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.


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


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Table 1.

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

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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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Table 2.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.