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

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

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

A.B.C. Radio National: Health Report Podcast
http://www.abc.net.au/rn/healthreport/
subscribe: http://www.abc.net.au/rn/healthreport/rss/hrt.xml

A.B.C. Radio National: Ockham's Razor Podcast
http://www.abc.net.au/rn/ockhamsrazor/
subscribe: http://www.abc.net.au/rn/podcast/feeds/ockham.xml

A.B.C. Radio National: The Science Show Podcast
http://www.abc.net.au/rn/scienceshow/
subscribe: http://www.abc.net.au/rn/scienceshow/rss/ssw.xml

A.B.C. Science: StarStuff Podcast
http://www.abc.net.au/science/starstuff/
subscribe: http://www.abc.net.au/science/podcast/starstuff.xml

BBC Radio 4: Material World Podcast
http://www.bbc.co.uk/radio4/science/thematerialworld.shtml
subscribe: http://downloads.bbc.co.uk/podcasts/radio4/material/rss.xml

BBC World Service: Science in Action Podcast
http://www.bbc.co.uk/worldservice/programmes/science_in_action.shtml
subscribe: http://downloads.bbc.co.uk/podcasts/worldservice/scia/rss.xml

Canadian Broadcasting Corporation: Quirks & Quarks Podcast
http://www.cbc.ca/quirks/
subscribe: http://www.cbc.ca/podcasting/includes/quirksaio.xml

Canadian Broadcasting Corporation: How To Think About Science Podcast
http://www.cbc.ca/ideas/features/science/index.html
subscribe: http://www.cbc.ca/podcasting/includes/thinkaboutscience.xml

Deutsche Welle Radio: Spectrum Podcast
http://www.dw-world.de/dw/article/0,2144,2789896,00.html
subscribe: http://rss.dw-world.de/xml/podcast_spectrum

Radio New Zealand: "Our Changing World" Podcast
http://www.radionz.co.nz/national/programmes/ourchangingworld
subscribe: http://www.radionz.co.nz/podcasts/ourchangingworld.rss

The American Society for Biochemistry and Molecular Biology (ASBMB) Podcast
http://www.asbmb.org/interactive.aspx
subcribe: http://www.asbmb.org/uploadedFiles/podcast/asbmb_podcast_rss.xml

Science Journal Podcast
http://www.sciencemag.org/about/podcast.dtl
subscribe: http://www.sciencemag.org/rss/podcast.xml

The Journal of Cell Biology and The Journal of Experimental Medicine Podcast
http://www.rupress.org/biobytes/
subscribe: http://www.jcb.org/biobytes/biobytesRSS.xml

Nature journal Podcast
http://www.nature.com/nature/podcast/
subscribe: http://www.nature.com/nature/podcast/rss/nature.xml

Nature journal: 'ChemPod' Podcast
http://www.nature.com/chemistry/podcast.html
subscribe: http://www.nature.com/chemistry/podcast/rss/chemistry.xml

Chemistry World Podcast
http://www.rsc.org/chemistryworld/podcast/CWpodcast.asp
subscribe: http://www.rsc.org/chemistryworld/CWLatestNews.xml

CSIRO Podcast
http://www.csiro.au/products/CSIROpod/index.html
subscribe: http://www.csiro.au/files/CSIROau/csiropod.xml

Museum of Science (Boston, USA) Podcast
http://www.mos.org/events_activities/podcasts
subscribe: http://www.mos.org/events_activities/podcasts&rss&podcast

Futures in Biotech Podcast
http://www.futuresinbiotech.com/
subscribe: http://leoville.tv/podcasts/fib.xml

MicrobeWorld Radio: Meet the Scientist Podcast
http://meetthescientist.wordpress.com/
subscribe: http://feeds.feedburner.com/meetthescientist

The Royal Society Podcast
http://royalsociety.org/page.asp?id=3966
subscribe: http://royalsociety.org/rss/audio.xml

Scientific American: "Science Talk" Podcast
http://www.sciam.com/podcast/podcasts.cfm?type=science-talk
subscribe: http://www.sciam.com/podcast/sciam_podcast_r.xml

The New Yoke Times: Science Times Podcast
http://www.nytimes.com/indexes/2008/11/17/science/index.html
subscribe: http://phobos.apple.com/WebObjects/MZStore.woa/wa/viewPodcast?id=120316173

Guardian News: Science Weekly Podcast
http://www.guardian.co.uk/science/series/science
subscribe: http://www.guardian.co.uk/science/series/science/podcast.xml

The role of tip-localized mitochondria in hyphal growth

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).


View Within Article



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.


--------------------------------------------------------------------------------




Full-size image (141K)


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.


View Within Article



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.


--------------------------------------------------------------------------------




Full-size image (26K)


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.


View Within Article



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).


--------------------------------------------------------------------------------




Full-size image (118K)


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.


View Within Article



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.


--------------------------------------------------------------------------------




Full-size image (170K)


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.


View Within Article



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).


--------------------------------------------------------------------------------




Full-size image (44K)


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.


View Within Article




--------------------------------------------------------------------------------


Full-size image (50K)


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.


View Within Article



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).


--------------------------------------------------------------------------------




Full-size image (40K)


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.


View Within Article



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.

References

Chada and Hollenbeck, 2004 S.R. Chada and P.J. Hollenbeck, Nerve growth factor signaling regulates motility and docking of axonal mitochondria, Curr. Biol. 14 (2004), pp. 1272–1276. Article | PDF (164 K) | View Record in Scopus | Cited By in Scopus (38)


Ciamporova et al., 2003 M. Ciamporova, K. Dekankova, Z. Hanackova, P. Peters, M. Ovecka and F. Baluska, Structural aspects of bulge formation during root hair initiation, Plant Soil 255 (2003), pp. 1–7. View Record in Scopus | Cited By in Scopus (8)


Collinge and Trinci, 1974 A.J. Collinge and A.P.J. Trinci, Hyphal tips of wild-type and spreading colonial mutants of Neurospora crassa, Arch. Microbiol. 99 (1974), pp. 353–368. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (26)


Cresti et al., 1977 M. Cresti, E. Pacini, F. Ciampolini and G. Sarfatti, Germination and early tube development in vitro of Lycopersicon peruvianum pollen: ultrastructural features, Planta 136 (1977), pp. 239–247. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (27)


Fuchs et al., 2002 F. Fuchs, H. Prokisch, W. Neupert and B. Westermann, Interaction of mitochondria with microtubules in the filamentous fungi Neurospora crassa, J. Cell Sci. 115 (2002), pp. 1931–1937.


Fuchs and Westermann, 2005 F. Fuchs and B. Westermann, Role of Unc104/KIF1-related motor proteins in mitochondrial transport in Neurospora crassa, Mol. Biol. Cell 6 (2005), pp. 153–161. View Record in Scopus | Cited By in Scopus (16)


Galway, 2000 M.E. Galway, Root hair ultrastructure and tip growth. In: R.W. Ridge and A.M.C. Emons, Editors, Root Hairs. Cell and Molecular Biology, Springer-Verlag, Tokyo (2000), pp. 1–15.


Garcia-Muse et al., 2003 T. Garcia-Muse, G. Steinberg and J. Perez-Martin, Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis, Eukaryot. Cell 2 (2003), pp. 494–500. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (20)


Geitmann and Emons, 2000 A. Geitmann and A.M.C. Emons, The cytoskeleton in plant and fungal cell tip growth, J. Microscopy 198 (2000), pp. 218–245. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (63)


Goldstein, 1978 D.A. Goldstein, Calculations of the concentrations of free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands, Biophys. J. 26 (1978), pp. 235–242.


Gooday, 1971 G.W. Gooday, An autoradiographic study of hyphal growth in some fungi, J. Gen. Microbiol. 67 (1971), pp. 125–133.


Grove et al., 1970 S.N. Grove, C.E. Bracker and D.J. Morre, An ultrastructural basis for hyphal tip growth in Pythium ultimum, Am. J. Bot. 57 (1970), pp. 245–266. Full Text via CrossRef


Gunter et al., 2004 T.E. Gunter, D.I. Yule, K.K. Gunter, R.A. Eliseev and J.D. Salter, Calcium and mitochondria, FEBS Lett. 567 (2004), pp. 96–102. Article | PDF (170 K) | View Record in Scopus | Cited By in Scopus (76)


Gupta and Heath, 2000 G.D. Gupta and I.B. Heath, A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa, Fungal Genet. Biol. 29 (2000), pp. 187–199. Abstract | PDF (544 K) | View Record in Scopus | Cited By in Scopus (9)


Haugland, 2002 R.P. Haugland, Handbook of Fluorescent Probes (ninth ed.), Molecular Probes, Eugene, Oregon (2002).


Heath and Kaminskyj, 1989 I.B. Heath and S.G.W. Kaminskyj, The organization of tip-growth-related organelles and microtubules revealed by quantitative analysis of freeze-substituted oomycete hyphae, J. Cell Sci. 93 (1989), pp. 41–52.


Heinhorst et al., 1985 S. Heinhorst, G. Cannon and A. Weissbach, Chloroplast DNA synthesis during the cell cycle in cultured cells of Nicotiana tabacum: inhibition by nalidixic acid and hydroxyurea, Arch. Biochem. Biophys. 239 (1985), pp. 475–479. Abstract | View Record in Scopus | Cited By in Scopus (8)


Holdaway-Clarke and Hepler, 2003 T.L. Holdaway-Clarke and P.K. Hepler, Control of pollen tube growth: role of ion gradients and fluxes, New Phytol. 159 (2003), pp. 539–563. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (91)


Howell et al., 1971 N. Howell, C.A. Zuiches and K.D. Munkres, Mitochondrial biogenesis in Neurospora crassa I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition, J. Cell Biol. 50 (1971), pp. 721–736. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)


Lew et al., 1986 R.R. Lew, D.P. Briskin and R.E. Wyse, Calcium uptake by endoplasmic reticulum from zucchini shoots. The use of chlortetracycline as a probe of calcium uptake, Plant Physiol. 82 (1986), pp. 47–53.


Lew, 1999 R.R. Lew, Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa, Eur. J. Cell. Biol. 78 (1999), pp. 892–902. View Record in Scopus | Cited By in Scopus (21)


Lew and Levina, 2004 R.R. Lew and N.N. Levina, Oxygen flux magnitude and location along growing hyphae of Neurospora crassa, FEMS Microbiol. Lett. 233 (2004), pp. 125–130. Abstract | View Record in Scopus | Cited By in Scopus (3)


Lew et al., 2004 R.R. Lew, N.N. Levina, S.K. Walker and A. Garrill, Turgor regulation in hyphal organisms, Fungal Genet. Biol. 41 (2004), pp. 1007–1015. Article | PDF (693 K) | View Record in Scopus | Cited By in Scopus (10)


Luck, 1963 D.J.L. Luck, Formation of mitochondria in Neurospora crassa. A quantitative radioautographic study, J. Cell Biol. 16 (1963), pp. 483–499. Full Text via CrossRef


Mattson, 1999 M.P. Mattson, Establishment and plasticity of neuronal polarity, J. Neurosci. Res. 57 (1999), pp. 577–589. View Record in Scopus | Cited By in Scopus (32)


McCluskey, 2003 K. McCluskey, The Fungal Genetics Stock Center: from molds to molecules, Adv. Appl. Microbiol. 52 (2003), pp. 245–262. Abstract | View Record in Scopus | Cited By in Scopus (46)


Mitchell and Moyle, 1969 P. Mitchell and J. Moyle, Estimation of membrane potential and pH difference across the cristae membrane of rat liver mitochondria, Eur, J. Biochem. 7 (1969), pp. 471–484. View Record in Scopus | Cited By in Scopus (41)


Morris and Hollenbeck, 1993 R.L. Morris and P.J. Hollenbeck, The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth, J. Cell Sci. 104 (1993), pp. 917–927. View Record in Scopus | Cited By in Scopus (92)


Neupert, 1997 W. Neupert, Protein import into mitochondria, Annu. Rev. Biochem. 66 (1997), pp. 863–917. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (743)


Nicholls and Chalmers, 2004 D.G. Nicholls and S. Chalmers, The integration of mitochondrial calcium transport and storage, J. Bioenerg. Biomemb. 36 (2004), pp. 277–281. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (23)


Pierson et al., 1990 E.S. Pierson, I.K. Lichtscheidl and J. Derksen, Structure and behaviour of organelles in living pollen tubes of Lilium longiflorum, J. Exp. Bot. 41 (1990), pp. 1461–1468.


Rumpal and Lnenicka, 2003 N. Rumpal and G.A. Lnenicka, Ca2+ clearance at growth cones produced by crayfish motor axons in an explant culture, J. Neurophysiol. 89 (2003), pp. 3225–3234. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (2)


Scaduto and Grotyohann, 1999 R.C. Scaduto Jr. and L.W. Grotyohann, Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives, Biophys. J. 76 (1999), pp. 469–477. View Record in Scopus | Cited By in Scopus (258)


Sebald et al., 1968 W. Sebald, Th. Bucher, B. Olbrich and F. Kaudewitz, Electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of Neurospora crassa wild type and MI-1 mutant, FEBS Lett. 1 (1968), pp. 235–240. Abstract | PDF (520 K) | View Record in Scopus | Cited By in Scopus (1)


Seiler et al., 1999 S. Seiler, M. Plamann and M. Schliwa, Kinesin and dyenin mutants provide insights into the roles of vesicle traffic during cell morphogenesis in Neurospora, Curr. Biol. 9 (1999), pp. 779–785. Article | PDF (140 K) | View Record in Scopus | Cited By in Scopus (65)


Silverman-Gavrila and Lew, 2000 L.B. Silverman-Gavrila and R.R. Lew, Calcium and tip growth in Neurospora crassa, Protoplasma 213 (2000), pp. 203–217. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (19)


Silverman-Gavrila and Lew, 2001 L.B. Silverman-Gavrila and R.R. Lew, Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa, Eur. J. Cell Biol. 80 (2001), pp. 379–390. Abstract | PDF (322 K) | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (15)


Silverman-Gavrila and Lew, 2002 L.B. Silverman-Gavrila and R.R. Lew, An IP3-activated Ca2+ channel regulates fungal tip growth, J. Cell Sci. 115 (2002), pp. 5013–5025. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (27)


Silverman-Gavrila and Lew, 2003 L.B. Silverman-Gavrila and R.R. Lew, Calcium gradient dependence of Neurospora crassa hyphal growth, Microbiology 149 (2003), pp. 2475–2485. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (22)


Srivastava et al., 1988 V.K. Srivastava, M.L. Pall and A.L. Schroeder, Deoxyribonucleoside triphosphate pools in Neurospora crassa: effects of histidine and hydroxyurea, Mutat. Res. 200 (1988), pp. 45–53. Abstract | View Record in Scopus | Cited By in Scopus (5)


Steinberg and Schliwa, 1993 G. Steinberg and M. Schliwa, Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules, J. Cell Sci. 106 (1993), pp. 555–564. View Record in Scopus | Cited By in Scopus (46)


Torralba and Heath, 2000 S. Torralba and I.B. Heath, Cytoskeletal and Ca2+ regulation of hyphal tip growth and initiation, Curr. Top. Dev. Biol. 51 (2000), pp. 135–187.


Torralba et al., 2001 S. Torralba, I.B. Heath and F.P. Ottensmeyer, Ca2+ shuttling in vesicles during tip growth in Neurospora crassa, Fungal Genet. Biol. 33 (2001), pp. 181–193. Abstract | PDF (805 K) | View Record in Scopus | Cited By in Scopus (12)


Uwate and Lin, 1980 W.J. Uwate and J. Lin, Cytological zonation of Prunus avium L. pollen tubes in vivo, J. Ultrastruct. Res. 71 (1980), pp. 173–184. Abstract | View Record in Scopus | Cited By in Scopus (4)


Vogel, 1956 H. Vogel, A convenient growth medium for Neurospora, Microb. Genet. Bull. 13 (1956), pp. 42–46.


Westermann and Prokisch, 2002 B. Westermann and H. Prokisch, Mitochondrial dynamics in filamentous fungi, Fungal Genet. Biol. 36 (2002), pp. 91–97. Article | PDF (110 K) | View Record in Scopus | Cited By in Scopus (15)


Yuan and Heath, 1991 S. Yuan and I.B.H. Heath, Chlortetracycline staining patterns of growing hyphal tips of the oomycete Saprolegnia ferax, Exp. Mycol. 15 (1991), pp. 91–102. Abstract | View Record in Scopus | Cited By in Scopus (15)


Zalokar, 1959 M. Zalokar, Growth and differentiation of Neurospora hyphae, Am. J. Bot. 46 (1959), pp. 602–610. Full Text via CrossRef

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

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).


--------------------------------------------------------------------------------


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
Full-size table

View Within Article



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).


--------------------------------------------------------------------------------




Full-size image (42K)


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.


View Within Article




--------------------------------------------------------------------------------


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
Full-size table

View Within Article



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.

References

Abu et al., 2004 S.M. Abu, G. Li and F.O. Asiegbu, Identification of Heterobasidion annosum (S-type) genes expressed during initial stages of conidiospore germination and under varying culture conditions, FEMS Microbiol. Lett. 233 (2004) (2), pp. 205–213. Abstract | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)


Akinsanmi and Ladipo, 2001 O.A. Akinsanmi and J.L. Ladipo, First report of soybean rust (Phakopsora pachyrhizi) in Nigeria, Plant Dis. 85 (2001), p. 97.


Altschul et al., 1997 Altschul, S.F., Madden, T.L., Schaffer, A.A., Jinghui Zhang, Zheng Zhang, Webb Miller, Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.


AVRDC, 1987 Asian Vegetable Research and Development Center, 1987. Bibliography of soybean rust, 1985–1986. AVRDC Asian Veg. Res. Dev. Cent. Library Bibliography Series 4. Publication No. 87-277.


AVRDC, 1992 Asian Vegetable Research and Development Center, 1992. Annotated bibliography of soybean rust (Phakopsora pachyrhizi Sydow). AVRDC (Asian Veg. Res. Dev. Cent.) Library Bibliography Series 4-1. Publication No. 92-372.


Austin et al., 2004 R. Austin, N.J. Provart, N.T. Sacadura, K.G. Nugent, M. Babu and B.J. Saville, A comparative genomic analysis of ESTs from Ustilago maydis, Funct. Integr. Genomics 4 (2004) (4), pp. 201–218.


Bonde et al., 1976 M.R. Bonde, J.S. Melching and K.R. Bromfield, Histology of the suscept-pathogen relationship between Glycine max and Phakopsora pachyrhizi, the cause of soybean rust, Phytopathology 66 (1976), pp. 1290–1294.


Bromfield, 1984 Bromfield, K.R., 1984. Soybean rust, Monograph (American Phytopathological Society), No. 11. American Phytopathological Society. St. Paul, MN.


Bromfield and Hartwig, 1980 K.R. Bromfield and E.E. Hartwig, Resistance to soybean rust (Phakopsora pachyrhizi) and mode of inheritance, Crop Sci. 20 (1980), pp. 254–255.


Cherry and Peet, 1966 E. Cherry and C.E. Peet, An efficient device for the rapid collection of fungal spores from infected plants, Phytopathology 56 (1966), pp. 1102–1103.


Choi and Dean, 2000 Choi, W., Dean, R.A., 2000. Sequence analysis and characterization of genes expressed during appressorium formation in rice blast fungus, Magnaporthe grisea. Unpublished. MAS1 Accession No. AF264035.1. MAS3 Accession No. AF3630651.


Dawe et al., 2003 A.L. Dawe, V.C. McMains, M. Panglao, S. Kasahara, B. Chen and D.L. Nuss, An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea, Microbiology 149 (2003) (9), pp. 2373–2384. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (22)


Diener et al., 2004 S.E. Diener, N. Dunn-Coleman, P. Foreman, T.D. Houfek, P.J. Teunissen, P. van Solingen, L. Dankmeyer, T.K. Mitchell, M. Ward and R.A. Dean, Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesi, FEMS Microbiol. Lett. 230 (2004) (2), pp. 275–282. Abstract | View Record in Scopus | Cited By in Scopus (9)


d’Enfert, 1997 C. d’Enfert, Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa, Fungal Genet. Biol. 21 (1997) (2), pp. 163–172.


Ebbole et al., 2004 D.J. Ebbole, Y. Jin, M. Thon, H. Pan, E. Bhattarai, T. Thomas and R. Dean, Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags, Mol. Plant Microbe Interact. 17 (2004) (12), pp. 1337–1347. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (33)


Frederick et al., 2002 R.D. Frederick, C.L. Snyder, G.L. Peterson and M.R. Bonde, Polymerase chain reaction assays for the detection and discrimination of the soybean rust pathogens Phakopsora pachyrhizi and P. meibomiae, Phytopathology 92 (2002), pp. 217–227. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (36)


Giavara et al., 2005 S. Giavara, E. Kosmidou, M. Prakash Hanfe, M.E. Bianchi, A. Morgan, F. d’Adda di Fagagna and S.P. Jackson, Yeast Nhp6A/B and mammalian Mmgb1 facilitate the maintenance of genome stability, Curr. Biol. 15 (2005) (1), pp. 68–72. Article | PDF (226 K) | View Record in Scopus | Cited By in Scopus (16)


Guettler et al., 2003 S. Guettler, E.N. Jackson, S.A. Lucchese, L. Honaas, A. Green, C.T. Hittinger, Y. Tian, W.W. Lilly and A.C. Gathman, ESTs from the basidiomycete Schizophyllum commune grown on nitrogen-replete and nitrogen-limited media, Fungal Genet. Biol. 39 (2003) (2), pp. 191–198. Article | PDF (156 K) | View Record in Scopus | Cited By in Scopus (8)


Hartmann et al., 2001 M. Hartmann, G. Heinrich and G.H. Braus, Regulative fine-tuning of the two novel DAHP isoenzymes aroFp and aroGp of the filamentous fungus Aspergillus nidulans, Arch. Microbiol. 175 (2001) (2), pp. 112–121. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (6)


Hartwig, 1986 E.E. Hartwig, Identification of a fourth major gene conferring resistance to soybean rust, Crop Sci. 26 (1986), pp. 1135–1136.


Hartwig and Bromfield, 1983 E.E. Hartwig and K.R. Bromfield, Relationships among three genes conferring specific resistance to rust in soybeans, Crop Sci. 23 (1983), pp. 237–239.


Horiuchi et al., 1999 H. Horiuchi, M. Fujiwara, S. Yamashita, A. Ohta and M. Takagi, Proliferation of intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans, J. Bacteriol. 181 (1999) (12), pp. 3721–3729. View Record in Scopus | Cited By in Scopus (45)


Justesen et al., 1996 A. Justesen, S. Somerville, S. Christiansen and H. Giese, Isolation and characterization of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f. sp. hordei, Gene 170 (1996) (1), pp. 131–135. Article | PDF (745 K) | View Record in Scopus | Cited By in Scopus (17)


Kamoun et al., 1999 S. Kamoun, P. Hraber, B. Sobral, D. Nuss and F. Govers, Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences, Fungal Genet. Biol. 28 (1999) (2), pp. 94–106. Abstract | PDF (197 K) | View Record in Scopus | Cited By in Scopus (86)


Karlsson et al., 2003 M. Karlsson, A. Olson and J. Stenlid, Expressed sequences from the basidiomycetous tree Heterobasidion annosum during early infection of scots pine, Fungal Genet. Biol. 39 (2003) (1), pp. 51–59. Article | PDF (208 K) | View Record in Scopus | Cited By in Scopus (26)


Keller and Hohn, 1997 N.P. Keller and T.M. Hohn, Metabolic pathway gene clusters in filamentous fungi, Fungal Genet. Biol. 21 (1997) (2), pp. 17–29. Abstract | PDF (174 K) | View Record in Scopus | Cited By in Scopus (185)


Keon et al., 2000 J. Keon, A. Bailey and J.A. Hargreaves, A group of expressed cDNA sequences from the wheat fungal leaf blotch pathogen, Mycosphaerella graminicola (Septoria tritici), Fungal Genet. Biol. 29 (2000) (2), pp. 118–133. Abstract | PDF (103 K) | View Record in Scopus | Cited By in Scopus (37)


Killgore and Heu, 1994 E. Killgore and R. Heu, First report of soybean rust in Hawaii, Plant Dis. 78 (1994), p. 1216.


Kim et al., 1998 Y.K. Kim, D. Li and P.E. Kolattukudy, Induction of Ca2+-calmodulin signaling by hard-surface contact primes Colletotrichum gloeosporioides conidia to germinate and form appressoria, J. Bacteriol. 180 (1998) (19), pp. 5144–5150. View Record in Scopus | Cited By in Scopus (43)


Kim et al., 2000 Y.K. Kim, Z.M. Liu, D. Li and P.E. Kolattukudy, Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia, J. Bacteriol. 182 (2000) (17), pp. 4688–4695. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (11)


Kim et al., 2001 S. Kim, I.-P. Ahn and Y.-H. Lee, Analysis of genes expressed during rice–Magnaporthe grisea interactions, Mol. Plant Microbe Interact. 14 (2001) (11), pp. 1340–1346. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (66)


Kuehl et al., 1984 L. Kuehl, B. Salmond and L. Tran, Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues, J. Cell Biol. 99 (1984) (2), pp. 648–654. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (14)


Künzler et al., 1992 M. Künzler, G. Paravicini, C.M. Egli, S. Irniger and G.H. Braus, Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-l-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae, Gene 113 (1992) (1), pp. 67–74. Abstract | View Record in Scopus | Cited By in Scopus (22)


Kupfer et al., 1997 D.M. Kupfer, C.A. Reece, S.W. Clifton and R.A. Prade, Multicellular ascomycetous fungal genomes contain more than 8000 genes, Fungal Genet. Biol. 21 (1997) (3), pp. 364–372. Abstract | PDF (176 K) | View Record in Scopus | Cited By in Scopus (62)


Lee et al., 2002 S.H. Lee, B.G. Kim, K.J. Kim, J.S. Lee, D.W. Yun, J.H. Hahn, G.H. Kim, K.H. Lee, D.S. Suh, S.T. Kwon, C.S. Lee and Y.B. Yoo, Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus, Fungal Genet. Biol. 35 (2002) (2), pp. 115–134. Abstract | PDF (112 K) | View Record in Scopus | Cited By in Scopus (32)


Levy, 2003 C. Levy, Measures to control soybean rust in Southern Africa and an initial investigation of the meteorological factors that favor its development, Phytopathology 93 (2003), p. S103.


Li et al., 2004 R. Li, R. Rimmer, L. Buchwaldt, A.G. Sharpe, G. Squin-Swartz, C. Coutu and D.D. Hegedus, Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis, Fungal Genet. Biol. 41 (2004) (8), pp. 735–753. Article | PDF (226 K) | View Record in Scopus | Cited By in Scopus (23)


Liu and Kolattukudy, 1999 Z.M. Liu and P.E. Kolattukudy, Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment, J. Bacteriol. 181 (1999) (11), pp. 3571–3577. View Record in Scopus | Cited By in Scopus (35)


Martinez et al., 2004 D. Martinez, L.F. Larrondo, N. Putnam, M.D. Gelpke, K. Huang, J. Chapman, K.G. Helfenbein, P. Ramaiya, J.C. Detter, F. Larimer, P.M. Coutinho, B. Henrissat, R. Berka, D. Cullen and D. Rokhsar, Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78, Nat. Biotechnol. 22 (2004) (6), pp. 695–700. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (119)


Masse et al., 2002 J.E. Masse, B. Wong, Y.M. Yen, F.H. Allain, R.C. Johnson and J. Feigon, The Saccharomyces cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding, J. Mol. Biol. 323 (2002) (2), pp. 263–284. Article | PDF (5230 K) | View Record in Scopus | Cited By in Scopus (21)


McLean and Byth, 1980 R.J. McLean and D. Byth, Inheritance of resistance to rust (Phakopsora pachyrhizi) in soybean, Aust. J. Agric. Res. 31 (1980), pp. 951–956. Full Text via CrossRef


Melching et al., 1983 J.S. Melching, K.R. Bromfield and C.H. Kingsolver, The plant pathogen containment facility at Frederick, Maryland, Plant Dis. 67 (1983), pp. 717–722. Full Text via CrossRef


Morel, 2001 Morel, W.P., 2001. Roya de la soja. Ministerio de Agricultura y Ganaderia, Subsecretaria de Agricultura, Direccion de Investigacion Agricola, Centro Regional de Investigacion Agricola-CRIA, Capitan Miranda, Itapua, Paraguay. Comunicado Tecnico-Report Official, Serie Fitopathologia, 1, Junio de 2001.


Nelson et al., 1997 M.A. Nelson, S. Kang, E.L. Braun, M.E. Crawford, P.L. Dolan, P.M. Leonard, J. Mitchell, A.M. Armijo, L. Bean, E. Blueyes, T. Cushing, A. Errett, M. Fleharty, M. Gorman, K. Judson, R. Miller, J. Ortega, I. Pavlova, J. Perea, S. Todisco, R. Trujillo, J. Valentine, A. Wells, M. Werner-Washburne, S. Yazzie and D.O. Natvig, Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa, Fungal Genet. Biol. 21 (1997) (3), pp. 348–363. Abstract | PDF (591 K) | View Record in Scopus | Cited By in Scopus (96)


Neuman and Dobinson, 2003 M.J. Neuman and K.F. Dobinson, Sequence tag analysis of gene expression during pathogenic growth and microsclerotia development in the vascular wilt pathogen Verticillium dahliae, Fungal Genet. Biol. 38 (2003) (1), pp. 54–62.


Nugent et al., 2004 K.G. Nugent, K. Choffe and B.J. Saville, Gene expression during Ustilago maydis filamentous growth: EST library creation and analysis, Fungal Genet. Biol. 41 (2004) (3), pp. 349–360. Article | PDF (346 K) | View Record in Scopus | Cited By in Scopus (17)


OBrian et al., 2003 G.R. OBrian, A.M. Fakhoury and G.A. Payne, Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus, Fungal Genet. Biol. 39 (2003) (2), pp. 118–127. Article | PDF (249 K) | View Record in Scopus | Cited By in Scopus (23)


Ono et al., 1992 Y. Ono, P. Buritica and J.F. Hennen, Delimitation of Phakopsora, Physopella, and Cerotelium and their species on leguminosae, Mycol. Res. 96 (1992), pp. 825–850.


Osherov and May, 2000 N. Osherov and G.S. May, Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis, Genetics 155 (2000) (2), pp. 647–656. View Record in Scopus | Cited By in Scopus (76)


Osherov and May, 2001 N. Osherov and G.S. May, The molecular mechanisms of conidial germination, FEMS Microbiol. Lett. 199 (2001) (2), pp. 153–160. Article | PDF (338 K) | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (51)


Ospina-Giraldo et al., 2000 M.D. Ospina-Giraldo, P.D. Collopy, C.P. Romaine and D.J. Royse, Classification of sequences expressed during the primordial and basidiome stages of the cultivated mushroom Agaricus bisporus, Fungal Genet. Biol. 29 (2000) (2), pp. 81–94. Abstract | PDF (136 K) | View Record in Scopus | Cited By in Scopus (17)


Park et al., 1999 I.C. Park, H. Horiuchi, C.W. Hwang, W.H. Yeh, A. Ohta, J.C. Ryu and M. Takagi, Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae, FEMS Microbiol. Lett. 170 (1999) (1), pp. 131–139. Article | PDF (991 K) | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (21)


Posada and Frederick, 2002 M.L. Posada and R.D. Frederick, Expressed sequence tags analysis from germinating urediniospores of the plant pathogen Phakopsora pachyrhizi, Phytopathology 92 (2002), p. S66.


Pretorius et al., 2001 Z.A. Pretorius, R.J. Kloppers and R.D. Frederick, First report of soybean rust in South Africa, Plant Dis. 85 (2001), p. 1288.


Qutob et al., 2000 D. Qutob, P.T. Hraber, B.W.S. Sobral and M. Gijzen, Comparative analysis of expressed sequences in Phytophthora infestans, Plant Physiol. 123 (2000), pp. 243–253. View Record in Scopus | Cited By in Scopus (93)


Randall et al., 2005 T.A. Randall, R.A. Dwyer, E. Huitema, K. Beyer, C. Cvitanich, H. Kelkar, A.M.V. Ah Fong, K. Gates, S. Roberts, E. Yatzkan, T. Gaffney, M. Law, A. Testa, T. Torto-Alalibo, M. Zhang, L. Zheng, E. Mueller, J. Windass, A. Binder, P.R.J. Birch, U. Gisi, F. Govers, N.A. Gow, F. Mauch, P. van West, M.E. Waugh, J. Yu, T. Boller, S. Kamoun, S.T. Lam and H.S. Judelson, Large-scale gene discovery in the Oomycete Phytophthora infestans reveals likely components of phytopathogenicity shared with true fungi, Mol. Plant Microbe Interact. 18 (2005) (3), pp. 229–243. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (43)


Rossi, 2003 R.L. Rossi, First report of Phakopsora pachyrhizi, the causal organism of soybean rust in the province of Misiones, Argentina, Plant Dis. 87 (2003), p. 102.


Rytter et al., 1984 J.L. Rytter, W.M. Dowler and K.R. Bromfield, Additional alternative hosts of Phakopsora pachyrhizi, causal agent of soybean rust, Plant Dis. 68 (1984), pp. 818–819. Full Text via CrossRef


Saksirirat and Hoppe, 1991 W. Saksirirat and H.H. Hoppe, Teliospore germination of soybean rust fungus (Phakopsora pachyrhizi Syd.), J. Phytopathol. 132 (1991), pp. 339–342. Full Text via CrossRef


Schneider et al., 2005 R.W. Schneider, C.A. Hollier, H.K. Whitam, M.E. Palm, J.M. McKemy, J.R. Hernandez, L. Levy and R. DeVries-Paterson, First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States, Plant Dis. 89 (2005), p. 773.


Sims et al., 2004 A.H. Sims, G.D. Robson, D.C. Hoyle, S.G. Oliver, R.A. Prade, H.H. Russell, N.S. Dunn-Coleman and M.E. Gent, Use of expressed sequence tag analysis and microarrays of the filamentous fungus Aspergillus nidulans, Fungal Genet. Biol. 41 (2004) (2), pp. 199–212. Article | PDF (342 K) | View Record in Scopus | Cited By in Scopus (23)


Sinclair, 1989 J.B. Sinclair, Threats to soybean production in the tropics: red leaf blotch and leaf rust, Plant Dis. 73 (1989) (7), pp. 604–606. Full Text via CrossRef


Sinclair and Hartman, 1996 Sinclair, J.B., Hartman, G.L. (Eds.), 1996. Proceedings of the Soybean Rust Workshop (1995). National Soybean Research Laboratory, Publication No. 1.


Soanes et al., 2002 D.M. Soanes, W. Skinner, J. Keon, J. Hargreaves and N.J. Talbot, Genomics of phytopathogenic fungi and the development of bioinformatic resources, Mol. Plant Microbe Interact. 15 (2002) (5), pp. 421–427. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (51)


Steen et al., 2003 B.R. Steen, S. Zuyderduyn, D.L. Toffaletti, M. Marra, S.J. Jones, J.R. Perfect and J. Kronstad, Cryptococcus neoformans gene expression during experimental cryptococcal meningitis, Eukaryot. Cell 2 (2003) (6), pp. 1336–1349. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (30)


Thomas et al., 2001 S.W. Thomas, S.W. Rasmussen, M.A. Glaring, J.A. Rouster, S.K. Christiansen and R.P. Oliver, Gene identification in the obligate fungal pathogen Blumeria graminis by expressed sequence tag analysis, Fungal Genet. Biol. 33 (2001) (3), pp. 195–211. Abstract | PDF (577 K) | View Record in Scopus | Cited By in Scopus (65)


Trail et al., 2003 F. Trail, J.R. Xu, P. San Miguel, R.G. Halgren and H.C. Kistler, Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum), Fungal Genet. Biol. 38 (2003) (2), pp. 187–197. Article | PDF (274 K) | View Record in Scopus | Cited By in Scopus (48)


Yang et al., 1991 X.B. Yang, W.M. Dowler and M.H. Royer, Assessing the risk and potential impact of an exotic plant disease, Plant Dis. 75 (1991), pp. 976–982.


Yeh et al., 1981 C.C. Yeh, A.T. Tschanz and J.B. Sinclair, Induced teliospore formation by Phakopsora pachyrhizi on soybeans and other hosts, Phytopathology 71 (1981), pp. 1111–1112. Full Text via CrossRef


Yen et al., 1998 Y.M. Yen, B. Wong and R.C. Johnson, Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine, J. Biol. Chem. 273 (1998) (8), pp. 4424–4435. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (52)


Yorinori et al., 2002 Yorinori, J.T., Paiva, W.M., Frederick, R.D., Fernandez, F.T.P., 2002. Ferrugem da soja (Phakopsora pachyrhizi) no Brasil e no Paraguai, nas safras 2000/01 e 2001/02, p. 94. In: Resumos Da II Congreso Brasilerio de Soja e Mercosoja 2002. Embrapa Soja, Londrina, 2002, 393 p. (Documentos/Embrapa soja; n. 181. Resumo 094).


Yorinori et al., 2005 J.T. Yorinori, W.M. Paiva, R.D. Frederick, L.M. Costamilan, P.F. Bertagnoli, G.L. Hartman, C.V. Godoy and J.J. Nunes, Epidemics of soybean rust (Phakopsora pachyrhizi) in Brazil and Paraguay from 2001 to 2003, Plant Dis. 89 (2005), pp. 675–677. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (24)


Zhu et al., 2001 H. Zhu, M. Nowrousian, D. Kupfer, H.V. Colot, G. Berrocal-Tito, H. Lai, D. Bell-Pedersen, B.A. Roe, J.J. Loros and J.C. Dunlap, Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes, Genetics 157 (2001), pp. 1057–1065. View Record in Scopus | Cited By in Scopus (48)


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.