protein M. grisea 4.00E−20 1 Filamentous fungus
Pp1610 AAK52794 MAS3 protein M. grisea 5.00E−05 1 Filamentous fungus
Pp0119 EAA55479 Hypothetical protein MG09286.4 M. grisea 1.10E−10 1 Filamentous fungus
Pp0222 EAA49745 Hypothetical protein MG09736.4 M. grisea 4.00E−06 1 Filamentous fungus
Pp0612 EAA48468 Hypothetical protein MG00126.4 M. grisea 3.30E−16 1 Filamentous fungus
Pp1045 EAA53245 Hypothetical protein MG07522.4 M. grisea 1.40E−31 1 Filamentous fungus
Pp2038 EAA51058 Hypothetical protein MG04818.4 M. grisea 2.00E−25 2 Filamentous fungus
Pp0225 XP_330149 Hypothetical protein N. crassa 3.40E−20 1 Filamentous fungus
Pp0534 XP_328580 Hypothetical protein N. crassa 1.30E−07 1 Filamentous fungus
Pp0704 XP_322643 Predicted protein N. crassa 1.10E−11 1 Filamentous fungus
Pp0809 XP_328793 Hypothetical protein N. crassa 5.20E−10 1 Filamentous fungus
Pp0829 XP_328520 Hypothetical protein N. crassa 9.60E−09 1 Filamentous fungus
Pp0925 XP_331047 Hypothetical protein N. crassa 1.00E−35 1 Filamentous fungus
Pp1006 XP_322643 Predicted protein N. crassa 1.00E−12 1 Filamentous fungus
Pp1013 XP_324202 Predicted protein N. crassa 2.60E−09 2 Filamentous fungus
Pp1027 XP_327468 Hypothetical protein N. crassa 2.00E−59 1 Filamentous fungus
Pp1325 XP_326398 Hypothetical protein N. crassa 2.70E−29 1 Filamentous fungus
Pp1411 XP_328221 Hypothetical protein N. crassa 1.70E−06 1 Filamentous fungus
Pp1615 XP_324693 Hypothetical protein N. crassa 3.00E−30 1 Filamentous fungus
Pp1835 XP_327028 Hypothetical protein N. crassa 5.00E−11 1 Filamentous fungus
Pp1925 CAD21504 conserved hypothetical protein N. crassa 5.00E−09 1 Filamentous fungus
Pp2010 XP_324370 Predicted protein N. crassa 1.00E−16 1 Filamentous fungus
Pp0334 NP_054890 Post-synaptic protein CRIPT; HSPC139 protein H. sapiens 8.80E−16 1 Mammal
Pp0748 BAA91611 Unnamed protein product H. sapiens 9.00E−09 1 Mammal
Pp0346 NP_001009405 PTPL1-associated RhoGAP 1 Rattus norvegicus 1.70E−06 1 Mammal
Pp1446 AAP78751 Ac1147 R. norvegicus 7.10E−34 1 Mammal
Pp1514 Q94480 VEG136 protein Dictyostelium discoideum 2.00E−31 1 Mycetozoan
Pp0516 T23541 Hypothetical protein K09C8.4 C. elegans 1.00E−05 1 Nematode
Pp0826 NP_917657 P0410E01.14 (hypothetical protein) O. sativa 7.00E−14 1 Plant
Pp1134 AAB49498 183 kDa protein Odontoglossum ringspot virus 3.00E−05 1 Virus
Pp1439 XP_332134 Hypothetical protein N. crassa 1.00E−39 1 Filamentous fungus
Pp0444 T37512 Hypothetical protein SPAC11D3.01c S. pombe 6.00E−14 1 Yeast
Pp0522 T38996 Hypothetical protein SPAC637.04 S. pombe 2.00E−07 1 Yeast
Pp0924 NP_596150 Hypothetical zinc finger protein S. pombe 2.50E−06 1 Yeast
Pp0931 NP_595085 Hypothetical glycine-rich protein S. pombe 6.80E−06 1 Yeast
Pp1019 NP_595449 Conserved hypothetical protein S. pombe 8.00E−21 1 Yeast
Pp1106 NP_595642 Hypothetical protein S. pombe 1.00E−07 2 Yeast
Pp1238 T41411 Hypothetical protein SPCC576.01c S. pombe 1.20E−06 1 Yeast
Pp1704 EAA50939 Hypothetical protein MG04698.4 M. grisea 1.00E−16 1 Filamentous fungus
Pp0406 EAA55557 Hypothetical protein MG01208.4 M. grisea 2.50E−44 1 Filamentous fungus
Pp1626 EAA49354 Hypothetical protein MG01012.4 M. grisea 3.00E−41 1 Filamentous fungus
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The best BLASTX score is reported for redundant clones (Table 1). A total of 189 putative genes were identified, of which 28.6% shared similarity to proteins from yeast, 50.8% to protein sequences from other fungi, while the rest exhibited similarity to proteins from a wide variety of organisms including bacteria, plants, mammals, insects, nematodes, and other invertebrates. The P. pachyrhizi cDNA library contained a broad range of genes, predominantly encoding putative proteins involved in primary metabolism, gene/protein expression, and cell structure (Table 1; Fig. 2). The ESTs with significant similarity to hypothetical proteins or proteins with unknown function were placed into the unclassified proteins category (Table 1; Fig. 2). The EST sequences with significant similarities (Evalue ≤ 10−15) to fungal and plant ESTs are shown in Table 2. Two different homologs of gEgh16, a protein expressed by Blumeria graminis f. sp. hordei during appressorium formation, were the most abundant ESTs in the P. pachyrhizi EST library (Table 1).

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Fig. 2. Classification of the 189 unique P. pachyrhizi ESTs from the germinating urediniospore library. The ESTs with significant matches (BLASTX Evalue < E−5) to the non-redundant database were classified into functional Expressed Gene Anatomy Database categories as described in Table 1. The percentage of ESTs in each of the eight categories is shown.

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Table 2.

EST clones displaying similarity (BLASTN, Evalue < 1E−15) to entries in the NCBI EST database

Clone Accession No. Description E value Organism
Pp0116 BI191959 l3h06fs.r1 Fusarium sporotrichioides Tri 10 overexpressed cDNA library F. sporotrichioides cDNA clone l3h06fs 5′, mRNA sequence 1.10E−24 Filamentous fungus
Pp0206 AU011975 AU011975 S. pombe late log phase cDNA S. pombe cDNA clone spc06169, mRNA sequence 4.90E−16 Yeast
Pp0406 BU060702 Fgr-C_1_H20_T3 Carbon-starved mycelia G. zeae cDNA, mRNA sequence 6.00E−35 Filamentous fungus
Pp0437 CB012207 Lb12C03 mycelium of L. bicolor grown for 3 weeks L. bicolor cDNA 5′, mRNA sequence 7.00E−32 Filamentous fungus
Pp1004 CF883485 Tric088xm20.b1 Trichoderma reesei mycelial culture, Version 6 October 2003 H. jecorina cDNA clone tric088xm20, mRNA sequence. 1.00E−51 Filamentous fungus
Pp1027 BU060160 Fgr-C_0_M05_T7 Carbon-starved mycelia G. zeae cDNA, mRNA sequence 0 Filamentous fungus
Pp1147 BG279541 b3h06np.r1 N. crassa sexual cDNA library, Uni-zap vector system N. crassa cDNA clone b3h06np 5′, mRNA sequence 1.00E−152 Filamentous fungus
Pp1318 CF190146 k7i06j2.r1 C. neoformans strain B3501 C. neoformans var. neoformans cDNA clone k7i06j2 5′, MRNA sequence 1.30E−21 Filamentous fungus
Pp1326 CF847171 psHB042xA02f USDA-IFAFS: expression of P. sojae genes during infection and propagation_sHB P. sojae cDNA clone sHB042A02 5′, mRNA sequence 3.00E−121 Oomycete
Pp1420 BQ110457 VD0108A10 VD01 Verticillium dahliae cDNA, mRNA sequence 1.00E−114 Filamentous fungus
Pp1432 AW324553 Basidiome and primordium cDNA libraries A. bisporus cDNA 5′ similar to β-tubulin, mRNA sequence 2.50E−46 Filamentous fungus
Pp1446 BU038322 LIT000228 root-induced cDNA library from L. bicolor L. bicolor cDNA, MRNA sequence 1.00E−124 Filamentous fungus
Pp1504 CF641217 D37_B10 Filamentous Forced Diploid Ustilago maydis cDNA 3′, mRNA sequence 5.00E−42 Filamentous fungus
Pp1547 CB898049 tric013xf01 Trichoderma reesei mycelial culture, Version 3 April H. jecorina cDNA clone tric013xf01, mRNA sequence 2.30E−51 Filamentous fungus
Pp1709 CF639134 D11_G01 Filamentous Forced Diploid U. maydis cDNA 3′, mRNA sequence 4.00E−27 Filamentous fungus
Pp1744 AI211414 p0b02a1.r1 A. nidulans 24 h asexual developmental and vegetative cDNA lambda zap library E. nidulans cDNA clone p0b02a1 5′, mRNA sequence 3.20E−28 Filamentous fungus
Pp1811 AW333990 S29A2 AGS-1 P. carinii cDNA 3′, mRNA sequence 4.80E−68 Filamentous fungus
Pp1843 CF644300 K19_B10 Filamentous Forced Diploid U. maydis cDNA 3′, mRNA sequence 3.00E−116 Filamentous fungus
Pp0127 BQ800593 EST 7628 Veraison Grape berries SuperScript Plasmid Library Vitis vinifera cDNA clone PT011A12 3′, mRNA sequence 6.90E−54 Plant
Pp0207 BQ464782 HU01I02T HU Hordeum vulgare subsp. vulgare cDNA clone HU01I02 5-PRIME, mRNA sequence 0 Plant
Pp0236 BQ907430 P006B08 Oryza sativa mature leaf library induced by M. grisea O. sativa cDNA clone P006B08, mRNA sequence 1.30E−53 Plant
Pp0404 CB643819 OSJNEb04L15.r OSJNEb O. sativa (japonica cultivar-group) cDNA clone OSJNEb04L15 3′, mRNA sequence 0 Plant
Pp0611 CA522045 KS11039D12 KS11 Capsicum annuum cDNA, mRNA sequence 1.10E−32 Plant
Pp0630 CD879728 AZO4.106C24F011012 AZO4 Triticum aestivum cDNA clone AZO4106C24, mRNA sequence 1.40E−15 Plant
Pp0713 CD879049 AZO4.104E06F010929 AZO4 T. aestivum cDNA clone AZO4104E06, mRNA sequence 2.10E−17 Plant
Pp0729 BI123652 I026P65P Populus leaf cDNA library Populus tremula x Populus tremuloides cDNA, mRNA sequence 7.50E−39 Plant
Pp0910 BQ908773 T015B01 Oryza sativa mature leaf library induced by M. grisea O. sativa cDNA clone T015B01, MRNA sequence 2.80E−45 Plant
Pp1219 CD878534 AZO4.102P17F011002 AZO4 T. aestivum cDNA clone AZO4102P17, mRNA sequence 3.00E−17 Plant
Pp1724 CA126740 SCVPLR1006B09.g LR1 Saccharum officinarum cDNA clone SCVPLR1006B09 5′, mRNA sequence 1.00E−80 Plant
Pp1729 CA253801 SCRLFL4105G02.g FL4 S. officinarum cDNA clone SCRLFL4105G02 5′, mRNA sequence 2.80E−91 Plant
Pp1848 CF811551 NA760 cDNA non-acclimated Bluecrop library Vaccinium corymbosum cDNA 5′, mRNA sequence 7.20E−24 Plant
Pp1924 BU672690 TR51 Leaf rust-infected wheat T. aestivum/P. triticina mixed EST library cDNA clone TR51, mRNA sequence 8.00E−35 Plant
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3.2. Gene families
Among the 908 ESTs analyzed, 18 potential gene families were identified by sequence similarity. Predicted function of these gene families could be ascribed to four groups, whereas 14 of the putative gene families did not show any significant similarity to entries in the databases. Eleven families contained two members, and four of them had three members. The remaining three putative gene families consisted of four, five, and nine members, respectively. The latter gene family contained four distinct homologs of gEgh16, one for the putative gEgh16 precursor isoform A, and one for the putative gEgh16 precursor isoform B from B. graminis f. sp. hordei. In addition, this group had one homolog for MAS1 and two homologs for MAS3 from Magnaporthe grisea. Three gene families showed similarity to homologs for DAHP synthase, conidiation protein 6 from Neurospora crassa, and the non-histone chromosomal proteins from Saccharomyces cerevisiae.

4. Discussion
Within the past decade, EST analyses have been conducted for several filamentous fungi and oomycetes such as: Agaricus bisporus (Ospina-Giraldo et al., 2000), Aspergillus flavus and Aspergillus parasiticus (OBrian et al., 2003), Aspergillus nidulans (Sims et al., 2004), B. graminis (Thomas et al., 2001), Cryphonectria parasitica (Dawe et al., 2003), Fusarium graminearum (Trail et al., 2003), Heterobasidium annosum (Abu et al., 2004 and Karlsson et al., 2003), M. grisea (Ebbole et al., 2004 and Kim et al., 2001), Mycosphaerella graminicola (Keon et al., 2000), N. crassa (Nelson et al., 1997 and Zhu et al., 2001), Pleurotus ostreatus (Lee et al., 2002), Schizophyllum commune (Guettler et al., 2003), Sclerotinia sclerotiorum (Li et al., 2004), Trichoderma reesi (Diener et al., 2004 and Steen et al., 2003), Ustilago maydis (Austin et al., 2004 and Nugent et al., 2004), Verticillium dahliae (Neuman and Dobinson, 2003), and Phytophthora infestans (Kamoun et al., 1999, Qutob et al., 2000 and Randall et al., 2005). In addition to the fungal ESTs available in the dbEST database at the NCBI, another EST database exists with sequences from 14 different phytopathogenic fungi and oomycetes (Soanes et al., 2002). In this study, we investigate the molecular genetics in the obligate soybean rust pathogen P. pachyrhizi. A total of 908 randomly chosen EST clones were sequenced and analyzed to identify which genes are expressed in germinating urediniospores. A relatively low level of redundancy was found among the P. pachyrhizi EST clones, similar to what has been observed in EST analyses from other filamentous fungi (Keon et al., 2000, Lee et al., 2002, Ospina-Giraldo et al., 2000, Thomas et al., 2001 and Trail et al., 2003). More than 52% of the EST clones showed no significant similarity to the entries in the public protein databases, which highlights the paucity in our knowledge of gene expression in filamentous fungi. The 432 P. pachyrhizi sequences that showed significant matches to sequences in the databases were classified into eight functional categories following the EGAD. Although proteins with unknown function or hypothetical proteins were the most prevalent, proteins involved in metabolism and in protein and gene expression were highly represented (Table 1).

Among the 908 cDNA clones, 488 unique ESTs were identified. These unigenes represent approximately 4–5% of the total 8000–12,000 expressed genes that are estimated in filamentous fungi (Kupfer et al., 1997 and Martinez et al., 2004). The remaining 420 sequences correspond to redundant cDNAs that form clusters ranging from 2 to 142 ESTs. Some P. pachyrhizi genes appear to be highly expressed during urediniospore germination, especially the two EST clones Pp0104 and Pp 0417, which share similarity to gEgh16 from B. graminis and appeared 36 and 142 times, respectively, among the clones sequenced in the library (Table 1). The function of gEgh16 is unknown (Justesen et al., 1996).

When the P. pachyrhizi ESTs were queried against the dbEST at NCBI, only 18 ESTs showed significant similarity (Evalue ≤ 10−15) to fungal or yeast entries, while 14 ESTs showed significant similarity to plant entries (Table 2). The low number of P. pachyrhizi ESTs with similarity to other fungi is due to the lack of gene expression studies that have been conducted in fungi. Two P. pachyrhizi EST clones, Pp1147 and Pp1420, have significant similarity to ribosomal proteins from N. crassa and S. cerevisiae, respectively, and these two ESTs also have high similarity to ESTs from other filamentous fungi. The EST Pp1027 shows significant similarity to a hypothetical protein from N. crassa and A. nidulans (Evalue < 10−30) and 93% identity (Evalue = 0) to an EST from Gibberella zeae, which suggests that it is a conserved gene.

Spore germination is an essential developmental stage in the life cycle of all filamentous fungi. It is a highly regulated process that responds to environmental stimuli via signaling cascades that are amenable to genetic and biochemical inquiry (Osherov and May, 2000 and Osherov and May, 2001). Three important steps can be distinguished during spore germination. First, the dormancy is broken in response to appropriate environmental conditions. Second, isotropic growth occurs, involving water uptake and the resumption of numerous metabolic activities. Third, polarized growth takes place and a germ tube is formed from which new mycelium originates (d’Enfert, 1997). Unlike most filamentous fungi in which low-molecular mass nutrients such as sugars, amino acids, and inorganic salts are required for conidial germination (Osherov and May, 2001), P. pachyrhizi urediniospores are capable of germinating on the surface of water. For some fungi, contact with a solid surface is required for conidial germination (Thomas et al., 2001). It is interesting to note that two ESTs identified in this analysis, Pp1527 and Pp0839, share very high similarity to Ca2+/calmodulin-dependent protein kinase and calmodulin kinase I, respectively. The expression of calmodulin is induced by contact with a hard surface in both Colletotrichum gloeosporioides and M. grisea (Kim et al., 1998, Kim et al., 2000 and Liu and Kolattukudy, 1999). The expression of these calmodulin kinase homologs suggests that a similar calcium-signaling pathway may regulate urediniospore germination in P. pachyrhizi.

In fungi, the cell wall undergoes significant modification during spore germination. Three P. pachyrhizi ESTs showed similarity to enzymes involved in the dissolution and formation of the cell wall. EST clones Pp0122, Pp0922, and Pp1605 share similarity to chitin deacetylase, acetylxylan esterase, and chitin synthase (csm1), respectively, from M. grisea. The csm1 gene product contains a myosin motor-like domain (Park et al., 1999). In A. nidulans, its homolog CsmA has an important role in polarized cell wall synthesis and maintenance of cell wall integrity, and the myosin motor-like domain has been shown to be required for these functions (Horiuchi et al., 1999).

DNA and RNA synthesis do not appear to be necessary during the early stages of spore germination, whereas protein synthesis is required (Osherov and May, 2001). They suggest that dormant conidia contain a pre-existing pool of mRNA and ribosomes that are primed for rapid activation and translation in the presence of nutrients. Our results indicate that increased protein synthesis activity occurs during spore germination in P. pachyrhizi. Three different homologs for translation initiation factors and two homologs for elongation factors were identified, as well as several genes involved in post-translational modification, protein modification, and metabolism of amino acids (Table 1). In their model of spore germination, DNA and RNA synthesis are required in the later stages of spore germination for hyphal development (Osherov and May, 2001). As the germination of the P. pachyrhizi urediniospores was asynchronous in our experiment, sequences similar to genes involved in both the early and later spore germination processes were found among the P. pachyrhizi ESTs.

Several putative gene families were identified among the ESTs analyzed in this study. The main group consists of nine different ESTs: four ESTs, Pp0104, Pp0417, Pp1033, and Pp1039, are homologs of gEgh16; Pp0103 is a homolog of the putative gEgh16 precursor isoform A; Pp0730 is a homolog of the putative gEgh16 precursor isoform B from B. graminis f. sp. hordei; Pp1429 is a homolog for MAS1; and two ESTs, Pp1044 and Pp1610, are homologs for MAS3 from M. grisea. The EST clones Pp0104 and Pp0417, which are similar to gEgh16, are highly redundant in the P. pachyrhizi library suggesting that they are highly expressed during urediniospore germination. The function of the gEgh16 protein has not been determined in B. graminis f. sp. hordei, but it is highly expressed during germ tube formation and hyphal growth. There is evidence that gEgh16 is a member of a gene family in B. graminis f. sp. hordei (Justesen et al., 1996). Although the function of MAS1 and MAS3 are unknown in M. grisea, the genes encoding for these proteins are expressed during appressorium formation (Choi and Dean, 2000).

Another potential gene family in P. pachyrhizi is comprised of five ESTs similar to DAHP synthase (Pp0323, Pp0425, Pp0744, Pp1336, and Pp1503). DAHP synthase catalyzes the first step in the shikimate pathway that leads to the biosynthesis of aromatic amino acids. In N. crassa and Escherichia coli, three isozymes of DAHP synthase have been characterized and each one is regulated by the three aromatic amino acids. In A. nidulans and S. cerevisiae, two DAHP synthase encoding genes have been described, and the enzymes are differentially regulated by tyrosine and phenylalanine (Hartmann et al., 2001 and Künzler et al., 1992). In addition to the DAHP synthase homologs, a P. pachyrhizi EST clone (Pp0134) was found to share similarity to dehydroshikimate dehydrogenase, which is also part of the shikimate pathway. It has been shown that quinate and shikimate, two metabolic intermediates of the shikimate pathway, can be metabolized by a variety of fungi as alternative carbon sources (Keller and Hohn, 1997).

Two ESTs (Pp1628 and Pp1812) share similarity to the non-histone chromosomal proteins NHP6A and NHP6B, respectively, from S. cerevisiae. NHP6A and NHP6B are high mobility group proteins, which are members of a family of heterogeneous chromatin-associated DNA-binding proteins in eukaryotic cells (Masse et al., 2002 and Yen et al., 1998). NHP6A is a member of the subclass HMG1/2 proteins that contain the HMG DNA-binding domain and are present at approximately 1 molecule per 2–3 nucleosomes (Kuehl et al., 1984). These proteins have been implicated in chromatin remodeling, DNA replication, transcription, and recombination (Giavara et al., 2005), and it will be interesting to determine their role in P. pachyrhizi ediniopsore germination.

In this study, approximately 39% of the unique ESTs appeared to be related to previously characterized genes. This highlights the scarcity of genomic information available from pathogenic fungi. The EST projects have been shown to be a valid and fast way to gain information on components that regulate vital processes in pathogenic fungi and the interaction with their hosts. In 2002, a Phakopsora genome sequencing project, funded by the U.S. Department of Agriculture-Agricultural Research Service and the Department of Energy (DOE), was initiated at the DOE-Joint Genome Institute to generate draft quality sequence of P. pachyrhizi and P. meibomiae. The ESTs identified in this study, along with the analyses of the cDNA libraries from P. pachyrhizi infected soybeans, will aid in the annotation of genes from the Phakopsora genome project. These data will facilitate our understanding of the biology and the evolution of obligate fungal pathogens and will also advance our efforts to develop effective means for soybean rust control.

Acknowledgments
We thank Connie Briggs at the USDA-ARS-ERRC-NAF for sequencing the EST clones. We are grateful to Drs. Seogchan Kang, William Schneider, Morris Bonde, Paul Tooley, and Douglas Luster for critical review of the manuscript. This work was supported by USDA-ARS CRIS Projects 1920-22000-23-00D and 1920-22000-27-00D. M.L.P.B. was supported by an USDA-ARS Administrator’s Postdoctoral Fellowship.

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Corresponding author. Fax: +1 301 619 2880.
1 Present address: DOE-Joint Genome Institute, Lawrence Berkeley National Laboratory, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA.

In vitro differentiation of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic acti

2008-11-23T21:07:00.001-08:00

Copyright © 2003 Elsevier Science (USA). All rights reserved.

In vitro differentiation of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic action of chemical and physical signals

Nicola Wiethölter, Susanne Horn, Katrin Reisige, Ursula Beike and Bruno M. Moerschbacher,

Department of Plant Biochemistry and Biotechnology, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, 48143, Münster, Germany

Received 17 July 2002; accepted 19 September 2002. ; Available online 11 March 2003.

Abstract
Biotrophic plant pathogenic fungi often develop a sophisticated series of infection structures for non-destructive host tissue penetration. In vitro, early infection structures of rust fungi—germ tube, appressorium, substomatal vesicle, infection hyphae—can easily be induced, but in vitro differentiation rates of late infection structures—haustorial mother cells (hmc), haustoria—are low at best. Under appropriate conditions (humid atmosphere), a combination of physical (mild heat shock) and chemical signals (trans-2-hexen-1-ol) induced the in vitro differentiation of hmc in the wheat stem rust fungus, Puccinia graminis f. sp. tritici. Around two thirds of the in vitro differentiated germlings developed up to three hmc which were cytologically identical to hmc formed in planta. Efficient in vitro differentiation of hmc will allow us to analyse in molecular detail the processes involved in the induction and differentiation of this critically important developmental stage of the economically important plant pathogenic rust fungi.

Author Keywords: Puccinia graminis f. sp. tritici; Wheat stem rust fungus; Haustorial mother cells; Biotrophic fungi; Leaf alcohol; Trans-2-hexen-1-ol; In vitro differentiation; Infection structures

Article Outline
1. Introduction
2. Materials and methods
2.1. Origin of fungal material
2.2. Induction of infection structures
2.3. Staining and microscopy
3. Results
4. Discussion
Acknowledgements
References
1. Introduction
Rust fungi are obligately biotrophic plant pathogens that are highly specialised for growth and development on and in their respective host plant tissues. Their biotrophic nature requires careful host tissue penetration and colonisation in order to prevent premature recognition by the host cells and the ensuing triggering of induced resistance mechanisms such as hypersensitive host cell death. Uredospore germlings of most rust fungi enter their host tissues via the natural openings of the plant stomates, thus preventing host tissue wounding (Mendgen et al., 1996).

Uredospores germinating on a plant surface produce a germ tube which tightly adheres to the cuticle, apparently a prerequisite for oriented germ tube growth towards the stomates (Dickinson, 1969; Maheshwari and Hildebrandt, 1967; Wynn, 1976). Upon reaching a stoma, tip growth of the germ tube is arrested and an appressorium is formed on top of the stoma ( Allen, 1923 and Allen, 1926). The appressorium containing the two fungal nuclei which emerged from the spore is separated from the germ tube by a septum, and a first round of synchronised mitoses occurs in the appressorium. From the appressorium, a narrow penetration peg grows through the stomatal opening and develops into a vesicle in the substomatal cavity.

The cell wall of penetration peg and vesicle differs from the cell wall of germ tube and appressorium (Harder et al., 1986; Littlefield and Heath, 1979). The cytoplasmic content of the appressorium is transferred into the substomatal vesicle where a second round of synchronised mitoses occurs, leading to a total of eight nuclei. Infection hyphae emanating from the vesicle start growing in the intercellular spaces of the host tissue, and pairs of fungal nuclei migrate into these hyphae ( Allen, 1923; Heath and Heath, 1976; Staples et al., 1975). When the tip of an infection hypha reaches the cell wall of an epidermal host cell, tip growth is arrested and a haustorial mother cell is formed. The haustorial mother cell usually containing 2–4 fungal nuclei is separated from the infection hypha by a septum ( Heath and Heath, 1975; Heath et al., 1996). From the haustorial mother cell, a narrow haustorial neck grows through the host plant’s cell wall and develops into a haustorium in the periplasmic space of the host cell. A second change in cell wall characteristics occurs upon differentiation of haustorial mother cells ( Chong and Harder, 1982; Chong et al., 1985 and Chong et al., 1986). Infection hyphae branch just proximal to the haustorial mother cell septum, and the branches develop new haustorial mother cells at their tips upon host mesophyll cell encounters. The infection hyphae of the rust mycelium remain extracellular throughout host tissue colonisation and eventual fungal sporulation. Only the terminally differentiated haustoria reach into the pericellular space of the host cells.

Quite apparently, the sophisticated series of infection structures—germ tube, appressorium, substomatal vesicle, infection hyphae, haustorial mother cells, haustoria—are a prerequisite for the obligately biotrophic rust fungi to penetrate, colonise, and feed from the host plant tissue with a minimum of host cell perturbation. The unique process of infection structure differentiation can be expected to be an absolute conditio sine qua non for the successful rust development and as such, to provide promising targets for the development of novel fungicides.

Rust uredospores readily germinate in the presence of liquid water, producing a germ tube tightly adhering to any hydrophobic surface. Also, the differentiation of infection structures can be induced in vitro when appropriate chemical or physical signals, such as an appropriately structured hydrophobic surface, are provided (Dickinson, 1969; Heath and Perumalla, 1988; Hoch and Staples, 1987; Mendgen et al., 1996; Read et al., 1992). Unlike the in vivo situation, where a series of independent signals can be assumed to regulate the development of infection structures on and in the host leaf ( Heath, 1997; Mendgen, 1982), a single trigger may induce the consecutive differentiation of appressorium, substomatal vesicle, infection hyphae and, albeit often at low frequency only, haustorial mother cells in vitro ( Deising et al., 1991; Heath and Perumalla, 1988). The cowpea rust fungus, Uromyces vignae, has even been reported to build some rare haustoria in vitro upon appropriate chemical triggers (Heath, 1990).

Topographical signals are less effective in triggering the differentiation of infection structures of rust fungi specialised on monocotyledonous host plants, such as the wheat stem rust fungus (Allen et al., 1991; Read et al., 1997). Instead, a number of other physical and chemical signals such as a mild heat shock ( Dunkle and Allen, 1971; Emge, 1958; Maheshwari et al., 1967), organic compounds ( Macko et al., 1978), host epicuticular waxes ( Daniels, 1996; Grambow, 1977; Grambow and Grambow, 1978; Grambow and Riedel, 1977), or leaf volatiles ( Daniels, 1996; Grambow, 1977; Grambow and Riedel, 1977) have been reported to trigger the sequential in vitro development of appressoria, substomatal vesicles, and infection hyphae of the wheat stem rust fungus. Recently, a combination of surface ridges of appropriate sizes and spacings with trans-2-hexen-1-ol was shown to act synergistically in inducing these infection structures (Collins et al., 2001). However, the wheat stem rust fungus does not easily differentiate haustorial mother cells in vitro. We here report on the reproducible, high frequency differentiation of haustorial mother cells of the wheat stem rust fungus in vitro, by the synergistic action of a physical and a chemical signal.

2. Materials and methods
2.1. Origin of fungal material
Uredospores of the wheat stem rust fungus Puccinia graminis f. sp. tritici Eriks. & Henn., race 32, were collected from fully susceptible wheat plants Triticum compactum L. cv. Little Club. Uredospores were frozen in liquid nitrogen and stored at −70 °C. Spores were reactivated for 3 min at 43 °C.

2.2. Induction of infection structures
For the induction of infection structures of the wheat stem rust fungus (appressoria, substomatal vesicles, infection hyphae and haustorial mother cells), 0.5 mg of uredospores were distributed with a brush in the lid of a Petri dish (Ø 6 cm, Greiner, Frickenhausen). Five milliliters of trans-2-hexen-1-ol (Aldrich, Taufkirchen) (0.5 mM in doubly distilled water) was filtered (pore Ø 0.22 μm, cellulose ester membrane, Fisherbrand, Schwerte; 30 ml Luer Lock syringe, Plastipak, Becton–Dickinson, Heidelberg) into the bottom of the Petri dishes. The spore-containing lids were replaced on the Petri dish bottoms and sealed with parafilm (American National Can, Baltimore, Maryland) to prevent evaporation. The Petri dishes were placed in a temperature controlled chamber (Heraeus, Hanau) at 23 °C where the uredospores started to germinate. When the average length of the germ tubes had reached the size of the spore diameter (approx. 50–60 min) the germlings were given a mild heat shock at 30 °C for 2 h (Emge, 1958; Maheshwari et al., 1967), followed by further incubation at 23 °C. All incubation steps were carried out in darkness.

2.3. Staining and microscopy
Fungal infection structures were analyzed using an Olympus BX 40 and a Leica DM RBE microscope both equipped with epifluorescence, and an Olympus IX 50 inverted microscope with phase contrast optics. The filter module U-MNV (excitation filter BP 400–410, dichroic mirror DM 455, barrier filter BA 455 nm) was used for fluorescence microscopy using the Olympus BX 40. Fluorescence microscopy using the Leica DM RBE was carried out with the excitation filter BP 450–490 nm, the dichroic mirror RKP 510 nm and the barrier filter LP 515 nm. Microphotographs were taken on Agfa CT Precisa slide film. Cell walls of the fungus were stained using Calcofluor White (Sigma, Taufkirchen) (Maeda and Ishida, 1967). For this procedure, a stock solution (2.5 mg ml−1 in water) was diluted 1:20 before use. The fungus was incubated with this staining solution for 30 s and washed 10 times with water. Fungal nuclei could be stained afterwards using DAPI (4′,6-Diamin-2′-phenylindol-dihydrochloride; Sigma, Taufkirchen) (0.01 μgml−1 DAPI in water, 10 s) then washed 10 times with water (Butt et al., 1989).

3. Results
Germlings of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, were treated with trans-2-hexen-1-ol, either as a volatile when the germlings were grown in a humid atmosphere, or in dissolved form when the germlings were grown immersed in a liquid medium. In both cases, trans-2-hexen-1-ol induced the differentiation of an appressorium, a substomatal vesicle, and one or two infection hyphae within 24 h. Fig. 1 shows that in a humid atmosphere, induction by trans-2-hexen-1-ol also led to the formation of haustorial mother cells in about 10% of the germlings within about three days. In contrast, no haustorial mother cells were formed when the germlings were grown submerged in a trans-2-hexen-1-ol solution (data not shown).

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Fig. 1. Time course of in vitro differentiation of infection structures by Puccinia graminis f. sp. tritici uredospores induced by the application of the volatile leaf alcohol trans-2-hexen-1-ol (0.5 mM). Around 20% of the sporelings produced infection structures within 24 h, and about half of these formed haustorial mother cells. Symbols represent appressoria (•), substomatal vesicles (▪), infection hyphae (), and haustorial mother cells (). Data given are means ± SD of three independent experiments, with a minimum of 80 sporelings counted per time point in each experiment.

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In vitro differentiated haustorial mother cells of the wheat stem rust fungus appeared as small (15 μm in length), granular structures at the end of the infection hypha, from which they were clearly separated by a septum (Fig. 2E). Their cell walls appeared thicker than those of the infection hypha, and the fluorescent brightener Calcofluor White bound more strongly, leading to bright fluorescence under UV-light. Invariably, two nuclei were observed in each haustorial mother cell (Fig. 2E). These typical characteristics easily allowed their unequivocal identification, e.g. as compared to stress-induced terminal swellings of infection hyphae ( Fig. 2D).

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Fig. 2. In vitro differentiation of infection structures including haustorial mother cells by the wheat stem rust fungus Puccinia graminis f. sp. tritici after application of a mild heat shock (2 h, 30 °C) and trans-2-hexen-1-ol (0.5 mM) in a humid atmosphere. (A) and (B) represent fully differentiated germlings using bright field and phase contrast optics, respectively (gt, germ tube; ap, appressorium; sv, substomatal vesicle; ih, infection hypha; hmc, haustorial mother cell) (bar: 15 μm). When fungal structures were stained with DAPI (E, F) and Calcofluor (C–F), nuclei, septa, and cell wall alterations were visible under fluorescent light, and haustorial mother cells were clearly distinguishable from terminal swellings of infection hyphae (D). When differentiation was induced by trans-2-hexen-1-ol alone, appressoria and subsequent infection structures formed at the end of a long and often branched germ tube (C). When trans-2-hexen-1-ol was combined with a heat shock, infection structures were formed much faster at the end of a short germ tube only (A, B, D–F). When germlings were immersed in liquid culture medium after infection structures had formed (A, F), an occasional outgrowth of haustorial mother cells was observed (F).

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One of the most effective ways of inducing infection structure differentiation in the wheat stem rust fungus is a mild heat shock given shortly after germination. This treatment is active with germlings growing submerged in liquid or in a humid atmosphere, but no haustorial mother cells formed even after prolonged times of incubation (data not shown). However, we found that a combination of the chemical signal trans-2-hexen-1-ol (0.5 mM) and the physical signal of a mild heat shock (30 °C, 2 h) effectively triggered the induction of haustorial mother cells at high frequency, when the germlings were grown in a humid atmosphere. Fig. 3 gives the time course of appearance of the different infection structures induced.

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Fig. 3. Time course of in vitro differentiation of infection structures by Puccinia graminis f. sp. tritici uredospores induced by a mild heat shock (2 h, 30 °C, grey bar) and the application of the volatile leaf alcohol trans-2-hexen-1-ol (0.5 mM). Appressoria (•) started to appear during the heat shock. Substomatal vesicles (▪) and infection hyphae () were first observed 2 and 8 h after the end of the heat shock, respectively. First haustorial mother cells () were formed between 21 and 45 h after the heat shock. Data given are means ± SD of three independent experiments, with a minimum of 80 germlings counted per time point in each experiment.

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Tip growth of the germ tube was stopped by the heat shock so that the germlings had a compact shape (Figs. 2A, B, D–F). Under these conditions, about 60% of the germinated uredospores had differentiated appressoria, substomatal vesicles, and infection hyphae 24 h after germination. Haustorial mother cells emerged 24–48 h after germination, and a maximum hmc-rate of 40% was reached 72 h after spore germination (Fig. 3), after which time no further increase was observed.

Often, haustorial mother cells developed at the tip of both infection hyphae of a rust germling. Some infection hyphae branched just proximal to the haustorial mother cell septum, and a third haustorial mother cell might then form at the tip of the branch (Figs. 2A and B). Fungal growth and development then stopped; we never observed more than three haustorial mother cells on a single germling. In order to test whether failure of further growth could be attributed to exhaustion of the nutrient pool of the uredospores, we dissolved the trans-2-hexen-1-ol in axenic culture medium (Fasters et al., 1993) instead of water, and inverted the Petri dishes at different times after germination, bringing the germlings into contact with the nutrient solution. As a control, Petri dishes with trans-2-hexen-1-ol in water were inverted at the same times. No substantial further growth was observed even in the axenic culture medium. Immersion of the germlings at earlier times after germination (7, 12, or 48 h) prevented or stopped further development of haustorial mother cells.

In axenic medium, we occasionally observed outgrowth of a fungal hypha from the differentiated haustorial mother cell (Fig. 2F). We never observed the differentiation of haustoria from the haustorial mother cells induced in vitro. Failure to resume sustained growth may be due to the trans-2-hexen-1-ol present in the medium, as growth did resume in pure culture medium. However, without the addition of trans-2-hexen-1-ol, no haustorial mother cells were formed.

4. Discussion
In vitro differentiation of haustorial mother cells has been reported previously for several rust species, including the bean rust fungus, Uromyces appendiculatus (Maheshwari et al., 1967), the cowpea rust fungus, U. vignae (Heath and Perumalla, 1988; Stark-Urnau and Mendgen, 1993), and the broad bean rust fungus, Uromyces viciae-fabae (Deising et al., 1991). In these cases, a single inductive signal (oil-collodion membranes or scratched polyethylene membranes) led to the formation of haustorial mother cells in 10–25% of the germlings.

In vitro induction of haustorial mother cell differentiation of the wheat stem rust fungus appears to be strictly dependent on (i) the development in a humid atmosphere—no haustorial mother cells were observed when the fungus grew submerged in water or liquid axenic medium—and (ii) on the presence of a suitable topographical signal (Read et al., 1997) or of a volatile chemical inductor, such as the leaf alcohol trans-2-hexen-1-ol. Under such conditions of a single inductive signal, haustorial mother cell differentiation was sporadic and occurred at low frequency only. A reproducible high frequency induction of haustorial mother cells was reached by the combination of a mild heat shock—which given alone induced appressoria, substomatal vesicles, and infection hyphae, but never haustorial mother cells—with the volatile inductor trans-2-hexen-1-ol. Under these conditions, about two thirds of the germlings developed infection structures, and about two thirds of these differentiated up to three haustorial mother cells. The high frequency of induction combined with the formation of multiple haustorial mother cells per germling allows an unprecedented high number of haustorial mother cells to be differentiated in vitro.

In planta, haustorial mother cells of the wheat stem rust fungus are oval, long and slender (Allen, 1923; Niks, 1986). They are separated from the infection hypha by a septum, they are characterised by an optically dense appearance, and they contain two nuclei ( Allen, 1923). The cell wall of in planta grown haustorial mother cells of the wheat stem rust fungus is more complex than that of the infection hyphae, containing additional layers which are also present in the septum, leading to increased stainability with Calcofluor ( Chong et al., 1985). Haustorial mother cells of the wheat stem rust fungus formed in vitro exhibited all of these typical characteristics.

Although the morphogenetically active signals inducing infection structure differentiation in different rust fungi differ in detail, the genetic program induced appears to share similarities. In vitro, a single trigger usually induces the sequential differentiation of appressoria, substomatal vesicles, and infection hyphae at high frequency, while the differentiation rarely extends to the building of haustorial mother cells. In planta, however, those rust fungi that do not penetrate closed stomata in the dark, e.g. Puccinia graminis f. sp. tritici, differentiate an appressorium upon encounter of a closed stoma, but the development of further infection structures is arrested until the stoma opens (Wynn and Staples, 1981). Moreover, the subsequent development of the infection hyphae appears to respond to additional host factors, as e.g. the intercellular infection hyphae of the oat crown rust fungus have been shown to grow directly into the mesophyll of an infected oat leaf, while they grow parallel to the leaf surface in an infected wheat leaf ( Moerschbacher et al., 1990). Clearly, the morphogenetic program of sequential infection structure differentiation is naturally triggered and regulated by a number of host derived signals.

The concept of multiple recognition extends to the differentiation of haustorial mother cells and, most likely at least, also of haustoria (Heath, 1997). Surface signals from the plant cell walls have been implicated in these differentiation steps ( Fasters et al., 1993; Heath, 1990; Mendgen, 1978 and Mendgen, 1982). While the exact nature of these signals acting in planta is not yet known, the wheat stem rust fungus appears to be a suitable object to study in vitro the combined action of different signals in the triggering of infection structure differentiation. It has been shown that the volatile leaf alcohol trans-2-hexen-1-ol acts synergistically with an inductive factor from epicuticular waxes of the host leaf (Grambow, 1977; Grambow and Grambow, 1978; Grambow and Riedel, 1977), and with topographical signals mimicking a gramineaceous stoma ( Collins et al., 2001). We are currently studying the combined action of all three of these factors on rust differentiation in vitro. We have shown in this study that a mild heat shock combined with the volatile inductor leads to the differentiation of haustorial mother cells. It will be difficult to identify the presumably chemical in planta equivalent of the thermal signal as haustorial mother cells did not develop in submerged culture so that application of putative signal molecules is difficult. We are currently developing methods to reproducibly deposit known amounts of putative non-volatile chemical inductors on appropriate surfaces so that we can study their morphogenetic effects on rust development in a humid atmosphere.

Acknowledgements
We gratefully acknowledge many fruitful discussions with Dr. Nick Read, University of Edinburgh, where some preliminary experiments to this study were carried out.

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Moerschbacher, B.M., Noll, U., Ocampo, C.A., Flott, B.E., Gotthardt, U., Wüstefeld, A. and Reisener, H.J., 1990. Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust. Physiol. Plant. 78, pp. 609–615. Full Text via CrossRef

Niks, R.E., 1986. Variation of mycelial morphology between species and formae speciales of rust fungi of cereals and grasses. Can. J. Bot. 64, pp. 2976–2983.

Read, N.D., Kellock, L.J., Collins, T.J. and Gundlach, A.M., 1997. Role of topography sensing for infection–structure differentiation in cereal rust fungi. Planta 202, pp. 163–170. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (32)

Read, N.D., Kellock, L.J., Knight, H. and Trewavas, A.J., 1992. Contact sensing during infection by fungal pathogens. In: Callow, J.A. and Green, J.R., Editors, 1992. Perspectives in Plant Cell Recognition, Cambridge University Press, Cambridge, pp. 137–172.

Staples, R.C., App, A.A. and Ricci, P., 1975. DNA synthesis and nuclear division during formation of infection structures by bean rust uredospore germlings. Arch. Microbiol. 104, pp. 123–127. View Record in Scopus | Cited By in Scopus (1)

Stark-Urnau, M. and Mendgen, K., 1993. Differentiation of aecidiospore- and uredospore-derived infection structures on cowpea leaves and on artificial surfaces by Uromyces vignae. Can. J. Bot. 71, pp. 1236–1242.

Wynn, W.K., 1976. Appressorium formation over stomates by the bean rust fungus: response to a surface contact stimulus. Phytopathology 66, pp. 136–146.

Wynn, W.K. and Staples, R.C., 1981. Tropism of fungi in host recognition. In: Staples, R.C. and Toenniessen, G.H., Editors, 1981. Plant Disease Control, Resistance and Susceptibility, Wiley/Interscience, New York, pp. 45–69.

Corresponding author. Fax. +49-251-832-83-71

Cloning and Characterization of a cDNA of cro rI from the White Pine Blister Rust Fungus Cronartium ribicola*1

2008-11-23T21:06:00.001-08:00

Cloning and Characterization of a cDNA of cro rI from the White Pine Blister Rust Fungus Cronartium ribicola*1

Xueshu Yu, Abul K. M. Ekramoddoullah2, Doug W. Taylor and Nina Piggott

Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, British Columbia, V8Z 1M5, Canada

Accepted 8 October 2001. ; Available online 4 March 2002.

Abstract
White pine blister rust (WPBR) is caused by the fungus Cronartium ribicola which has five spore stages on two unrelated hosts, the five-needle pines and Ribes spp. Recently, during the molecular analysis of the proteins and genes involved in host–pathogen interaction, the WPBR fungal protein Cro rI was identified in infected white pine tissues. To further characterize Cro rI, an expression cDNA library from poly(A)+ mRNA of C. ribicola axenic mycelial culture was constructed and immunoscreened and the cDNA was cloned. Sequence analysis indicated an open reading frame of 462 bases, which encodes a protein of 153 amino acid residues with a molecular mass of 16.7 kDa and a predicted isoelectric point (pI) of 8.93. Based on the N-terminal amino acid sequences of Cro rI, the secreted portion of Cro rI protein should be 136 amino acids long with several putative posttranslational modification sites and a molecular mass of 14.8 kDa. The predicted pI for the secreted portion was 9.34. The predicted N-terminal signal peptide was 17 amino acids long. The N-terminal 42-amino acid sequence of the predicted mature protein (secreted portion) was identical to the amino terminal sequence of Cro rI that was previously determined. Southern blot hybridizations indicated that the C. ribicola genome contained at least two copies of the cro rI gene. Isolation of the genomic PCR fragment, which was approximately 400 bp longer than the cDNA, and subsequent cloning and sequencing analyses confirmed that there were three introns within the coding regions. Western immunoblot analyses revealed that Cro rI protein accumulated in large amounts only in the infected white pine tissues while no trace was detectable in the alternate Ribes stage or the five different spores, suggesting a critical role of Cro rI in the haploid stage of the fungus (in pine). The translocation of Cro rI was only found to occur in cankered trees, and not in the young infected seedlings. The implications of Cro rI in pathogenesis are discussed.

Author Keywords: amplification of genomic DNA by PCR; gene structure; secretory signal; translocation; spore; susceptible tree

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25. J. A. Muir and R. S. Hunt, Assessing potential risks of white pine blister rust on western white pine from increased cultivation of currants. HortTechnology 10 (2000), pp. 523–527. View Record in Scopus | Cited By in Scopus (2)

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*1 The nucleotide sequence data reported in this paper have been submitted to the GenBank Nucleotide Sequence Database under the Accession Nos. AF232039, AF232040, AF232041, AF232042, and AF232043.

2 To whom correspondence should be addressed. Fax: (250) 363-0775. E-mail: aekramoddoul@pfc.forestry.ca.

Plasma Membrane H+-ATPase Activity in Spores, Germ Tubes, and Haustoria of the Rust FungusUromyces viciae-fabae

2008-11-23T21:05:00.001-08:00

Regular Article
Plasma Membrane H+-ATPase Activity in Spores, Germ Tubes, and Haustoria of the Rust FungusUromyces viciae-fabae*1

Christine Struck, Matthias Hahn and Kurt Mendgen 1

Lehrstuhl für Phytopathologie, Fakultät für Biologie, Universität Konstanz, 78 434, Konstanz, Germany

Accepted 9 December 1995. ; Available online 19 April 2002.

Abstract
Using plasma membrane-enriched vesicles, the properties of the H+-ATPase (EC 3.6.1.35) from the rust fungusUromyces viciae-fabaewere studied. The enzyme is strictly Mg2+-dependent and is inhibited by vanadate. The pH-optimum is at 6.7. By Western blot analysis using a monoclonal antibody against corn plasma membrane H+-ATPase a polypeptide of approximately 104 kDa could be detected. The vanadate-sensitive H+-ATPase activity of microsomal vesicles obtained from different stages of rust development was determined. Uredospores had only a very low enzyme activity (1.9 μmol Pi × mg−1protein × h−1). In germ tubes the ATPase activity was about twofold higher (4.0 μmol Pi × mg−1protein × h−1). An eightfold higher ATPase activity (16.1 μmol Pi × mg−1protein × h−1) was found in microsomal vesicles from haustoria which had been isolated from rust-infectedVicia fabaleaves. These results suggest, that the electrochemical gradient generated by the H+-ATPase of haustoria plays an important role for their function, possibly by promoting nutrient uptake from host cells.

Author Keywords: ATPase; biotrophic fungi; haustorium; nutrient uptake; plant pathogen

*1 This paper was accepted under the editorship of Robert Brambl.

Introductory mycology

2008-11-23T20:17:00.000-08:00

Author Alexopoulos, Constantine John, 1907-
Title Introductory mycology / C.J. Alexopoulos, C.W. Mims, M. Blackwell.
Publication Details New York : Wiley, 1996.
589.2 5 /4
Edition 4th ed.
Description x, 869 p. : ill. ; 25 cm.
ISBN 0471522295 (cloth : alk. paper)
9780471522294
Subject Mycology.
Other Author Mims, Charles W.,
Blackwell, Meredith.

Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses.

2008-11-20T17:08:00.000-08:00

J Virol Methods. 2008 Nov 6; : 18996414 (P,S,G,E,B,D) [Cited?]Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses.

[My paper] C F Gambley, A D W Geering, J E Thomas
Department of Primary Industries and Fisheries, Horticulture and Forestry Science, 80 Meiers Road, Indooroopilly, Queensland 4068, Australia; School of Integrative Biology, The University of Queensland, St. Lucia, Queensland 4072, Australia.
A semi-automated, immunomagnetic capture-reverse-transcription PCR (IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

Banana contains a diverse array of endogenous badnaviruses

2008-11-20T17:00:00.001-08:00

Banana contains a diverse array of endogenous badnaviruses
Andrew D. W. Geering1, Neil E. Olszewski2, Glyn Harper3, Benham E. L. Lockhart4, Roger Hull3 and John E. Thomas1

1 Department of Primary Industries and Fisheries, 80 Meiers Road, Indooroopilly, Queensland 4068, Australia
2 Department of Plant Biology, University of Minnesota, St Paul, MN 55108, USA
3 John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
4 Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA

Correspondence
Andrew D. W. Geering
andrew.geering@dpi.qld.gov.au

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars ‘Obino l'Ewai’ and ‘Klue Tiparot’, were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.

The GenBank/EMBL/DDBJ accession numbers reported in this paper are AY189378–AY189383, AY189384–AY189392, AY189444–AY189453, AY189393–AY189400, AY189401–AY189419, AY189420–AY189435 and AY189436–AY189443

First record of Nematospora coryli in Australia and its association with dry rot of Citrus

2008-11-20T16:47:00.001-08:00

First record of Nematospora coryli in Australia and its association with dry rot of Citrus

Roger G. Shivas A , E , Malcolm W. Smith B , Thomas S. Marney A , Toni K. Newman B , Debra L. Hammelswang B , Anthony W. Cooke C , Ken G. Pegg C and Ian G. Pascoe D

A Plant Science, Queensland Department of Primary Industries and Fisheries, 80 Meiers Road, Indooropilly, Qld 4068, Australia.
B Horticulture and Forestry Science, Queensland Department of Primary Industries and Fisheries, Bundaberg Research Station, 49 Ashfield Road, Kalkie, Qld 4670, Australia.
C Horticulture and Forestry Science, Queensland Department of Primary Industries and Fisheries, 80 Meiers Road, Indooropilly, Qld 4068, Australia.
D Primary Industries Research Victoria, Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Vic. 3156, Australia.
E Corresponding author. Email: roger.shivas@dpi.qld.gov.au

Abstract
Nematospora (Eremothecium) coryli was isolated from Citrus and identified for the first time in Australia. This insect-transmitted yeast was associated with dry rot in cultivated and native Citrus fruits. Although N. coryli is known as a serious seed pathogen of many tropical and sub-tropical plants, evidence is presented that it has been present and undetected in Queensland for at least ninety years.

Australasian Plant Pathology 34(1) 99–101

Submitted: 1 July 2004 Accepted: 26 July 2004 Published: 22 March 2005

Full text DOI: 10.1071/AP04075

© CSIRO 2005

Fungi defence against quarantine threats

2008-11-20T16:42:00.000-08:00

Fungi defence against quarantine threats
7/05/2008 10:36:00 AM
A key diagnostic tool which could help speedily resolve threats to Australian quarantine or export trade crises has been developed by Queensland and international scientists.
Qld Department of Primary Industries principal plant pathologist Dr Roger Shivas has co-authored the book, ‘Fungi of Australia: The Smut Fungi’ with German mycologist and world expert Dr Kálmán Vánky.

The book details long-term research into Australian smut fungi in the first comprehensive guide of these important plant pathogens for almost 100 years.

The book and a CD provide interactive keys that allow quick and accurate identification of all known species of smut fungi in Australia.

The CD was co-authored with DPI&F senior research scientist Dr Dean Beasley who has crafted over 1000 images in the first ever diagnostic key of this kind for a group of plant pathogens.

Dr Shivas anticipates that the book will become an essential resource for resolving quarantine and trade issues, as well as identifying smut fungi and the diseases they cause.

He said the rapid and accurate identification of new and unusual smut fungi will allow industry to move quickly to stop new invasions.

Dr Shivas says that the book and CD take the taxonomy of fungi into the 21st Century.

“Now anyone with basic training in plant health will be able to quickly and with reasonable confidence identify all the Australian species of smut fungi.

“There are 296 species of smut fungi in Australia from covered smut which attacks barley to the recently arrived sugarcane smut.

“Smuts are parasites usually of cereals and grasses that form black powdery masses of spores which spread the disease in the air, seeds and soil.” Dr Shivas said.

Smut fungi can cause diseases in cereal crops that could devastate yields if left untreated.

Using effective fungicidal seed treatments and the development of smut resistant varieties has reduced the importance of smut diseases in recent years.

Some exotic smut fungi still threaten Australian crops however.

“The seriousness of the threat posed by smuts to Australia’s billion dollar wheat industry was highlighted in 2004 when a shipment of Australian wheat was rejected by an importing country because it allegedly contained spores of the Karnal bunt smut fungus.

“The issue was only resolved when common contaminant spores were shown to have been confused with those of Karnal bunt.”

Dr Shivas said that event convinced him that an easy-to-use and reliable means of identifying smut fungi was needed.

* The book and CD are available from CSIRO Publishing, PO Box 1139, Collingwood VIC 3066 (sales@publish.csiro.au).

SOURCE: Queensland Country Life

Identification and systematics of rust fungi in Queensland

2008-11-20T16:24:00.000-08:00

Topic: Identification and systematics of rust fungi in Queensland
Supervisors: Tanya Scharaschkin (NRS), Roger Shivas (QDPI&F), Andrew Geering (QDPI&F)
If interested, contact: Dr Tanya Scharaschkin, School of Natural Resource Sciences. Email – t.scharaschkin@qut.edu.au
Duration: Mid November 2008 to mid February 2009 (start and end dates are flexible)
Description:
The rust fungi (Uredinales) are destructive pathogens of a range of plants including cereals, legumes, forest trees and native plants. They have caused famines and destroyed the economies of entire countries. Rusts usually attack the leaves of plants where they can produce up to five different types of spores. Most rusts are limited to specific host plant families, genera or even species.
There are approximately 7,000 species of rust fungi worldwide, with over 500 species reported from Australia. The QDPI&F Plant Pathology Herbarium (Indooroopilly) has a large collection of specimens of rust fungi from Australia. Amongst these specimens are many unidentified and undescribed species of rust fungi, particularly those found on native Australian plants.
Traditional taxonomy of the rusts has been based primarily on the morphology of teliospores. An opportunity exists for a student to work with this collection under the supervision of experienced mycologists and molecular biologists. The student will use morphological and molecular techniques to gain an understanding of the diversity and phylogeny of rust fungi in Queensland. This project has potential to result in publications (e.g., description of new species) and could be extended into an honours research project.

DNA replication cycle in parthenogenetically developing eggs of the starfish Asterina pectinifera

2008-11-07T16:23:00.000-08:00

DNA replication cycle in parthenogenetically developing eggs of the starfish Asterina pectinifera

Author(s): Nomura A, Nemoto S
Source: DEVELOPMENT GROWTH & DIFFERENTIATION Volume: 40 Issue: 4 Pages: 377-386 Published: AUG 1998
Times Cited: 4 References: 16 Citation Map
Abstract: Starfish oocytes artificially activated by a calcium ionophore will develop normally if the formation of polar bodies is suppressed. In the present paper, schedules of the DNA replication period (S phase) of these parthenogenotes were explicitly timed using 5-bromo-2'-deoxyuridine (BrdU) and anti-BrdU monoclonal antibody. Their schedule of S phase was identical to that of fertilized eggs. Consequently an S phase regulation system is triggered even in parthenogenotes raised by dual treatment of egg activation and polar body suppression. The S phase schedule of parthenogenotes confirms the temporal pattern of chromosome duplication, observed by other researchers, leading to tetraploid parthenogenotes. The S phase determination also provides a basis for argument concerning the number of centrioles participating in parthenogenetic development. If polar body formation of activated eggs was not suppressed, the first S phase was normal, but the second S phase did not recur on time. A rigidly regulated system of DNA replication cycle, which should be an essential prerequisite for parthenogenesis, thus requires the content of polar bodies.

Biology of Wolbachia

2008-11-07T16:21:00.000-08:00

Biology of Wolbachia
Author(s): Werren JH
Source: ANNUAL REVIEW OF ENTOMOLOGY Volume: 42 Pages: 587-609 Published: 1997
Times Cited: 414 References: 132 Citation Map
Abstract: Wolbachia are a common and widespread group of bacteria found in reproductive tissues of arthropods. These bacteria are transmitted through the cytoplasm of eggs and have evolved various mechanisms for manipulating reproduction of their hosts, including induction of reproductive incompatibility, pathenogenesis, and feminization. Wolbachia are also transmitted horizontally between arthropod species. Significant recent advances have been made in the study of these interesting microorganisms. In this paper, Wolbachia biology is reviewed, including their phylogeny and distribution, mechanisms of action, population biology and evolution, and biological control implications. Potential directions for future research are also discussed.

Chronic pubertal, but not adult chronic cannabinoid treatment impairs sensorimotor gating, recognition memory, and the performance in a progressive ra

2008-10-07T22:00:00.000-07:00

Chronic pubertal, but not adult chronic cannabinoid treatment impairs sensorimotor gating, recognition memory, and the performance in a progressive ratio task in adult rats

Source: NEUROPSYCHOPHARMACOLOGY Volume: 28 Issue: 10 Pages: 1760-1769 Published: OCT 2003

Abstract: There is evidence from studies in humans and animals that a vulnerable period for chronic cannabinoid administration exists during certain phases of development. The present study tested the hypothesis that long-lasting interference of cannabinoids with the developing endogenous cannabinoid system during puberty causes persistent behavioral alterations in adult rats. Chronic treatment with the synthetic cannabinoid agonist WIN 55,212-2 (WIN) (1.2 mg/kg) or vehicle was extended over 25 days either throughout the rats' puberty or for a similar time period in adult rats. The rats received 20 injections intraperitoneally (i.p.), which were not delivered regularly. Adult rats were tested for object recognition memory, performance in a progressive ratio (PR) operant behavior task, locomotor activity, and prepulse inhibition (PPI) of the acoustic startle response (ASR). PPI was significantly disrupted only by chronic peripubertal cannabinoid treatment. This long-lasting PPI deficit was reversed by the acute administration of the dopamine antagonist haloperidol. Furthermore, we found deficits in recognition memory of pubertal-treated rats and these animals showed lower break points in a PR schedule, whereas food preference and locomotion were not affected. Adult chronic cannabinoid treatment had no effect on the behaviors tested. Therefore, we conclude that puberty in rats is a vulnerable period with respect to the adverse effects of cannabinoid treatment. Since PPI deficits, object recognition memory impairments, and anhedonia/avolition are among the endophenotypes of schizophrenia, we propose chronic cannabinoid administration during pubertal development as an animal model for some aspects of the etiology of schizophrenia.
Document Type: Article
Language: English
Author Keywords: WIN 55,212-2; recognition memory; prepulse inhibition; reward; schizophrenia; puberty
KeyWords Plus: SPATIAL WORKING-MEMORY; OBJECT-RECOGNITION; PREFRONTAL CORTEX; PREPULSE INHIBITION; DOPAMINE NEURONS; CHRONIC DELTA(9)-TETRAHYDROCANNABINOL; SCHIZOPHRENIC-PATIENTS; RECEPTOR ANTAGONIST; ACOUSTIC STARTLE; BRAIN
Reprint Address: Schneider, M (reprint author), Univ Bremen, Dept Neuropharmacol, Brain Res Inst, POB 33 04 40, D-28334 Bremen, Germany
Addresses:
1. Univ Bremen, Dept Neuropharmacol, Brain Res Inst, D-28334 Bremen, Germany
Publisher: NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND
Subject Category: Neurosciences; Pharmacology & Pharmacy; Psychiatry
IDS Number: 729XG
ISSN: 0893-133X
DOI: 10.1038/sj.npp.1300225

MARIJUANA AND REPRODUCTION - EFFECTS ON PUBERTY AND PREGNANCY IN FEMALE RATS - EXPERIMENTAL RESULTS

2008-10-07T21:58:00.000-07:00

MARIJUANA AND REPRODUCTION - EFFECTS ON PUBERTY AND PREGNANCY IN FEMALE RATS - EXPERIMENTAL RESULTS

Source: ANNALES D ENDOCRINOLOGIE Volume: 53 Issue: 1 Pages: 37-43 Published: 1992

Abstract: The main psychoactive component of marihuana, delta-9-tetrahydrocannabiol (THC), was investigated at low doses (1-mu-g/kg/day) on the onset of puberty, on the reproductive functions in female rats up to the seventy fifth to eightieth day of life as well as during the pregnancy.
The administration of THC caused a delay of the onset of puberty, and the number of ova on the day of first estrus was significantly lower in treated animals.

After puberty, alterations occured in the neuroendocrine functions of animals received THC: estrous cycles were irregular, serum LH level was decreased.

When THC was injected during the third week of pregnancy it caused a significant prolongation of the gestation period. There was 30 % stillbirths (v.s. 3 % in physiological saline treated rats). No teratological effects were observed. Serum LH, progesterone and prostaglandin contents were decreased.

The results indicate that marihuana causes alterations in reproductive functions. The importance of fight against drug abuse is emphasized.

Document Type: Article
Language: French
KeyWords Plus: DELTA-9 TETRAHYDRO-CANNABINOL; ADULT MALE-RATS; LUTEINIZING-HORMONE; SERUM LH; DELTA-9-TETRAHYDROCANNABINOL; ENDOCRINE; EXPOSURE; ALCOHOL; FSH
Reprint Address: WENGER, T (reprint author), FAC MED BUDAPEST, ANAT LAB 2, BUDAPEST, HUNGARY
Addresses:
1. FAC MED LILLE, HISTOL EMBRYOL & BIOL REPROD LAB, F-59045 LILLE, FRANCE
2. UNIV BORDEAUX 1, NEUROCYTOCHIM FONCTIONNELLE LAB, F-33405 TALENCE, FRANCE
3. INSERM, U156, F-59045 LILLE, FRANCE
Publisher: MASSON EDITEUR, 120 BLVD SAINT-GERMAIN, 75280 PARIS 06, FRANCE
Subject Category: Endocrinology & Metabolism
IDS Number: JA805
ISSN: 0003-4266

Calvin Cycle pathway

2008-08-16T23:54:00.000-07:00

Overview of the Calvin Cycle pathway. Original work by Mike Jones

2008-08-05T17:44:00.001-07:00

Suberin: waxy, waterproof chemical in some plant cells, notably cork (in stems) and endodermis cells (in roots). Suberin is an extremely complex and irregular material, like lignin -- with which it shares some similarities. Suberin is composed of two physically separated domains: the aliphatic and phenolic. The phenolic domain is rather lignin-like, but with even greater variability, and built on the same basic unit of a di- or tri-hydroxyphenyl group attached to a three-carbon chain, variously oxidized and integrated with the carbohydrates of the cell walls. Perhaps the most common building block is ferulic acid: formally, 3-(2'-methoxy-3'-hydroxyphenyl)-propenoic acid. Distally, the phenolic domain is attached at points by ester linkages to glycerol. The remaining hydroxyls of the glycerol molecule are ester-linked to some strange-looking C-18 to C-30 lipids. These lipids are substituted at C9-10 with one or two hydroxyls, or even with an epoxide link between the two carbons. Finally the ω- position may be oxidized to a carboxylate (alone or esterized to glycerol) or hydroxyl (alone or esterised to ferulic acid). Variations allow for cross-linkage to other suberin molecules via the 9-10 or ω positions. Image adapted from Bernards (2002).

Suberin is a plant cell wall modification that involves the deposition of both a poly(phenolic) and a poly(aliphatic) domain within the same tissue. As such, the deposition of suberin represents the coordinate regulation of both fatty acid metabolism (giving rise to suberin-specific aliphatic compounds) and phenylpropanoid metabolism (givin rise to suberin-specific phenolic compounds). Over the past decade, we have used chemical analyses to identify target metabolites unique to suberin and biochemical analyses to identify unique steps in their biosynthesis. We are now in a position to clone and characterize the genes that encode the enzymes for these unique biochemical steps. With these genes in hand, we will be able to ask questions about the regulation of suberization and study the mechanism(s) by which disparate pathways converge to make the unique material known as suberin.

In addition, we are interested in the role(s) for which plants use secondary metabolites to interact with other organisms. Recently, we have focused on the role of ginsenosides (e.g., Rb1 at right) in the interaction between ginseng and one of its major pathogens, Pythium irregulare.

Our research group is located on the fourth floor of the North Campus Building, forming part of the Environmental Stress Biology Group. Other members of the ESBG include Dr. Norman Huner, Dr. Denis Maxwell and Dr. Charlie Trick.

Dyeing the cross-sections for lignin

2008-07-28T04:00:00.000-07:00

Dyeing the cross-sections

2 - Phloroglucinol/HCl is used to dye lignified cell walls red
Phloroglucinol/HCl (figure 2) is used to dye lignified cell walls red. This dye works best for herb-chronology because it pronounces the structures you are interested in (the vessels and lignified parenchyma that often adds to the visualization of growth rings in the roots). To prepare the dye dissolve 1-2 points of a spattle of the phenol derivative phloroglucinol (also known as phloroglucin) in 20 ml of 75% alcohol. Use HCl conc. (≥32%) as the other reagent. Using dropper bottles you first apply one drop of phloroglucinol/EtOH to the cutting and, after 5-10 seconds or so, add one drop of HCl. In most cases this produces instantaneous reddish colouring of the lignified tissue. For thin cuttings where only the vessels are lignified colouring may take a little while. In this case do not take the photographs too quickly or you might end up with photos of low contrast. To help speed up colouring you might add a second drop of HCl. On the other hand, thicker cuttings or those that have a lignified parenchyma may colour quickly and deeply so that you should take the photo quickly to prevent dark photographs. In this case you should reduce the amounts of dye applied to the cuttings. If you do not produce satisfactory colouring try to add some more phloroglucinol to the alcoholic solution.

"The Fungal Kingdom: Diverse and Essential Roles in Earth's Ecosystem," June 2008

2008-07-28T03:48:00.000-07:00

"The Fungal Kingdom: Diverse and Essential Roles in Earth's Ecosystem," (June 2008)
Prepared by Arturo Casadevall, Joe Heitman, and Merry Buckley

Fungi can cause a number of life-threatening diseases but they also are becoming increasingly useful to science and manufacturing every year. However, many people, scientists among them, are largely unaware of the roles fungi play in the world around us. Research on fungi and fungal diseases are seriously neglected as a result – a situation with grave negative repercussions for human health, agriculture, and the environment. The Fungal Kingdom explores the roles fungi play in the world around us.

http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004018/Fungal_Kingdom.pdf

Prehistoric mystery organism verified as giant fungus

2008-07-23T22:23:00.000-07:00

Prehistoric mystery organism verified as giant fungus
‘Humongous fungus’ towered over all life on land

April 23, 2007
Scientists at the University of Chicago and the National Museum of Natural History in Washington, D.C., have produced new evidence to finally resolve the mysterious identity of what they regard as one of the weirdest organisms that ever lived.

Their chemical analysis indicates that the organism was a fungus, the scientists report in the May issue of the journal of Geology, published by the Geological Society of America. Called Prototaxites (pronounced pro-toe-tax-eye-tees), the organism went extinct approximately 350 million years ago.

Prototaxites has generated controversy for more than a century. Originally classified as a conifer, scientists later argued that it was instead a lichen, various types of algae or a fungus. Whatever it was, it stood in tree-like trunks more than 20 feet tall, making it the largest-known organism on land in its day.

“No matter what argument you put forth, people say, well, that’s crazy. That doesn’t make any sense,” said C. Kevin Boyce, an Assistant Professor in Geophysical Sciences at Chicago. “A 20-foot-tall fungus doesn’t make any sense. Neither does a 20-foot-tall algae make any sense, but here’s the fossil.”

The Geology paper adds a new line of evidence indicating that the organism is a fungus. The fungus classification first emerged in 1919, with Francis Hueber of the National Museum of Natural History in Washington, D.C., reviving the idea in 2001. His detailed studies of internal structure have provided the strongest anatomical evidence that Prototaxites is not a plant, but a fungus.

“Fran Hueber has contributed more to our understanding of Prototaxites than anyone else, living or dead,” said Carol Hotton, also of the National Museum of Natural History. “He built up a convincing case based on the internal structure of the beast that it was a giant fungus, but agonized over the fact that he was never able to find a smoking gun in the form of reproductive structures that would convince the world that it was indeed a fungus,” Hotton said.

Co-authoring the Geology paper with Boyce, Hotton and Hueber himself were Marilyn Fogel, George Cody and Robert Hazen of the Carnegie Institution of Washington, and Andrew Knoll of Harvard University. Their work was funded by NASA’s Astrobiology Institute and by the American Chemical Society Petroleum Fund.

Prototaxites lived worldwide from approximately 420 million to 350 million years ago. During this period, which spans part of the Silurian and Devonian periods of geologic time, terrestrial Earth looked quite alien in comparison to the modern world.

Simple vascular plants, the ancestors of the familiar conifers, ferns and flowering plants of today, began to diversify on land during the Devonian Period. “Initially, they’re just stems. They don’t have roots. They don’t have leaves. They don’t have anything like that,” Boyce said.

Millipedes, wingless insects and worms were among the other organisms making a living on land by then, but no backboned animals had yet evolved out of the oceans. “That world was a very strange place,” Boyce said.

Although vascular plants had established themselves on land 40 million years before the appearance of Prototaxites, the tallest among them stood no more than a couple feet high. By the end of the Devonian, approximately 345 million years ago, large trees, ferns, seeds, leaves and roots had all evolved. “They’re all there. They just exploded over this one time period,” Boyce said.

Canadian paleontologist Charles Dawson published the first research on Prototaxites in 1859, based on specimens found along the shores of Gaspé Bay in Quebec, Canada. Hueber pored through Dawson’s field notebooks, written “in a completely illegible scrawl,” Hotton said.

“Fran spent months deciphering them for clues about the localities where specimens had been collected, how Dawson interpreted them and other information that helped understand this humongous fungus,” she said.

Hueber also traveled to Canada, Australia and Saudi Arabia to collect specimens. He tediously sliced them into hundreds of thin sections and made thousands of images taken through microscopes to determine the organism’s identity.

Now Boyce, Hotton and their colleagues have produced independent evidence that supports Hueber’s case. The team did so by analyzing two varieties—isotopes—of carbon contained in Prototaxites and the plants that lived in the same environment approximately 400 million years ago.

The metabolism of plants is limited by photosynthesis. Deriving their energy from the sun and their carbon from carbon dioxide in the air, any given type of plant will typically contain a similar ratio of carbon-12 to carbon-13 as another plant of the same type. “But if you’re an animal, you will look like whatever you eat,” Boyce said. And Prototaxites displayed a much wider variation in its ratio of carbon-12 to carbon-13 content than would be expected in any plant.

Geological processes can alter the isotopic composition of fossils, but Boyce and his colleagues conducted tests to verify that the carbon isotopic composition of the specimens they analyzed stemmed from organic rather than geologic factors.

As for why these bizarre organisms grew so large, “I’ve wondered whether it enabled Prototaxites to distribute its spores widely, allowing it to occupy suitable marshy habitats that may have been patchily distributed on the landscape,” Hotton said.

The relatively simple Devonian ecosystems certainly seemed to contain nothing to prevent them from growing slowly for a long time. Plant-eating animals had not yet evolved, Boyce said. But even if Prototaxites hadn’t been eaten by the dinosaurs and elephants that came much later, they probably grew too slowly to rebuild from regular disturbances of any kind, Boyce said.

“It’s hard to imagine these things surviving in the modern world,” he said.
from:
http://www-news.uchicago.edu/releases/07/070423.fungus.shtml

Circumventing the PDR pathway in fungi.

2008-07-23T16:32:00.001-07:00

Fig. 1. Circumventing the PDR pathway in fungi. (A) In cells with clinically important resistance to azole drugs, high-level transcription of PDR efflux-pump genes involves the recruitment of RNA polymerase II, which depends on a drug-induced interaction between the ScPdr1p/Pdr3p and mediator complexes. The efflux pumps reduce the intracellular concentration of the drug below that required to inhibit the azole target Erg11p, allowing normal cell growth. (B) Binding of a multifunctional azole to the XBD domain of ScPdr1p/Pdr3p blocks expression of the drug pumps responsible for multidrug efflux and inhibits drug efflux by occupying a binding site in residual efflux pumps. The intracellular concentration of the drug is thus sufficient to block ergosterol biosynthesis in the endoplasmic reticulum. Reduced ergosterol content of membranes, production of toxic methylated sterols, and oxidative damage kill the fungal cell. Alternatively, other antifungals directly inhibit the electrogenic plasma membrane H+-ATPase Pma1p, preventing the uptake of nutrients driven by the plasma membrane electrochemical gradient. The cells die rapidly because of a limited cellular energy supply and a loss of ion balance. Partial inhibition of Pma1p activity compromises the activity of both MFS and ABC transporters and increases the potency of azole drugs. [View Larger Version of this Image (55K GIF file)]

Outwitting Multidrug Resistance to Antifungals

2008-07-23T16:29:00.000-07:00

Science 18 July 2008:
Vol. 321. no. 5887, pp. 367 - 369
DOI: 10.1126/science.1159746
Prev | Table of Contents | Next

Perspective
Outwitting Multidrug Resistance to Antifungals
Brian C. Monk and Andre Goffeau*
The economic cost of fungal infection and its mortality associated with multidrug resistance remain unacceptably high. Recent understanding of the transcriptional regulation of plasma membrane efflux pumps of modest specificity provides new avenues for the development of broad-spectrum fungicides. Together with improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux, we envisage multifunctional azole analogs that inhibit not only ergosterol biosynthesis and drug efflux-pump activity but also activation of the transcriptional machinery that induces drug efflux-pump expression.

Department of Oral Sciences, Faculty of Dentistry, University of Otago, Post Office Box 647, Dunedin, New Zealand; and Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, 1348, Belgium.

* To whom correspondence should be addressed. E-mail: andre.goffeau@uclouvain.be

Eight hundred million years of evolution have generated 1.5 million fungal species that occupy many distinct ecological niches, yet only 300 fungi cause disease in humans (1). The identification of antifungals that act specifically against these pathogens is a particular challenge because of fungal diversity, individualized pathways for infection, and fungal use of multiple mechanisms that circumvent exogenous toxins. These highly regulated mechanisms include innate resistance to specific antifungal drugs, formation of biofilms, selection of spontaneous mutations that increase expression or decrease susceptibility of the drug target (2), stress-related tolerance that enhances short-term survival (3, 4), modification of chromosomal ploidy (5), and overexpression of multidrug efflux pumps (6). Fortunately, compared with infections caused by drug-resistant bacteria, those caused by resistant fungal pathogens and their spread to other patients occur relatively infrequently. However, the economic cost of fungal infection and its associated mortality, especially in debilitated and high-investment patients, remain unacceptably high.

A Clinical Perspective

The most prominent fungal pathogens affecting humans include Aspergillus fumigatus, Candida albicans, C. glabrata, C. parasilosis, C. tropicalis, C. krusei, and Cryptococcus neoformans (7). Although the skin, mucosal surfaces, and immune system usually provide robust defenses, weakened immunodefenses dramatically increase susceptibility to debilitating and life-threatening opportunistic fungal infections. Fungal infections are normally treated with a modest repertoire of drugs derived from five antifungal classes that target DNA and RNA synthesis, ergosterol, the ergosterol biosynthetic pathway, or the biosynthesis of the cell-wall component 1,3-β-D-glucan (Table 1). Unfortunately, the prophylactic use of fungistatic azoles such as fluconazole has been associated with an increased frequency of innate or acquired drug resistance in clinical isolates and the selection of non-albicans Candida, non-fumigatus Aspergillus, opportunistic yeastlike fungi, zygomycetes, and hyaline molds. Despite the fact that broader-spectrum third-generation azole drugs and the more expensive echinocandin class of antifungals prevent an increased proportion of life-threatening infections, Candida species remain the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients, whereas disseminated Aspergillus infections kill up to 80% of affected patients.

Mechanisms of Multidrug Resistance

Because of its economic and clinical impact, a focus on multidrug resistance rather than resistance to specific antifungals in pathogenic fungi is timely. Multidrug resistance, called pleiotropic drug resistance (PDR) in Saccharomyces cerevisiae, is an ancient phenomenon that preceded the modern use of antifungals (8). The adenosine triphosphate (ATP)–binding cassette (ABC) and major facilitator superfamily (MFS) transporter families responsible for multidrug resistance operate in all fungi. We distinguish among the transporters that belong to different species by using the prefix Sc for S. cerevisiae, Cg for C. glabrata, or Ca for C. albicans.

Saccharomyces cerevisiae. PDR in S. cerevisiae is the best-understood multidrug resistance mechanism in fungi. Point mutations conferring resistance to chemically diverse drugs (including azoles) have been mapped in genes encoding the zinc-finger transcription factors ScPdr1p or ScPdr3p (9, 10). These gain-of-function mutations activate over 20 target genes, the major ones being either ATP-driven (ABC transporter genes ScPDR5, ScSNQ2, and ScYOR1) orproton motive force–driven (MFS transporter genes ScTPO1 and ScFLR1) efflux pumps (11, 12). Resistance to a wide spectrum of drugs is conferred via the activation of efflux-pump gene expression, which involves the binding of Pdr1p/Pdr3p to the consensus binding element PDRE (13, 14). Mechanisms regulating PDR in S. cerevisiae include mutation of PDR1/3, plasma membrane sphingolipid homeostasis, ScPdr3p autoregulation, ScPdr3p-specific activation due to loss of mitochondrial respiration, chaperone-specific differential regulation of ScPdr1p and ScPdr3p (15), and ScPdr1p-dependent compensatory expression of efflux pumps (16). Yeast cells incubated with antifungals and other drugs transiently activate ScPdr1p/Pdr3p (16). Drugs such as itraconazole and progesterone bind to a 250–amino acid hydrophobic xenobiotic binding domain (XBD) of ScPdr1p/Pdr3p, enabling a specific association with the KIX domain of the Gal11p subunit of the mediator complex that recruits RNA polymerase II for expression of the ScPdr1p/Pdr3p-controlled genes (17) (Fig. 1A). Other transcription factors such as Yrr1p, Stb5p, Rdr1p, Yrm1p, and Yap1p also contribute to the expression of the various efflux transporter genes (17, 18).

Pathogenic fungi. The human pathogen C. glabrata uses the transcription factor CgPdr1p to control expression of the ABC multidrug efflux pumps CgCdr1p and CgCdr2p through mechanisms very similar to those of its close relative S. cerevisiae. The pumps are induced by treatment with diverse drugs and are highly expressed in respiration-defective mutants. Antifungal binding to the CgPdr1p XBD induces multidrug resistance via a KIX domain from the C. glabrata mediator complex (19). Mutants overexpressing CgPdr1p coordinately regulate 11 genes homologous to ScPdr1/ScPdr3p targets (20). These similarities support the use of S. cerevisiae in developing tools that are directly applicable to antifungal resistance in C. glabrata.

Multiple azole resistance stemming from longterm prophylaxis is frequently found in clinical isolates of the more distant pathogen C. albicans (21). The resistance mechanisms of this diploid species are complex and include mutations in single genes, loss of heterozygosity, chromosomal rearrangements, and selective segregation of chromosomal fragments (22). About 85% of fluconazole-resistant clinical isolates show multidrug resistance due to overexpression of the ABC transporters CaCdr1p and CaCdr2p (homologs of the S. cerevisiae ScPdr5p) and the major facilitator superfamily (MFS) pump CaMdr1p (homolog of ScFlr1p). The efflux functions of these transporters can be cloned in S. cerevisiae (23, 24). Expression of the CaCdr1p and CaCdr2p pumps is controlled by the transcription factor CaTac1p (25), which shares about 20% identity with ScPdr1/ScPdr3p. CaTac1p and ScPdr1p/ScPdr3p recognize substantially different PDREs (25), and CaTac1p causes more focused transcription than ScPdr1p/Pdr3p (26, 27). High doses of the female steroid hormone progesterone transiently upregulate, via steroid-specific PDREs, the same core of ABC transporters induced by antifungal intervention or gain-of-function mutations in the transcription factors (26, 28). Fluconazole-resistant clinical isolates often constitutively overexpress the MFS transporter CaMdr1p, either by itself or in combination with the azole target CaErg11p and/or the CaCdr1p and CaCdr2p ABC pumps. Although the MFS transporter CaMdr1p seems more efficient than its homolog ScFlr1p, it is overexpression of ABC transporters that confers clinically important, high-level azole resistance.

Preliminary data on non-albicans Candida species, Cryptococcus neoformans, and A. fumigatus (29) suggest that various resistance phenomena identified in C. albicans may operate in these pathogenic fungi and that PDR-related transcriptional mechanisms may contribute to their multidrug resistance.

Prospects

The long-awaited structural resolution of antifungal binding sites in the azole target ScErg11p as well as in the drug efflux pumps related to ScPdr5p would undoubtedly provide insight into multidrug resistance and guide strategies for impairing their activities. Meanwhile, and despite molecular mechanisms of differing complexity contributing to multidrug resistance in pathogenic fungi, a newly detected Achilles heel may be the transcriptional control of the antifungal efflux pumps. Of particular interest is the discovery that PDR transcriptional activators bind substrates of the efflux pumps they induce. We therefore may anticipate the development of novel multifunctional azole analogs. Erg11p would still be their primary target. Inclusion of a novel substituent would then enable inhibition of XBD-dependent coupling of Pdr1p/Pdr3p with its cognate mediator complex plus physical blockade of efflux via PDR transporters (Fig. 1B). The structures of itraconazole and fluconazole suggest that a fluconazole-like scaffold could be modified to antagonize not only Erg11p but also the transcriptional XBD and the active site from efflux pumps. Similarly, the dependence of the transient steroid response on interactions with the XBD domain of CgPdr1p, and possibly CaTac1p, indicates that a steroid hormone antagonist could increase the potency of azoles used against vaginal infections. Functional overexpression of Erg11p and both MFS and ABC drug efflux pumps from pathogenic fungi has been accomplished with an activated PDR5 promoter in a S. cerevisiae host whose major PDR genes had been deleted (24). This approach has allowed the assessment of innate and overexpression-related resistance to antifungals and the discovery of efflux-pump inhibitors. Similarly, ScPdr1p-regulated overexpression of functional ScPdr5p-related pump homologs is expected to provide screens for the identification of the novel broad-spectrum azoles or narrower-spectrum steroid antagonists hypothesized above (30). By minimizing pump expression, drug pump activity, and the opportunity for stress responses, these drugs should transform the fungistatic azoles into potent fungicides.

A complementary strategy is the identification of new targets whose dysfunction kills fungi rapidly, thus avoiding the emergence of both drug tolerance and efflux-mediated resistance. About 250 genes deemed essential in S. cerevisiae encode products that are at least 40% conserved across a broad range of fungi, including the fungal pathogens C. glabrata, C. albicans, C. neoformans, and A. fumigatus (31). Only about 50 of these gene products show less than 40% homology with human proteins. One of these is the plasma membrane proton pump (Pma1p), which generates the electrochemical gradient that fungi require for ion balance, nutrient uptake, and energy production. Pma1p inhibitors are fungicidal, indirectly block the activity of both ABC and MFS drug efflux pumps (32), and substantial resistance to them has yet to be detected.

Finally, diagnosis of disseminated fungal infections is too slow because conventional identification requires phenotypic examination of colonies grown for at least 48 hours on selective medium. The identification of drug resistance often requires a further step. Polymerase chain reaction amplification of ribosomal RNA intervening transcribed sequences followed by DNA pyrosequencing should halve the time needed for species-level fungal identification (33, 34). Translation of this technology into the clinic will allow the early identification of fungal species, including innately resistant species or those susceptible to the development of multidrug resistance. The application of appropriate prophylaxis with existing and novel antifungals and of ongoing surveillance will save many lives.

References and Notes

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35. Supported by NIH grant DE016885.

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Science 321 (5887), 355. [DOI: 10.1126/science.321.5887.355]
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Effects of psilocybin on time perception and temporal control of behaviour in humans.

2008-07-14T04:17:00.000-07:00

Effects of psilocybin on time perception and temporal control of behaviour in humans.

J. Psychopharmacol. 2007 Jan;21(1):50-64. Epub 2006 May 19.

Wittmann M, Carter O, Hasler F, Cahn BR, Grimberg U, Spring P, Hell D, Flohr H, Vollenweider FX.
Generation Research Programme, Human Science Centre, Ludwig-Maximilian University Munich, Bad Tölz, Germany, and Heffter Research Centre, University Hospital of Psychiatry, Zürich, Switzerland.
Hallucinogenic psilocybin is known to alter the subjective experience of time. However, there is no study that systematically investigated objective measures of time perception under psilocybin. Therefore, we studied dose-dependent effects of the serotonin (5-HT)2A/1A receptor agonist psilocybin (4-phosphoryloxy-N, N-dimethyltryptamine) on temporal processing, employing tasks of temporal reproduction, sensorimotor synchronization and tapping tempo. To control for cognitive and subjective changes, we assessed spatial working memory and conscious experience. Twelve healthy human volunteers were tested under placebo, medium (115 microg/kg), and high (250 microg/kg) dose conditions, in a double-blind experimental design. Psilocybin was found to significantly impair subjects' ability to (1) reproduce interval durations longer than 2.5 sec, (2) to synchronize to inter-beat intervals longer than 2 sec and (3) caused subjects to be slower in their preferred tapping rate. These objective effects on timing performance were accompanied by working-memory deficits and subjective changes in conscious state, namely increased reports of 'depersonalization' and 'derealization' phenomena including disturbances in subjective 'time sense.' Our study is the first to systematically assess the impact of psilocybin on timing performance on standardized measures of temporal processing. Results indicate that the serotonin system is selectively involved in duration processing of intervals longer than 2 to 3 seconds and in the voluntary control of the speed of movement. We speculate that psilocybin's selective disruption of longer intervals is likely to be a product of interactions with cognitive dimensions of temporal processing -presumably via 5-HT2A receptor stimulation.
PMID: 16714323 [PubMed - indexed for MEDLINE]

Psilocybin can occasion mystical-type experiences having substantial and sustained personal meaning and spiritual significance.

2008-07-14T03:51:00.000-07:00

Psilocybin can occasion mystical-type experiences having substantial and sustained personal meaning and spiritual significance.

Psychopharmacology (Berl). 2006 Aug;187(3):268-83; discussion 284-92. Epub 2006 Jul 7.

Griffiths RR, Richards WA, McCann U, Jesse R.
Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 5510, Nathan Shock Drive, Baltimore, MD 21224-6823, USA. rgriff@jhmi.edu
RATIONALE: Although psilocybin has been used for centuries for religious purposes, little is known scientifically about its acute and persisting effects. OBJECTIVES: This double-blind study evaluated the acute and longer-term psychological effects of a high dose of psilocybin relative to a comparison compound administered under comfortable, supportive conditions. MATERIALS AND METHODS: The participants were hallucinogen-naïve adults reporting regular participation in religious or spiritual activities. Two or three sessions were conducted at 2-month intervals. Thirty volunteers received orally administered psilocybin (30 mg/70 kg) and methylphenidate hydrochloride (40 mg/70 kg) in counterbalanced order. To obscure the study design, six additional volunteers received methylphenidate in the first two sessions and unblinded psilocybin in a third session. The 8-h sessions were conducted individually. Volunteers were encouraged to close their eyes and direct their attention inward. Study monitors rated volunteers' behavior during sessions. Volunteers completed questionnaires assessing drug effects and mystical experience immediately after and 2 months after sessions. Community observers rated changes in the volunteer's attitudes and behavior. RESULTS: Psilocybin produced a range of acute perceptual changes, subjective experiences, and labile moods including anxiety. Psilocybin also increased measures of mystical experience. At 2 months, the volunteers rated the psilocybin experience as having substantial personal meaning and spiritual significance and attributed to the experience sustained positive changes in attitudes and behavior consistent with changes rated by community observers. CONCLUSIONS: When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences. The ability to occasion such experiences prospectively will allow rigorous scientific investigations of their causes and consequences.
PMID: 16826400 [PubMed - indexed for MEDLINE

Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later

2008-07-14T03:38:00.000-07:00

Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later

R R Griffiths1*, W A Richards2, M W Johnson3, U D McCann3, and R Jesse4
1 Department of Psychiatry and Behavioral Sciences and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
4 Council on Spiritual Practices, San Francisco, California, USA

* To whom correspondence should be addressed.

Abstract

Psilocybin has been used for centuries for religious purposes; however, little is known scientifically about its long-term effects. We previously reported the effects of a double-blind study evaluating the psychological effects of a high psilocybin dose. This report presents the 14-month follow-up and examines the relationship of the follow-up results to data obtained at screening and on drug session days. Participants were 36hallucinogen-naïve adults reporting regular participation in religious/spiritual activities. Oral psilocybin (30 mg/70 kg) was administered on one of two or three sessions, with methylphenidate (40 mg/70 kg) administered on the other session(s). During sessions, volunteers were encouraged to close their eyes and direct their attention inward. At the 14-month follow-up, 58% and 67%, respectively, of volunteers rated the psilocybin-occasioned experience as being among the five most personally meaningful and among the five most spiritually significant experiences of their lives; 64% indicated that the experience increased well-being or life satisfaction; 58% met criteria for having had a ‘complete’ mystical experience. Correlation and regression analyses indicated a central role of the mystical experience assessed on the session day in the high ratings of personal meaning and spiritual significance at follow-up. Of the measures of personality, affect, quality of life and spirituality assessed across the study, only a scale measuring mystical experience showed a difference from screening. When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences that, at 14-month follow-up, were considered by volunteers to be among the most personally meaningful and spiritually significant of their lives.

Key Words: entheogen, hallucinogen, humans, mystical experience, psilocybin, psychedelic, religion, spiritual

First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108094300
© 2008 British Association for Psychopharmacolo

Human hallucinogen research: guidelines for safety

2008-07-14T03:35:00.000-07:00

Human hallucinogen research: guidelines for safety

M W Johnson1, W A Richards2, and R R Griffiths3*
1 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

* To whom correspondence should be addressed.

Abstract

There has recently been a renewal of human research with classical hallucinogens (psychedelics). This paper first briefly discusses the unique history of human hallucinogen research, and then reviews the risks of hallucinogen administration and safeguards for minimizing these risks. Although hallucinogens are relatively safe physiologically and are not considered drugs of dependence, their administration involves unique psychological risks. The most likely risk is overwhelming distress during drug action (‘bad trip’), which could lead to potentially dangerous behaviour such as leaving the study site. Less common are prolonged psychoses triggered by hallucinogens. Safeguards against these risks include the exclusion of volunteers with personal or family history of psychotic disorders or other severe psychiatric disorders, establishing trust and rapport between session monitors and volunteer before the session, careful volunteer preparation, a safe physical session environment and interpersonal support from at least two study monitors during the session. Investigators should probe for the relatively rare hallucinogen persisting perception disorder in follow-up contact. Persisting adverse reactions are rare when research is conducted along these guidelines. Incautious research may jeopardize participant safety and future research. However, carefully conducted research may inform the treatment of psychiatric disorders, and may lead to advances in basic science.

Key Words: 5-HT2A agonists, adverse reactions, DMT, entheogens, hallucinogens, human research, LSD, mescaline, psilocybin, psychedelics, safety guidelines

First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108093587
© 2008 British Association for Psychopharmacology

A Very Memorable Trip

2008-07-14T03:26:00.000-07:00

A Very Memorable Trip

By Greg Miller
ScienceNOW Daily News
1 July 2008

More than a year after taking a hallucinogenic drug in a carefully controlled experiment, most people rate the experience among the most personally meaningful and spiritually significant of their lives, researchers report online today in the Journal of Psychopharmacology. Such findings are helping to renew interest in research with hallucinogens, a field whose reputation long suffered from the psychedelic excesses of the 1960s.
The new study follows up with 36 volunteers who participated in earlier experiments led by psychopharmacologist Roland Griffiths of Johns Hopkins University in Baltimore, Maryland. The researchers monitored the mostly middle-aged subjects while they took a strong dose of psilocybin, the active ingredient in hallucinogenic mushrooms. All of the volunteers had indicated at least some participation in religious or spiritual activities--such as meditating or going to church--and the researchers instructed them to direct their attention inward while under the drug's sway. None had previous experience with hallucinogens. On questionnaires completed after the drug had worn off, and again 2 months later, they rated the experience as highly significant, the researchers reported in a 2006 paper in Psychopharmacology. Volunteers frequently described a sense of greater truth or a sense of the unity of all things while on the drug, for example.

The experience remained highly significant to most of the volunteers 14 months later, the researchers now report: 58% rated it among the five most personally meaningful experiences of their lives and 67% rated it among the five most spiritually significant. And 64% said the experience had improved their sense of well-being or life satisfaction. It's remarkable, Griffiths says, that people continued to rate their 8-hour experience in the lab as similar in significance to life events such as the birth of a first child.

The findings suggest to Griffiths that hallucinogenic drugs may provide a way to investigate the neurobiology of religious experiences by evoking in the lab the kinds of mystical experiences traditionally achieved by prayer, meditation, or fasting. Would the drug have the same effect on a group of atheist or agnostic subjects? "We're dying to do that study," he says.

In the meantime, Griffiths's team is recruiting volunteers for a clinical trial to test whether similar psilocybin experiences can reduce anxiety and depression in cancer patients. A few studies in the late 1960s and early 1970s suggested that the hallucinogen LSD might ease suffering in terminal cancer patients, but that line of investigation was dropped and largely forgotten, says David Nichols, a psychopharmacologist at Purdue University in West Lafayette, Indiana. Although such patients often receive heavy doses of pain drugs along with antidepressants and anxiety drugs, Nichols says hallucinogens might provide a better alternative. "If you could change their perception of death and reduce their stress in that way, it would improve their quality of life because their consciousness wouldn't be dulled by sedatives or narcotics," he says.

Identification of a serotonin/glutamate receptor complex implicated in psychosis

2008-07-05T16:50:00.000-07:00

Identification of a serotonin/glutamate receptor complex implicated in psychosis

The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception1, 2. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR)3, 4. Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia5, 6, 7. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR8, 9, 10 and resemble some of the core symptoms of schizophrenia10, 11, 12. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR–mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR–mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.

Letter
Nature 452, 93-97 (6 March 2008) | doi:10.1038/nature06612; Received 2 November 2007; Accepted 20 December 2007; Published online 24 February 2008

FIGURE 1. Receptor interaction.

2008-07-05T16:27:00.000-07:00

González-Maeso et al.2 find that the metabotropic glutamate receptor 2 (mGluR2) and serotonin 5-HT2A receptor (2AR) physiologically bind each other, leading to reciprocal regulation of their functions. Agonists that stimulate mGluR2 are antipsychotic, whereas 2AR agonists, such as hallucinogens, have the opposite effect. It is conceivable that the clinically significant anti-schizophrenic effects of LY2140023, an mGluR2 agonist1, derive from reducing the excessive — and hence hallucinogen-like — activity of 2AR. DOI, 2,5-dimethoxy-4-iodoamphetamine.

From the following article:
Neuroscience: A complex in psychosis
Solomon H. Snyder
Nature 452, 38-39(6 March 2008)
doi:10.1038/452038a

Neuroscience: A complex in psychosis

2008-07-05T16:24:00.000-07:00

Neuroscience: A complex in psychosis

Solomon H. Snyder1

Abstract
The molecular basis of psychoses such as schizophrenia remains largely mysterious. The interaction between two of the brain receptors involved adds to evidence that will help in the search for explanations.

This is a story that involves three types of receptor in the brain that influence human perception and behaviour (those for the neurotransmitters dopamine, serotonin and glutamate), and the drugs that block or enhance their activity. Such drugs are used by researchers to investigate the causes of psychotic disorders such as schizophrenia, and by clinicians to treat patients.

Nature 452, 38-39 (6 March 2008) | doi:10.1038/452038a; Published online 5 March 2008

Picky Plants: Do They "Choose" The Best Fungal Partner?

2008-06-29T23:01:00.001-07:00

Picky Plants: Do They "Choose" The Best Fungal Partner?

ScienceDaily (Aug. 9, 2001) — MADISON, Wis. --- Every time we make a choice, whether between job offers in two different cities or about what to have for dinner, evaluating the costs and benefits of each option is part of the process. Researchers at the University of Michigan are finding that the ability to actively select one option over another may no longer be reserved for higher animals; in fact, plants may make choices too.

Many plants form partnerships with fungi that live in the soil. Attached to the plant's roots, the fungus provides the plant with nutrients needed for growth---usually phosphorous—and the plant provides the fungus with something it needs, usually carbon. Many plants show increased growth when they team up with a fungus, but all fungi are not created equal. Depending on the environment, one fungus may cost the plant more or less carbon in exchange for the nutrients the fungus makes available to the plant.

And according to a paper to be presented at the annual meeting of the Ecological Society of America on Aug. 8 by U-M doctoral student Miroslav Kummel, "plants may be actively 'choosing' the species of fungus that supports the highest growth for the plant."

Depending on environmental factors such as soil type or amount of light, fungi differ in their effects on plant growth, and a plant living in the shade may be better off with a different fungus than a plant living in the sun. "Of course this is the result of long-term selection," says Deborah Goldberg, a professor of ecology and evolutionary biology and one of Kummel's faculty advisers, "but the consequences are the same as if it were a cognitive choice, and that's pretty cool."

Kummel looked at the distribution pattern of different types of fungi growing on balsam fir seedlings in an area with light conditions ranging from full sun to full shade. He found that a fir seedling living in the shade associates with a different fungus than a fir seedling living in the sun, and that it teams up with the fungus that "costs" the least, while still benefiting the plant.

The mechanism by which the plant "chooses" the fungus is not yet known. It could result from the plant selectively aborting roots that associate with the more "expensive" fungus or from selective growth of new root tips. By isolating pure cultures of different fungi to more closely examine the exchange of nutrients between plant and fungus, Kummel hopes to unravel this mechanism. These experiments are in progress. Ultimately, Kummel's work could have implications for the timber industry, as many of our pulp crops and commercial hardwoods also form associations with fungi.

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Adapted from materials provided by University Of Michigan.

Plant-Fungal Symbiosis Found In High-Heat Extreme Environment

2008-06-29T22:59:00.001-07:00

Plant-Fungal Symbiosis Found In High-Heat Extreme Environment

ScienceDaily (Nov. 27, 2002) — ARLINGTON, Va. -- Researchers examining plants growing in the geothermal soils of Yellowstone National Park and Lassen Volcanic National Park have found evidence of symbiosis between fungi and plants that may hold clues to how plants adapt to and tolerate extreme environments.

The research was funded in part through the National Science Foundation's (NSF) Microbial Observatories Program and published in the Nov. 22 issue of the journal Science.

Biologists Regina Redman of the University of Washington and Joan Henson of Montana State University and their colleagues examined 200 samples of Dichanthelium lanuginosum, also called "Geyser's Dichanthelium," for fungal colonization. They found what may be a new species of the fungus Curvularia that survives only in temperatures greater than 98 degrees when it associates with plants.

The researchers suggest that thermotolerance may occur through symbiotic mechanisms like heat dissipation by pigment, such as melanin, or the activation of a "biological trigger" that tells the plant to react to temperature changes more rapidly or strongly than plants that lack the fungus.

The researchers grew sample plants with and without the symbiotic fungus in a laboratory and heated the soil to test thermal resistance. The plants without the fungus shriveled at 122 degrees, whereas those plants with the fungus tolerated the heat for three days. The plants were also subjected to intermittent temperatures as high as 149 degrees. The fungus-free plants died, but the fungus-bearing plants survived for 10 days.

The researchers also demonstrated that the plants provide thermal protection to the fungus by isolating it in plant roots that had a field soil temperature of 113 degrees.

"Scientific understanding of how life can thrive in such extreme environments is at its infancy," said Microbiologist Matt Kane, NSF's Microbial Observatories Program Director. "Research funded by NSF's Microbial Observatories Program is demonstrating that when you look in interesting places, you discovery interesting life forms and interrelationships, such as these fungi and their plant partners."

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Adapted from materials provided by National Science Foundation.

Enzyme Revealed That Is Key To Fungus's Ability To Breach Immune System

2008-06-29T22:56:00.001-07:00

Enzyme Revealed That Is Key To Fungus's Ability To Breach Immune System

ScienceDaily (Nov. 13, 2003) — DURHAM, N.C. – A newly discovered mechanism by which an infectious fungus evades the immune system could lead to novel methods to fight the fungus and other disease-causing microbes, according to Howard Hughes Medical Institute investigators at Duke University Medical Center.

Disruption of a key enzyme in the fungus Cryptococcus neoformans – a common cause of infection of the central nervous system in patients such as organ transplant recipients who lack a functioning immune system -- led to a significant loss of fungal virulence in mice, the team found. That loss of virulence stemmed from the fungus's inability to launch a counterattack against components of the innate immune system, the body's first line of defense against infection, the study showed.

The Duke-based team -- led by HHMI geneticist Joseph Heitman, M.D., director of Duke's Center for Microbial Pathogenesis, and HHMI biochemist Jonathan Stamler, M.D. -- reported their findings in the Nov. 11, 2003, issue of Current Biology. The work was funded by the National Institutes of Allergy and Infectious Diseases and the Burroughs Wellcome Fund.

The "fungal defense" enzyme, called flavohemoglobin, is prevalent among many bacterial and fungal pathogens, Heitman said, which suggests that the findings in Cryptococcus are likely relevant to other infectious microbes. New drugs that target these enzymes might therefore represent effective treatments for a wide range of infectious diseases, he said.

The human immune system uses a two-pronged mechanism to fight infection: a rapid innate response and a slower adaptive response that depends on the production of antibodies. Key components of the innate immune system are "search-and-destroy" cells called macrophages that engulf and kill invading pathogens. Macrophages kill infectious microbes using a combination of oxidants, including hydrogen peroxide, nitric oxide and related molecules.

"The body must rely on macrophages of the innate immune system to protect itself before the adaptive immune system can respond to invasion," Heitman said. "While much is known about how pathogens defend themselves against hydrogen peroxide produced by the macrophages, this study is the first biologically relevant test of what microbes do to counteract nitric oxide and promote infection."

The researchers found that a mutant C. neoformans strain lacking the flavohemoglobin enzyme failed to break down nitric oxide in laboratory cultures. Fungus with the enzyme deficiency also ceased to grow when in the presence of nitric oxide, whereas ordinary fungus survived normally.

Mice infected with the flavohemoglobin-deficient C. neoformans survived for five days longer than those infected with the normally virulent strain. In contrast, the normal and mutant fungal strains were equally virulent in mice whose immune cells could not produce nitric oxide, the team reported.

The mutant fungus also failed to grow normally in laboratory dishes containing macrophage cells, further implicating the innate immune system in the loss of virulence exhibited by fungi lacking flavohemoglobin.

The team discovered a second enzyme, known as GSNO reductase, which also plays a role in defending the fungus against nitric oxide-related molecules produced by macrophages. Mutant fungal strains deficient in both enzymes were more severely impaired than those lacking flavohemoglobin only.

"By disabling either the fungal nitric oxide defense system or the immune system's ability to produce nitric oxide, we were able to tip the balance one way or the other – in favor of the fungal infection or the host," Heitman said. "That raises the possibility that we could treat infectious disease with drugs that either inhibit fungal defense enzymes or increase the innate immune system's ability to mount a nitrosative attack."

Collaborators on the study include Marisol de Jesus-Berrios, Ph.D., Gary Cox, M.D., Limin Liu, Ph.D., and Jesse Nussbaum, all of Duke.

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Adapted from materials provided by Duke University Medical Center.

Efficient Consumption Of Copper Allows Fungus To Infect The Brain

2008-06-29T22:46:00.002-07:00

Efficient Consumption Of Copper Allows Fungus To Infect The Brain

ScienceDaily (Feb. 9, 2007) — Infection with the fungus Cryptococcus neoformans is a problem for individuals whose immune system is compromised (for example individuals with HIV and individuals who are taking chemotherapeutics to treat cancer). It can cause either cryptococcal pneumonia or, more seriously, meningoencephalitis.

In a study that appears online on February 8 in advance of publication in the March print issue of the Journal of Clinical Investigation, researchers from the University of Illinois at Chicago show that in mice, the infecting fungus must be adapted to grow in the presence of low levels of copper if it is to efficiently infect the brain and cause meningoencephalitis.

Peter Williamson and colleagues showed that C. neoformans lacking a protein that is essential for it to take up copper from its environment (Cuf1) are impaired in their ability to infect the brain and cause fatal meningoencephalitis.

By contrast, these mutant C. neoformans infect the lung as efficiently as C. neoformans expressing Cuf1. Consistent with this, bacteria expressing high levels of a protein controlled by Cuf1 (Ctr4) were found in the brain of mice and humans infected with C. neoformans.

This study indicates that one factor that can limit the growth of C. neoformans in the brain of mice and humans is low levels of copper, but that this is not a factor limiting growth in the lung. The authors therefore suggest that determining the level of Ctr4 expressed by the C. neoformans infecting an individual might help determine that individual’s risk of developing meningoencephalitis.

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Adapted from materials provided by Journal of Clinical Investigation, via EurekAlert!, a service of AAAS.

Taking The Fungal Route Through The Soil

2008-06-29T22:46:00.001-07:00

Taking The Fungal Route Through The Soil

ScienceDaily (Feb. 21, 2007) — Fungal hyphae play a greater role in the spread of bacteria in the soil than was previously suspected. This is the finding reported by scientists from the Helmholtz Centre for Environmental Research (UFZ) in the scientific journal Environmental Science & Technology. For the first time, scientists have been able to prove that bacteria are able to travel through the soil on the mucous membrane of living fungi.

The experiments could help speed up the remediation of contaminated land using bacteria that break down harmful substances. Air and a lack of moisture create a barrier to the mobility of bacteria in the soil, preventing them from spreading and delaying the breakdown of pollutants.

Everything is just a question of contacts

It looks like a giant green ball of wool. With a bit of imagination the photo could also be likened to a huge motorway interchange with countless roads and junctions passing over and under each other on different levels. But what Leipzig-based microbiologist Dr Lukas Y. Wick is observing so intently on his screen is in fact a photograph of a mycelium taken with a confocal laser scanning microscope. The thread-like hyphae have a diameter of just 10 micrometres – one-seventh of the diameter of a human hair.

Nevertheless, fungi are some of the world’s greatest biomass producers. A single gram of field soil can contain up to 100 metres of mycelium. Wick’s actual research objects are much smaller still. He is interested in soil bacteria. Bacteria can weaken the human organism, but they can also be useful, e.g. by breaking down pollutants.

“For the bacterium a harmful substance is not harmful,” explains Wick. “It simply breaks down the carbon compounds, producing the energy and substances that it needs to live.” But before it can do this it has to get at its ‘food’. Air and lack of moisture present insurmountable obstacles. “This is why certain pollutants are broken down so slowly in the soil. Often it is not a lack of biochemical capacity, but rather a lack of contacts.” The scientists at the UFZ are therefore studying the paths followed by the bacteria.

Probably the world’s largest motorway network

Mycelia appear to act as a kind of underground highway for bacteria. This is the conclusion reached by Lukas Wick and his team. In the laboratory experiment they succeeded in demonstrating that the bacteria move through the soil on the mycelium. The ingredients: one pollutant, separating layers made of glass pellets, uncontaminated soil and a bacterium called Pseudomonas putida. The bacteria have to fight their way through all these layers to reach the phenanthrene, their ‘food’. This polycyclic aromatic hydrocarbon is a widespread pollutant produced during every combustion process: at petrol stations, in car exhausts, during forest fires, in cigarette smoke and in old municipal gas works.

“We deliberately make the bacteria work their way upwards against gravity so that people can’t say there could be a small amount of water trickling down and carrying the bacteria with it,” says Wick. “We have tried to rule out any doubts and objections from potential critics.” The bacteria made it to the top only in places where there was a mycelium running through the soil. In the identical parallel experiment without a mycelium the bacteria were unable to surmount the barriers. “With this paper we have shown that there is an infrastructure.”

Just follow your nose

The bacteria in this laboratory experiment are so-called chemotactic bacteria. This means that they measure the concentration of their ‘target chemical’ and then move towards where the concentration is higher – as if on autopilot.

“A bacterium is not a stupid creature – it has adapted to its environment and goes where there is food.” Only one type of bacteria was used in the model experiment. In nature, however, there are countless different bacteria, which gives rise to new questions: for which of them is it an advantage to be mobile and for which is it not? It will, therefore, be some time before the processes in the soil are fully understood.

The future aim of the Helmholtz researchers is to model microbial landscapes and to investigate what happens under the influence of different factors. For this they will make use of a tool that has already helped to predict the spread of rabies and the spread of resettled animal species – ecological modelling, which in future will also be able to provide forecasts about the spread of bacteria. This knowledge will make it easier to remediate contaminated soil, perhaps making the ‘fungal highway’ not only the largest in the world, but also the only one to help return nature to its original state.

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Adapted from materials provided by Helmholtz Centre For Environmental Research - UFZ.

Mutualism: Fungus Found That Needs Bacteria In Cytoplasm To Reproduce

2008-06-29T22:40:00.000-07:00

Mutualism: Fungus Found That Needs Bacteria In Cytoplasm To Reproduce

ScienceDaily (Apr. 6, 2007) — Endosymbiotic relationships--in which one organism lives within another--are striking examples of mutualism, and can often significantly shape the biology of the participant species.

In new findings that highlight the extent to which a host organism can become dependent on its internal symbiont, researchers have identified a case in which the reproduction of a fungus has become dependent on bacteria that live within its cytoplasm. The findings, which appear online in the journal Current Biology on April 5th, are reported by Laila Partida and Christian Hertweck from the Leibniz Institute for Natural Product Research and Infection Biology in Jena, Germany.

The particular partnership under study is the symbiosis of the fungus Rhizopus microsporus and Burkholderia bacteria that live within its cells. The two species effectively team up to break down young rice plants for their nutrients, causing a plant disease known as rice seedling blight. Past work from the research group had revealed that the Burkholderia bacteria play a critical role in the virulence of the fungus against rice seedlings: The bacteria produce a plant poison known as rhizoxin, which has been shown to be the causative agent in rice seedling blight.

The researchers now report a second, striking benefit conferred on the fungus by its intracellular symbiont. When the bacteria are eliminated from the fungus with antibiotic treatment, the fungal cells are no longer able to form spores, suggesting that the bacterial symbiont is in fact required for this mode of fungal reproduction. Spore formation in fungi is a universal process that allows the rapid distribution of fungal cells. The new findings appear to represent the first known case in which spore formation--also known as vegetative reproduction--depends on the presence of another organism.

The researchers found that when both organisms were brought together to re-establish the symbiosis, sporulation was restored in the fungus.

In collaboration with researchers at the Leibniz Institute for Age Research, Jena, the team also made progress in understanding how the endosymbiotic bacteria influence reproduction by their host. Using a laser gun to introduce Burkholderia that had been specially labeled with a marker known as green fluorescent protein, the researchers were able to detect the bacteria within both mycelium--the vegetative portion of the fungus--and fungal spores.

On the basis of their findings, the authors conclude that the symbiont-dependent spore formation they observe is a means to maintain the symbiosis between the two species. Although the fungus has lost control over its reproduction, the endofungal bacteria in return provide a highly potent toxin for defending the habitat and accessing nutrients from decaying plants.

Partida-Martinez et al.: "Endosymbiont-Dependent Host Reproduction Maintains Bacterial-Fungal Mutualism." Publishing in Current Biology 17, 1--5, May 1, 2007. DOI 10.1016/j.cub.2007.03.039.

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Adapted from materials provided by Cell Press, via EurekAlert!, a service of AAAS.

2008-06-29T22:37:00.000-07:00

Fungi Respond To Climate Change

ScienceDaily (Apr. 25, 2007) — Climate change is dramatically altering the growing patterns of mushrooms, toadstools and other fungi, new research has found.

There are around 18,000 different species of fungi in the UK -- three times as many as all plants put together. They provide vital ecosystem services for the welfare of native trees and other plants, and are the natural recyclers of the planet, but until now their response to global climate change has not been examined.

A team from Cardiff University’s School of Biosciences working on a project led by Royal Holloway, University of London and with the Natural Environment Research Council Centre for Ecology and Hydrology studied more than 52,000 fungal fruiting records from nearly 1,400 localities collected in southern England between 1950 - 2005.

The study found that fungi are fruiting significantly earlier and for a longer period than ever before. In the 1950s fungi fruited over a period of around 33 days but this has more than doubled to nearly 75 days in the current decade.

Professor Lynne Boddy, Cardiff School of Biosciences said: "The increase in the overall fruiting period is dramatic, and much higher than equivalent spring data reported for plants, insects or birds."

The study found that the alteration in fungal fruiting mirrors changes in British temperatures that have occurred since 1975. The increase in late summer temperatures and autumnal rains has caused early season species to fruit earlier and late season species to continue to fruit later. Furthermore, climate warming seems to have caused significant numbers of species to begin fruiting in spring as well as autumn, suggesting increases in decay rates in forests.

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Adapted from materials provided by Cardiff University.

Secrets Of Cooperation Between Trees And Fungi Revealed

2008-06-29T22:33:00.001-07:00

Secrets Of Cooperation Between Trees And Fungi Revealed

ScienceDaily (Mar. 6, 2008) — Plants gained their ancestral toehold on dry land with considerable help from their fungal friends. Now, millennia later, that partnership is being exploited as a strategy to bolster biomass production for next generation biofuels. The genetic mechanism of this kind of symbiosis, which contributes to the delicate ecological balance in healthy forests, also provides insights into plant health that may enable more efficient carbon sequestration and enhanced phytoremediation, using plants to clean up environmental contaminants.

These prospects stem from the genome analysis of the symbiotic fungus Laccaria bicolor, generated by the U.S. Department of Energy Joint Genome Institute (DOE JGI) and collaborators from INRA, the National Institute for Agricultural Research in Nancy, France, and published March 6 in the journal Nature. This international team effort also involved contributions from 16 institutions, including Oak Ridge National Laboratory; Ghent University, Belgium; Lund University, Sweden; Goettingen University, Germany; CNRS-Aix-Marseille University, France; Nancy University, France; and the University of Alabama, Huntsville.

In a manner of speaking, trees are the lungs of the earth. They draw CO2 from the atmosphere and convert it into sugars, which then become a source of energy. In the process they breathe O2 back into the atmosphere. This "green" production of biomass -- trees account for 90% of the planet's land-based biomass -- is a major influence on the health of our planet.

Trees' ability to generate large amounts of biomass or store carbon is underpinned by their interactions with soil microbes known as mycorrhizal fungi, which excel at procuring necessary, but scarce, nutrients such as phosphate and nitrogen. Most of these nutrients are transferred to the growing tree. When Laccaria bicolor establishes a partnership with plant roots, a mycorrhizal root is created. The fungus within the root is protected from competition with other soil microbes and gains preferential access to carbohydrates within the plant. Thus, the mutualistic relationship is established.

"Forests around the world rely on the partnership between plant roots and soil fungi and the environment they create, the rhizosphere," said Eddy Rubin, DOE JGI Director. "The Laccaria genome represents a valuable resource, the first of a series of tree community genomics projects to have passed through our production sequencing line. These community resources promise to advance a systems approach to forest genomics."

Rubin indicates that by using DNA sequence to survey the forest ecosystem, from the plants to symbiotic and pathogenic fungi, researchers can ultimately optimize the conditions under which a biomass plantation would thrive. "We now have the opportunity to gain fundamental insights into plant development and growth as related to their intimate interaction which symbiotic fungi. These insights will lead to bolstered biomass productivity and improved forests."

Laccaria bicolor occurs frequently in the birch, fir, and pine forests of North America and is a common symbiont of Populus, the poplar tree whose genome was determined by the JGI in 2006 The analysis of the 65-million-base Laccaria genome, the largest fungal genome sequenced to date, yielded 20,000 predicted protein-encoding genes, almost as many as in the human genome. In sifting through these data, researchers have discovered many unexpected features, including an arsenal of small secreted proteins (SSPs), several of which are only expressed in tissues associated with symbiosis. The most prominent SSP accumulates in the extending hyphae, the tips of the fungus that colonize the roots of the host plant.

"We believe that the proteins specific to this host/fungus interface play a decisive role in the establishment of symbiosis," said Francis Martin, the Nature study's lead author. This genome exploration led Martin and his CNRS-Marseille University and DOE JGI colleagues to the unexpected observation that the genome of Laccaria lacks the enzymes involved in degradation of the carbohydrate polymers of plant cell walls but maintains the ability to degrade non-plant cell walls, which may account for Laccaria's protective capacity. These observations point towards the dual life that mycorrhizal fungi like Laccaria possess, that is, the ability to grow in soil fending off pathogens and using decaying organic matter while serving as a custodian of living plant roots.

The genome, Martin said, shows a large number of new and expanded gene families compared with other fungi. Many of these families are involved in signaling and other processes that drive the complex transition between two distinct lifestyles of Laccaria: the benign saprotroph, able to use decaying matter of animal and bacterial origins, versus the symbiont, living in mutually profitable harmony with plant roots.

The team also discovered new classes of genes that may be candidates for the complex communication that must occur between the players in the host/plant subsoil arena during fungal development. They report that fungi play a critical role in plant nutrient use efficiency by translocating nutrients and water captured in soil pores inaccessible to roots of the host plant.

"The Laccaria genome sequence, its analysis, associated genomics, and bioinformatics tools provide an unprecedented opportunity to identify the key components of organism-environment interactions that modulate ecosystem responses to global change and increased nutrient input needed for faster growth, said Martin. "By examining and manipulating patterns of gene expression, we can identify the genetic control points that regulate plant growth and plant-mutualist response in an effort to better understand how these interactions control ecosystem function."

Mycorrhizae are critical elements of the terrestrial ecosystems, Martin said, since approximately 85 percent of all plant species, including trees, are dependent on such interactions to thrive. Mycorrhizae significantly improve photosynthetic carbon assimilation by plants.

"Host trees like Populus are able to harness this formidable web of mycorrhizal hyphae that permeates the soil and leaf litter and coax a relationship for their mutual nutritional benefit," said co-author DOE JGI and Oak Ridge National Laboratory researcher Jerry Tuskan. "This process is absolutely critical to the success of the interactions between the fungi and the roots of the host plant so that an equitable exchange of nutrients can be achieved." The DOE JGI and its collaborators have now embarked on characterizing several other poplar community symbionts that will provide a more comprehensive understanding of the biological community of the poplar forest. These include Glomus, a second plant symbiotic fungus, Melampsora, a leaf pathogen, and several plant endophytes, bacteria and fungi that live inside the poplar tree.

"DOE JGI's expanding portfolio of community genomes provides the researchers with a set of resources that can be used to map out the processes by which fungi colonize wood and soil litter. These fungi interact with living plants within their ecosystem in order to perform vital functions in the carbon and nitrogen cycles that are so fundamental to sustainable plant growth," said Tuskan.

The DOE JGI Laccaria effort was led by Igor Grigoriev. Other authors include Andrea Aerts, Erika Lindquist, Asaf Salamov, Harris Shapiro, Peter Brokstein, Chris Detter (Los Alamos National Laboratory), the DOE JGI Production Genomics Facility sequencing team led by Susan Lucas, and partners at the Stanford Human Genome Center, Jane Grimwood and Jeremy Schmutz.

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Adapted from materials provided by DOE/Joint Genome Institute.

Self-fertility In Fungi: The Secrets Of 'DIY Reproduction'

2008-06-29T22:31:00.002-07:00

Self-fertility In Fungi: The Secrets Of 'DIY Reproduction'

ScienceDaily (Aug. 17, 2007) — Research from The University of Nottingham sheds new light on a fascinating phenomenon of the natural world — the ability of some species to reproduce sexually without a partner.

Scientists have been trying to determine how individuals of a key fungus, Aspergillus nidulans, are able to have sex without the need for a partner.

In new findings published in the journal Current Biology on August 2, they reveal that the fungus has evolved to incorporate the two different sexes into the same individual.

This means that when sex occurs the fungus activates its internal sexual machinery and in essence 'mates with itself' to produce new offspring, rather than bypassing the sexual act.

This is a significant discovery as it helps scientists to understand how fungi reproduce in general. Fungi can cause health problems in humans and other serious animal and plant diseases, but are also useful as sources of pharmaceuticals and food products.

The long-term aim of the research is to be able to manipulate fungal sex to our own advantage, to prevent disease and help produce better strains for use in the food and biotech industries.

Dr Paul Dyer, of the School of Biology, was lead author of the study. He said: “When we think of sex in the animal world we normally associate it with males and females attracting each other and then coming together for the sexual act.”

“But things are different in the fungal and plant kingdoms, where a lot of species are 'self fertile'. This means that they are able to have sex to produce spores and seeds without the need for a compatible partner. Our findings show that Aspergillus nidulans provides a true example of 'DIY sex'.”

Self-fertilisation is thought to have developed in some plant and fungal species as a response to a scarcity of compatible mating partners. It also allows species to maintain a combination of genes — called a genotype — that is well adapted to surviving in a certain environment.

Aspergillus nidulans is often used as a model organism for scientists studying a wide range of subjects including basic genetic problems that are also applicable to humans, including recombination, DNA repair and cell metabolism.

The work was supported by a grant from the Biotechnology and Biological Sciences Research Council (BBSRC) and also involved researchers at Northern Illinois University and CNRS in France.

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Adapted from materials provided by University of Nottingham.

Evolution Of The Sexes: What A Fungus Can Tell Us

2008-06-29T22:31:00.001-07:00

Evolution Of The Sexes: What A Fungus Can Tell Us

ScienceDaily (Jan. 10, 2008) — Fungi don't exactly come in boy and girl varieties, but they do have sex differences. In fact, a new finding from Duke University Medical Center shows that some of the earliest evolved forms of fungus contain clues to how the sexes evolved in higher animals, including that distant cousin of fungus, the human.

A team lead by Joseph Heitman, M.D. has isolated sex-determining genes from one of the oldest known types of fungi, Phycomyces blakesleeanus, findings which appear in the Jan. 10 issue of Nature.

Fungi do not have entire sex chromosomes, like the familiar X and Y chromosomes that determine sexual identity in humans. Instead, they have sex determining sequences of DNA called "mating-type loci."

Mating-type loci have been found in a number of higher-level fungal species, and exhibit an unusual amount of diversity. These differences occur even among similar fungal species leading scientists to wonder how they evolved.

Heitman's group hypothesized that the sex-determining arrangement found in one of earliest forms of fungi might reveal the ancestral structure of mating-type loci, serving as a sort of molecular fossil.

"Fungi are good model systems for the evolution of human sexual differentiation because the genetic sequences responsible for sex are smaller versions of chromosomal sex-determining regions in people," Heitman said.

To identify the mating-type loci in Phycomyces, the researchers used a computer search to compare known mating-type loci in the genomes of other fungal lineages and then genetic mapping. "We employed a usual-suspects approach, comparing proteins between fungal types before identifying a candidate that appeared related in all lineages," says Heitman.

Within this stretch of DNA, they were able to isolate two versions of a gene that regulates mating, which they dubbed sexM, (sex minus) and sexP (sex plus). Strains of fungi with opposite versions of the sex genes are able to mate with each other.

Both versions of the gene, sexM and sexP, encode for a single protein called a high mobility group (HMG)-domain protein that leads to sex differentiation through an unknown process. This protein is very similar to one encoded by the human Y chromosome, called SRY, that when turned on leads a developing fetus to exhibit male characteristics. Heitman said this similarity suggests that HMG-domain proteins may mark the evolutionary beginnings of sex determination in both fungi and humans.

Heitman's team proposes that sexM and sexP were once the same gene that went through a mutation process called inversion. The new versions then evolved into two separate sex genes. The same process is most likely responsible for the evolution of the male Y chromosome, Heitman suggests.

Heitman hopes to next identify the sex region in another fungus, Rhizopus oryzae in order to better understand how HMG-domain proteins control sex determination in fungi. Rhizopus' genes can be cultured and chemically altered in a way that Phycomyces' sex genes can not.

"Rhizopus can be used to understand the influences of certain genes in lesser studied fungi much in the way we use mice to understand genetic effects in humans," explained Alexander Idnurm, Ph.D., the primary author on the study and recently appointed assistant professor at the University of Missouri-Kansas City.

Another troubling mystery for Heitman is that certain younger fungal species lack HMG-domain proteins. He proposes that these proteins have been replaced with alternative transcription factors, which are proteins that turn genes on and off.

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Adapted from materials provided by Duke University Medical Center.

Fungi Can Tell Us About The Origin Of Sex Chromosomes

2008-06-29T22:30:00.001-07:00

Fungi Can Tell Us About The Origin Of Sex Chromosomes

ScienceDaily (Mar. 18, 2008) — Fungi do not have sexes, just so-called mating types. A new study shows that there are great similarities between the parts of DNA that determine the sex of plants and animals and the parts of DNA that determine mating types in certain fungi. This makes fungi interesting as new model organisms in studies of the evolutionary development of sex chromosomes.

In the plant and animal kingdoms there are individuals of different sexes, that is, bearers of either many tiny sex cells (males) or a few large ones (females). In the third eukaryote kingdom (organisms with DNA gathered in the cell nucleus), the fungi kingdom, there are no sexes but rather a simpler and more primitive system of different so-called mating types. These are distinguished by different variants of a few specific genes.

There are many ways to determine sex. In humans it is done by sex chromosomes. It is thought that this sex difference arose in the plant and animal kingdom from the simpler system of mating types and that this happened several times independently of each other throughout evolution. The change is believed to have happened with the inhibition of a step in the copying process in DNA, which led to two separate chromosomes. These then developed further over a long period of time.

"In humans, sex chromosomes are believed to have developed over the last 300 million years from a common 'proto-sex chromosome,'" says Hanna Johannesson, who directed the study.

The new study shows for the first time that even though fungi do not have sexes, there are many similarities between the parts of the genome that determine sex in plants and animals and the parts of the genome that control mating types in certain fungi. The research group specifically studied a spore sac fungus (Neurospora tetrasperma) and can show that the similarities are great, regarding both present-day structure and the way in which it arose.

"It's hard to study the evolution of sex chromosomes, partly because so many different and important sex-specific characters are tied to them. But much of this can be avoided if we use simpler systems, like fungi, as models."

This research was published in the journal PLoS March 17, 2008.

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Adapted from materials provided by Uppsala University, via EurekAlert!, a service of AAAS.