Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici)

  • Peng Ling1, 2,

    Affiliated with

    • Meinan Wang2, 3,

      Affiliated with

      • Xianming Chen1, 2Email author and

        Affiliated with

        • Kimberly Garland Campbell1, 4

          Affiliated with

          BMC Genomics20078:145

          DOI: 10.1186/1471-2164-8-145

          Received: 24 November 2006

          Accepted: 04 June 2007

          Published: 04 June 2007

          Abstract

          Background

          Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogeniCity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi.

          Results

          A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%). In addition, 36 clones (18.4%) had significant homology to hypothetical proteins, 37 clones (18.9%) had some homology to genes in other fungi, and the remaining 50 clones (25.5%) did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection.

          Conclusion

          The full-length cDNA library is useful in identifying functional genes of P. striiformis.

          Background

          Puccinia striiformis Westend., a fungus in Pucciniacea, Uredinales, Basidiomycotina, Eumycota, causes stripe (yellow) rust. Based on specific pathogeniCity on cereal crops and grasses, the fungal species consists of various formae speciales, such as P. striiformis f. sp. tritici on wheat (Triticum aestivum), P. striiformis f. sp. hordei on barley (Hordeum vulgare), P. striiformis f. sp. poae on bluegrass (Poa pratensis) and P. striiformis f. sp. dactylidis on orchard grass (Dactylis glomerata) [9, 32]. Among the various formae speciales, the wheat and barley stripe rust pathogens are most economically important. Wheat stripe rust has been reported in more than 60 countries and all continents except Antarctica [6]. Devastating epidemics of wheat stripe rust often occur in many countries in Africa, Asia, Australia, Europe, North America and South America [6, 32]. In the U. S., stripe rust of wheat has existed for more than 100 years [19, 25]. The disease had been primarily a major problem in western US before 2000, but has become increasingly important in the south central and the Great Plains since 2000 [6, 11, 25]. Barley stripe rust is a relatively new disease in the west hemisphere. It has caused severe damage in some locations since it was introduced to Colombia in 1975 from Europe [14], and spread to Mexico in 1987 [1] and the U. S. in 1991 [5, 9, 29]. In spite of its importance, very little is known about the molecular biology and the genomics of the stripe rust fungus.

          The life cycle of the stripe rust fungus consists of the dikaryotic uredial and diploid telial stages in the nature [24, 32]. Teliospores can germinate to form haploid basidiniospores. Unlike the stem rust (P. graminis) and leaf rust (P. triticina) pathogens, the stripe rust pathogen does not have known alternate hosts for basidiniospores to infect, and thus, it does not have known sexual pycnial and aecial stages. Therefore, isolates of the fungus cannot be crossed through sexual hybridization, which makes it impossible to study the fungal genes through classic genetic approaches. The fungus reproduces and spreads through urediniospores and survives as mycelium in living host plants. Because urediniospores cannot keep their viability for very long, living plants (volunteers of wheat and barley crops and grasses, or crops and grasses in cool regions in the summer and in warm regions in the winter) are essential to keep the fungus alive from season to season. Although the pathogen does not have known sexual reproduction, there is a high degree of variation in virulence and DNA polymorphism in the natural populations of the stripe rust pathogens [5, 6, 8, 9, 11, 25]. More than 100 races of P. striiformis f. sp. tritici and more than 70 races of P. striiformis f. sp. hordei have been identified in the U. S. [5, 6] based on virulence/avirulence patterns produced on differential cultivars by isolates of the pathogens. The avirulence or virulence phenotypes have not been associated with any specific genes or DNA sequences due to the factors that the pathogen can not be studied by conventional analyses.

          The expressed sequence tag (EST) technology is an approach to identify genes in organisms that are difficult to study using classic genetic approaches and gene mutation by insertional mutagenesis. Liu et al. [26] analyzed abundant and stage-specific mRNA from P. graminis. Lin et al. [23] isolated and studied the expression of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions. Recently, EST libraries have been constructed for various fungal species including P. triticina [18], the probably most closely related fungal species to P. striiformis. ESTs provide valuable putative gene sequence information for genomic studies of targeted organisms. However, EST data has its own limitations such as incomplete cDNA sequence. Because ESTs are typically generated from the 3' end sequences of cDNA clones, EST libraries tend to be incomplete at the 5' end of the transcripts. The cDNA libraries constructed by conventional methods [17] normally contain a high percentage of 5' truncated clones due to the premature stop of reverse transcription (RT) of the template mRNA, particularly for cDNA clones derived from large mRNA molecules and those with the potential to form secondary structures. The size bias against large fragments commonly exists in conventional cDNA cloning procedures. Certain limitations also apply to the end products of the automatic EST assemblies, which may be composed of ESTs generated from different tissues or different developmental stages and may not reflect the accurate transcripts.

          Several methods have been developed to construct cDNA libraries that are enriched for full-length cDNAs, including RNA oligo ligation to the 5' end of mRNA [21, 33], 5' cap affinity selection via eukaryotic initiation factor [15], or 5' cap biotinylation followed by biotin affinity selection [2]. These methods can be used to improve the full-length cDNA clone content of the cDNA library, but they are all very laborious and involve several enzymatic steps that must be performed on mRNA. Therefore, they are prone to quality loss through RNA degradation. Furthermore, they all require high amounts of starting mRNA at μg level for reverse transcription and cloning processes. Comprehensive sets of accurate, full-length cDNA sequences would address many of the current limitations of the EST data. Genome-scale collections of full-length cDNA become important for analyses of the structures and functions of expressed genes and their products [31]. Full-length cDNA library is a powerful tool for functional genomics and is widely used as physical resources for identifying genes [36].

          A full-length cDNA library should be an important resource for studying important genes of the P. striiformis pathogen, for sequencing the whole genome, and for determining its interaction with host plants. The objectives of the present study were to construct a full-length cDNA library for P. striiformis f. sp. tritici and characterize selected cDNA sequences in the library to identify putative functional genes of P. striiformis f. sp. tritici.

          Results

          Full-length cDNA library generation and characterization

          Total RNA was extracted from 30 mg urediniospores of race PST-78 of P. striiformis f. sp. tritici and yielded approximately 7.5 μg total RNA of high purity. Full-length cDNA was synthesized by reverse transcription and enriched by subsequent long distance PCR (LD PCR). Only non-truncated first strand cDNAs were tagged by the SMART IV oligonucleotide sequence : 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3' during the initial reverse transcription. The PCR amplification products were digested with restriction enzyme sfiI to generate directional cloning ends. The agarose gel analysis of the digestion showed a significant amount of double stranded cDNA that appeared as a smear ranging from 300 bp to 12 kb. The sfiI-digested double strand cDNA was obtained from 5 fractionated gel zones. The gel zones containing smaller cDNA fragments (ranging from 500 bp to 4 kb) yielded approximately 800 ng to 1 μg of cDNA while the gel zones containing large cDNA fragments (ranging from 5 kb to 10 kb) had relatively lower cDNA yields in the 50 - 100 ng range. Although the large cDNA fragment output was relatively low, it was adequate for the subsequent ligation reaction for cloning.

          Fractionated cDNA was cloned into the sfiI sites of the pDNR-LIB cloning vector and transformed into DH10B competent cells. One microliter of ligation yielded a range of 1,000 to 2,000 recombinant clones for cDNA inserts within the large fractionated gel zone. More than 3,000 recombinant clones were obtained for cDNA inserts from the medium and smaller fractionated gel zones. The clone evaluation of random samples revealed cDNA insert length ranging from 200 bp up to 9 kb across all the fractionation inserts. In general, most of the inserts were in the length range of 500 bp to 4 kb. Large scale transformation was conducted using ligation reactions from each of the fractions, and clones were picked in a mixed fashion using an automated robotic clone picker. A total of 42,240 cDNA clones were arrayed in 112 micro-plates of 384-wells each. An additional copy of the cDNA library was generated by manual duplication.

          The average cDNA insert size and their distribution were analyzed by random sampling of cDNA clones from randomly selected plates. A total of 320 cDNA clones were double-digested by HindIII/EcoRI. The average cDNA insert size was 1.9 kb. Approximately, 96% of the clones had inserts longer than 500 bp, 54% of the cDNA clones had inserts longer than 1.5 kb, and 15% of the clones contained inserts longer than 3 kb. Only 3% of the clones had inserts smaller than 500 bp (Fig. 1). Therefore, the size fractionation procedure used in this library construction was effective for obtaining cDNA inserts of different lengths.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-145/MediaObjects/12864_2006_Article_858_Fig1_HTML.jpg
          Figure 1

          The insert size distribution of urediniospore cDNA clones ofPuccinia striiformisf. sp.tritici. The insert sizes of 320 randomly picked cDNA clones were determined by HindIII/EcoRI double digestion.

          cDNA sequence analysis

          A total of 198 cDNA clones were sequenced with a single pass reading from both ends of the cloning sites. Sequence reads of 800 - 1,000 bp were achieved for most of the clones. For each sampled cDNA clone, two sequence reads from both ends were aligned and were comparatively edited to generate a consensus sequence contig. Of the 196 clones, we obtained a completed cDNA sequence for 149 clones. The remaining 47 cDNA clones had two partial sequences because they had insert sizes that exceeded the single pass sequencing capability. The 243 single sequences were deposited in the EST sequence database of the GenBank (Accession numbers EG374272 - EG374514).

          All edited sequence contigs were searched against the NCBI fungal gene databases and the all-organism gene databases with their translated amino acid sequences. We consider that if a cDNA clone of P. striiformis f. sp. trtici and a gene in the fungal database share homology significant at an e-value of <1.00E-5, they likely belong to the same gene family and should share a similar broad sense function. A total of 73 cDNA clones (36.9%) met this requirement, and therefore, were considered with functions identified, of which 50 clones had completed sequences, 13 clones had partial sequences that hit the same or similar genes, and 10 clones had one partial sequence hitting a characterized gene (Table 1). These genes represented 51 different protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism and virulence/infection. Examples of these genes are glycine hydroxymethyltransferase, saccharopine dehydropine, mitogen-activated protein kinase (MAPK), serine/threonine kinase, β-tubulin, deacetylase, mitochondrial ATPase alpha-subunit, fatty acid oxidoreductase, phosphatidyl synthase, endopeptidase, elongation factor, ribosomal RNA unit, glucose-repressible protein, transaldolase, TATA-box binding protein, cell wall glucanase and pectin lyase. Thirty-seven clones (18.9%) had certain levels of homology to genes in other fungi, but the significance levels were not adequate for considering the functions identified (Table 2). Sequences of 36 clones (18.4%) were homologous to fungal genes with functions unclassified and the most of them were hypothetical proteins. Although many of the hypothetical protein genes had e-value < 1.00E-05, they are listed in Table 2 because of their unclear functions. Some of the hypothetical protein genes were homologous to genes in other plant pathogens, such as Ustilago maydis, Gibberella zeae and Magnapothe grisea. These genes could be related to plant infection. Many of the cDNA clones had homology of various levels to genes from plants (12%), other eukaryotes (34%), or to proteins of bacterial origin (11%) (data not shown). There were 50 clones (25.5%) with full-length sequences resulting in no-hit, indicating that they had no homology to any sequence available in the current NCBI databases (Table 3). These genes could be unique to P. striiformis f. sp. tritici. Alternatively, similar genes in other fungi have not been identified or desposited into the databases.
          Table 1

          Putative genes idenitified in cDNA clones of Puccinia striiformis f. sp. tritici based on their sequence comparison with other fungal genes through Blastx search of the NCBI databases

          Category & clone no.

          GenBank accession

          Size (bp)

          Full length or partiala

          Best hit in the NCBI fungal databases

              

          Protein

          Accession

          Organism

          e-value

          1. Amino acid metabolism

          65N4

          EG374380

          2044

          F

          Glycine hydroxymethyltransferase

          gb|AAW45780.1

          Cryptococcus neoformans

          1.00E-156

          60J18a

          EG374421

          1142

          P

          Potential kynurenine 3-monooxygenase

          gb|EAK98864.1

          Candida albicans

          2.00E-06

          60J18b

          EG374422

          1220

          P

          Potential kynurenine 3-monooxygenase

          gb|EAK98864.1

          Candida albicans

          1.00E-12

          58D15a

          EG374299

          897

          P

          Saccharopine dehydrogenase

          gi|70993695

          Aspergillus fumigatus

          2.00E-55

          58D15b

          EG374300

          780

          P

          Spermidine synthase

          emb|CAD71251.1

          Neurospora crassa

          3.00E-78

          2. Cell Defense

          35A16

          EG374447

          1351

          F

          Related to stress response protein

          emb|CAD21425.1

          Neurospora crassa

          2.00E-23

          3. Cell division/cycle

          80F12

          EG374389

          1560

          F

          Cell division control protein

          gb|AAB69764.1

          Candida albicans

          2.00E-28

          65O23

          EG374383

          2037

          F

          Cyclin c homolog 1

          ref|NP_596149.1

          Schizosaccharomyces pombe

          3.00E-07

          4. Cell signaling/cell communication

          40D3

          EG374466

          1534

          F

          Autophagy-related protein

          gb|AAW43831.1

          Cryptococcus neoformans

          6.00E-45

          70C17a

          EG374441

          1206

          P

          Fasciclin I family protein

          gi|44890027

          Aspergillus fumigatus

          3.00E-06

          58J15b

          EG374311

          807

          P

          GTPase activating protein

          gb|AAW43777.1

          Cryptococcus neoformans

          2.00E-09

          55B10a

          EG374277

          861

          P

          MAP kinase 1

          gb|AAO61669.1

          Cryptococcus neoformans

          3.00E-19

          55B10b

          EG374278

          932

          P

          MAP kinase

          gb|AAU11317.1

          Alternaria brassicicola

          7.00E-74

          65M20

          EG374379

          1098

          F

          Nucleoside-diphosphate kinase

          emb|CAD37041.1

          Neurospora crassa

          9.00E-53

          70E5

          EG374404

          1766

          F

          Serine/threonine kinase

          gi|58262703

          Cryptococcus neoformans

          3.00E-61

          10D13a

          EG374414

          1122

          P

          Serine palmitoyl transferase subunit

          gb|AAP47107.1

          Aspergillus nidulans

          4.00E-27

          10D13b

          EG374416

          1170

          P

          Serine palmitoyl transferase subunit

          gb|AAP47107.1

          Aspergillus nidulans

          2.00E-18

          30G12

          EG374337

          1131

          F

          Signal peptidase 18 KD subunit

          emb|CAE76335.1

          Neurospora crassa

          3.00E-10

          5. Cell Structure and growth

          58H22a

          EG374306

          920

          P

          Beta-tubulin

          emb|CAC83953.1

          Uromyces viciae-fabae

          3.00E-72

          58H22b

          EG374307

          859

          P

          Beta-tubulin

          emb|CAC83953.1

          Uromyces viciae-fabae

          5.00E-68

          10I12

          EG374325

          1105

          F

          Conidiation protein 6

          emb|CAD70456.1

          Neurospora crassa

          2.00E-10

          30J9

          EG374343

          1302

          F

          Deacetylase

          emb|CAD10036.1

          Cryptococcus neoformans

          2.00E-43

          60C15

          EG374348

          1456

          F

          Deacetylase

          gb|AAW47023.1

          Cryptococcus neoformans

          6.00E-35

          65D17

          EG374372

          1449

          F

          Deacetylase

          emb|CAD10036.1

          Cryptococcus neoformans

          4.00E-36

          40F18

          EG374469

          1117

          F

          Deacetylase

          emb|CAD10036.1

          Cryptococcus neoformans

          2.00E-31

          55D17

          EG374475

          1619

          F

          Deacetylase

          emb|CAD10036.1

          Cryptococcus neoformans

          5.00E-18

          35C19b

          EG374494

          836

          P

          Deacetylase

          emb|CAD10036.1

          Cryptococcus neoformans

          6.00E-18

          10C3

          EG374321

          1479

          F

          Deacetylase

          gb|AAW47023.1

          Cryptococcus neoformans

          6.00E-26

          35N24

          EG374461

          783

          F

          Hydrophobin

          emb|CAD42710.1

          Davidiella tassiana

          5.00E-34

          32H21a

          EG374436

          1176

          P

          Intraorganellar peroxisomal translocation component Pay32p (PAY32) gene

          gi|5821763

          Yarrowia lipolytica

          4.00E-32

          40B22

          EG374465

          1708

          F

          Nuclear filament-containing protein

          emb|CAA93293.1|

          Schizosaccharomyces pombe

          5.00E-16

          35G11a

          EG374497

          819

          P

          Pria_lened pria protein

          emb|CAA43289.1

          Lentinula edodes

          2.00E-12

          65M2

          EG374413

          2097

          F

          UDP-glucose dehydrogenase

          gb|AAS20528.1

          Cryptococcus neoformans

          1.00E-145

          6. Energy/TCA cycle

          35D23b

          EG374496

          629

          P

          64 kDa mitochondrial NADH dehydrogenase

          gb|AAW44492.1

          Cryptococcus neoformans

          1.00E-07

          40H12

          EG374471

          1249

          F

          Iron-sulfur cluster Isu1-like protein

          gb|AAQ98966.1

          Cryptococcus neoformans

          8.00E-56

          55E23a

          EG374279

          957

          P

          Mitochondrial ATPase alpha-subunit

          gb|AAA33560.1

          Neurospora crassa

          6.00E-78

          55E23b

          EG374280

          870

          P

          Mitochondrial ATPase alpha-subunit

          gb|AAA33560.1

          Neurospora crassa

          1.00E-101

          90M15

          EG374409

          1570

          F

          Mitochondrial carrier family protein

          gb|EAK95613.1

          Candida albicans

          1.00E-46

          30N15a

          EG374419

          1078

          P

          Succinate dehydrogenase flavoprotein subunit precursor

          gb|AAW45324.1

          Cryptococcus neoformans

          1.00E-63

          30N15b

          EG374420

          1143

          P

          Succinate dehydrogenase flavoprotein subunit precursor

          gb|AAW45324.1

          Cryptococcus neoformans

          1.00E-136

          10A2

          EG374481

          1114

          F

          V-type ATPase subunit G

          gb|AAB41886.1|

          Neurospora crassa

          6.00E-15

          7. Lipid metabolism

          65D3

          EG374370

          1809

          F

          Diacylglycerol O-acyltransferase

          gi|58268157

          Cryptococcus neoformans

          1.00E-84

          65G21a

          EG374424

          1078

          P

          Fatty acid oxidoreductase

          gb|AAW46114.1

          Cryptococcus neoformans

          2.00E-05

          65G21b

          EG374425

          1149

          P

          Fatty acid oxidoreductase

          gb|AAW46114.1

          Cryptococcus neoformans

          3.00E-32

          58J11b

          EG374309

          732

          P

          Phosphatidyl synthase

          gi|70999337

          Aspergillus fumigatus

          2.00E-20

          8. Nucleotide metabolism

          58C19a

          EG374297

          827

          P

          Uracil DNA N-glycosylase

          gb|AAW41098.1

          Cryptococcus neoformans

          7.00E-16

          58C19b

          EG374298

          857

          P

          Uracil DNA N-glycosylase

          gb|AAW41098.1

          Cryptococcus neoformans

          1.00E-19

          9. Protein modification

          65B1

          EG374366

          1847

          F

          Carboxypeptidase

          gi|19115337

          Schizosaccharomyces pombe

          7.00E-06

          66B11a

          EG374437

          1145

          P

          Endopeptidase

          gb|AAW41068.1

          Cryptococcus neoformans

          2.00E-69

          66B11b

          EG374438

          1200

          P

          Endopeptidase

          gb|AAW41068.1

          Cryptococcus neoformans

          1.00E-48

          80N15

          EG374397

          1944

          F

          Translation elongation factor eEF-1 alpha chain

          pir||S57200

          Puccinia graminis

          0.00E+00

          10. Protein translational modification

          55N13

          EG374483

          833

          F

          Ubiquitin-conjugating enzyme

          ref|NP_594859.1

          Schizosaccharomyces pombe

          7.00E-21

          11. Ribosomal protein complex

          55B4

          EG374472

          770

          F

          16S small subunit ribosomal RNA

          gi|52699765

          Xanthoria elegans

          2.00E-08

          35O22

          EG374462

          938

          F

          18S ribosomal RNA

          gi|21702995

          Gymnosporangium libocedri

          1.00E-154

          60E22

          EG374352

          1117

          F

          18S ribosomal RNA

          gi|34493860

          Puccinia graminis f. sp.tritici

          3.00E-142

          65C12

          EG374368

          1136

          F

          18S ribosomal RNA

          gi|34493860

          Puccinia graminis f. sp.tritici

          2.00E-66

          90D5a

          EG374432

          1119

          P

          18S ribosomal RNA

          gi|21724233

          Puccinia striiformis f. sp.tritici

          6.00E-102

          90D5b

          EG374431

          1147

          P

          ITS1, ITS2 and 5.8S ribosomal RNA

          gi|3668067

          Tricholoma matsutake

          9.00E-54

          58E11b

          EG374302

          831

          P

          25S ribosomal RNA

          gi|169606

          Puccinia graminis f. sp. dactylis

          1.00E-09

          23H10b

          EG374283

          1921

          F

          28S ribosomal RNA

          gi|37703614

          Puccinia allii

          1.00E-83

          35M12a

          EG374458

          763

          F

          28S ribosomal RNA

          gi|21724230

          Puccinia graminis f. sp. tritici

          2.00E-14

          35N2

          EG374460

          917

          F

          28S ribosomal RNA

          gi|46810582

          Fuscoporia viticola

          4.00E-06

          35P13

          EG374463

          888

          F

          28S ribosomal RNA

          gi|86160913

          Melampsora epitea

          2.00E-16

          40A4

          EG374464

          951

          F

          28S ribosomal RNA

          gi|58532805

          Puccinia carthami

          4.00E-05

          55J11

          EG374479

          957

          F

          28S ribosomal RNA

          gi|21724233

          Puccinia striiformis f. sp. tritici

          2.00E-26

          35I10b

          EG374502

          422

          P

          28S ribosomal RNA

          gi|21914221

          Puccinia graminis

          5.00E-77

          35I22a

          EG374505

          716

          P

          28S ribosomal RNA

          gi|21914221

          Puccinia graminis

          2.00E-70

          35I22b

          EG374504

          878

          P

          ITS1, ITS2 and 5.8S ribosomal RNA

          gi|21724233

          Puccinia striiformis f. sp.tritici

          5.00E-134

          10G18

          EG374323

          1108

          F

          28S ribosomal RNA

          gi|84452427

          Cladosporium cladosporioides

          1.00E-59

          30C19

          EG374333

          1117

          F

          28S ribosomal RNA

          gi|62005831

          Puccinia ferruginosa

          2.00E-13

          30H3

          EG374340

          1052

          F

          28S ribosomal RNA

          gi|21724233

          Puccinia striiformis f. sp. tritici

          3.00E-71

          30I12

          EG374341

          1067

          F

          28S ribosomal RNA

          gi|21724233

          Puccinia striiformis f. sp. tritici

          2.00E-39

          30M20

          EG374347

          1008

          F

          28S ribosomal RNA

          gi|21914221

          Puccinia graminis

          1.00E-93

          60J23

          EG374357

          2112

          F

          calnexin

          gb|AAS68033.1

          Aspergillus fumigatus

          1.00E-133

          12. Sugar/glycolysis metabolism

          30I15b

          EG374418

          617

          P

          Glucose-repressible protein

          emb|CAC28672.1

          Neurospora crassa

          2.00E-14

          90C20

          EG374401

          1130

          F

          Glucose-repressible protein

          gi|70996962

          Aspergillus fumigatus

          7.00E-18

          55J22b

          EG374287

          887

          P

          Glyoxal oxidase precursor

          gb|AAW44259.1

          Cryptococcus neoformans

          2.00E-90

          55J22a

          EG374286

          764

          P

          Glyoxal oxidase precursor

          gb|AAW41343.1

          Cryptococcus neoformans

          3.00E-30

          90H16

          EG374405

          1753

          F

          Phosphopyruvate hydratase

          gi|1086120

          Cladosporium herbarum

          1.00E-139

          30K8

          EG374344

          1547

          F

          Transaldolase

          gb|AAW46393.1

          Cryptococcus neoformans

          3.00E-95

          13. Transcription factor

          58E6

          EG374485

          1310

          F

          TATA-box binding protein

          gb|AAB57876.1

          Emericella nidulans

          7.00E-63

          14. Transport metabolism

          65M6

          EG374378

          1119

          F

          Cation transport-related protein

          gb|AAW42114.1

          Cryptococcus neoformans

          3.00E-13

          15. virulence/infection related protein

          70I2

          EG374433

          1952

          F

          Cell wall glucanase

          gi|70998053

          Aspergillus fumigatus

          2.00E-25

          30M9

          EG374345

          1162

          F

          Differentiation-related/infection protein

          gb|AAD38996.1

          Uromyces appendiculatus

          7.00E-11

          80C7

          EG374385

          1180

          F

          Differentiation-related/infection protein

          gb|AAD38996.1

          Uromyces appendiculatus

          1.00E-10

          60E18

          EG374351

          2147

          F

          Pectin lyase

          gb|AAA21817.1

          Glomerella cingulata

          2.00E-06

          a F = full-length sequence and P = partial sequence.

          Table 2

          cDNA clones showing homology to genes with characterized or unclassified proteins through Blastx search of the NCBI fungal databases

          Category & clone no.

          GenBank accession

          Size (bp)

          Full length or partiala

          Best hit in the NCBI databases

              

          Protein

          Accession

          Organism

          e-value

          1. Amino acid metabolism

          35I14

          EG374455

          766

          F

          Cystathionine beta-lyase

          gi|6636350

          Botryotinia fuckeliana

          5.70E+00

          2. Cell Defense

          66C24a

          EG374440

          1175

          P

          88 kDa immunoreactive mannoprotein MP88

          gb|AAL87197.1

          Cryptococcus neoformans

          1.00E-03

          3. Cell Division/cycle

          10F19

          EG374412

          1877

          F

          g1/s-specific cyclin pcl1 (cyclin hcs26)

          gb|AAW44590.1

          Cryptococcus neoformans

          2.00E-04

          4. Cell signaling/cell communication

          65G15

          EG374514

          1106

          P

          Protein kinase

          gi|15072451

          Cryphonectria parasitica

          1.20E+00

          30E21

          EG374336

          1128

          F

          Serine/threonine kinase

          gi|22531808

          Ustilago maydis

          3.90E-01

          65C6

          EG374367

          1649

          F

          Serine/threonine phosphatase

          gi|33087517

          Hypocrea jecorina

          3.90E-01

          80G5b

          EG374428

          1230

          P

          Mitogen-activated protein kinase

          gi|57227328

          Cryptococcus neoformans

          1.70E-00

          5. Cell Structure and growth

          58G9

          EG374486

          1714

          F

          Beta tubulin

          gi|47834278

          Penicillium flavigenum

          6.40E-00

          40G6b

          EG374274

          888

          P

          Cell wall protein

          gi|68471254

          Candida albicans

          4.60E-01

          58C4b

          EG374296

          819

          P

          Cell surface protein

          gi|70983232

          Aspergillus fumigatus

          2.60E-02

          10D19

          EG374322

          1212

          F

          Cell wall mannoprotein

          ref|NP_012685.1

          Saccharomyces cerevisiae

          1.00E-03

          90I19

          EG374406

          1240

          F

          Cell wall mannoprotein

          gi|6322611

          Saccharomyces cerevisiae

          1.50E-02

          90C22

          EG374402

          1641

          F

          Cytoplasm protein

          gb|AAW42379.1

          Cryptococcus neoformans

          1.00E-04

          10I15

          EG374326

          1088

          F

          Mitochondrial outer membrane beta-barrel protein

          gi|45758780

          Neurospora crassa

          1.70E-01

          60H1

          EG374354

          1035

          F

          Nuclear pore complex subunit

          gi|46437749

          Candida albicans

          5.00E-00

          70I19a

          EG374443

          1132

          P

          Nucleoskeletal-like protein

          gi|172053

          Saccharomyces cerevisiae

          1.30E-01

          6. Differentiation- related protein

          70A18

          EG374371

          1207

          F

          Differentiation-related protein

          gb|AAD38996.1

          Uromyces appendiculatus

          6.00E-03

          7. Mating type

          30M10

          EG374346

          1025

          F

          Mating type alpha locus

          gi|73914085

          Cryptococcus gattii

          6.80E+00

          30C22

          EG374334

          1110

          F

          Mating type alpha locus

          gi|73914085

          Cryptococcus gattii

          7.50E+00

          8. Nucleotide metabolism

          35K8

          EG374456

          1572

          F

          Ribonuclease H2 subunit

          gi|6320485

          Saccharomyces cerevisiae

          9.00E+00

          9. Protein translational modification

          100C10

          EG374490

          1179

          F

          Non-ribosomal peptide synthetase

          gi|62006079

          Hypocrea virens

          1.20E+00

          10. Ribosomal protein complex

          35L17

          EG374457

          585

          F

          18S ribosomal RNA

          gi|51102377

          Microbotryum dianthorum

          4.20E-02

          40C19a

          EG374512

          706

          P

          18S ribosomal RNA

          gi|28412377

          Leotiomycete sp.

          5.40E-01

          35H2b

          EG374500

          786

          P

          26S large subunit ribosomal RNA

          gi|30313824

          Pichia guilliermondii AjvM13

          1.00E-03

          35E4

          EG374451

          897

          F

          28S ribosomal RNA

          gi|46810582

          Fuscoporia viticola

          5.00E-03

          35P11a

          EG374506

          667

          P

          28S ribosomal RNA

          gi|62005826

          Puccinia artemisiae-keiskeanae

          1.00E-04

          55B15

          EG374473

          954

          F

          28S ribosomal RNA

          gi|84794517

          Puccinia striiformoides

          3.60E-01

          58B3

          EG374484

          884

          F

          28S ribosomal RNA

          gi|46810582

          Fuscoporia viticola

          3.30E-01

          58N22

          EG374488

          996

          F

          28S ribosomal RNA

          gi|20452324

          Rhodotorula pilati

          3.30E-01

          66I12

          EG374338

          1167

          F

          28S ribosomal RNA

          gi|46810582

          Fuscoporia viticola

          3.00E-04

          80G5a

          EG374427

          1106

          P

          Calnexin

          gi|45551624

          Aspergillus fumigatus

          2.30E-00

          11. Sugar/glycolysis metabolism

          58G18b

          EG374304

          796

          P

          Pyruvate decarboxylase

          gi|68480982

          Candida albicans

          1.40E+00

          10N6

          EG374330

          1029

          F

          Pyruvate kinase

          gi|168073

          Aspergillus nidulans

          6.00E+00

          12. Transport metabolism

          30G15

          EG374339

          1087

          F

          Membrane zinc transporter

          gi|47156070

          Aspergillus fumigatus

          5.70E-01

          40H8a

          EG374275

          656

          P

          amino acid transporter

          gi|70985369

          Aspergillus fumigatus

          3.10E+00

          80K19

          EG374395

          1728

          F

          Na+-ATPase

          gi|1777377

          Zygosaccharomyces rouxii

          2.00E-04

          55L18b

          EG374289

          845

          P

          Peptide transporter

          gi|70982509

          Aspergillus fumigatus

          5.30E-01

          13. Unclassified

          80G10

          EG374391

          1132

          F

          Genomic sequence

          gi|48056381

          Phakopsora pachyrhizi

          7.00E-53

          04F9

          EG374470

          1127

          F

          Hypothetical protein

          gi|71006713

          Ustilago maydis

          1.00E-06

          10N10

          EG374331

          1106

          F

          Hypothetical protein

          gi|58258450

          Cryptococcus neoformans

          6.00E-22

          30I21

          EG374342

          1906

          F

          Hypothetical protein

          gi|71023234

          Ustilago maydis

          1.00E-21

          35B6

          EG374449

          1060

          F

          Hypothetical protein

          gb|EAA67250.1

          Gibberella zeae

          1.00E-03

          35C10

          EG374450

          1465

          F

          Hypothetical protein

          gi|71004383

          Ustilago maydis 521

          2.00E-08

          35G21

          EG374454

          1332

          F

          Hypothetical protein

          gb|EAK81105.1

          Ustilago maydis

          5.00E-09

          35H2a

          EG374499

          758

          P

          Hypothetical protein

          gi|71021872

          Ustilago maydis

          1.80E+00

          40B2a

          EG374508

          603

          P

          Hypothetical protein

          gi|85114517

          Neurospora crassa

          3.00E-05

          40C12a

          EG374510

          792

          P

          Hypothetical protein

          gi|71019552

          Ustilago maydis

          4.00E-01

          55L8

          EG374491

          1417

          F

          Hypothetical protein

          gi|71004813

          Ustilago maydis

          1.50E-01

          58C4a

          EG374296

          764

          P

          Hypothetical protein

          MGG_09875.5b

          Magnaporthe grisea

          6.00E-12

          60D4

          EG374350

          1123

          F

          Hypothetical protein

          gi|50259357

          Cryptococcus neoformans

          7.00E-04

          60I14

          EG374356

          1565

          F

          Hypothetical protein

          gi|58263159

          Cryptococcus neoformans

          2.00E-09

          60L15

          EG374359

          2073

          F

          Hypothetical protein

          gb|EAA47832.1

          Magnaporthe grisea

          7.00E-10

          60N2

          EG374363

          1109

          F

          Hypothetical protein

          gi|46096746

          Ustilago maydis

          7.00E-03

          60N6

          EG374364

          1071

          F

          Hypothetical protein

          gi|49642978

          Kluyveromyces lactis

          8.00E-17

          65H5

          EG374374

          1390

          F

          Hypothetical protein

          gi|85095053

          Neurospora crassa

          1.40E+00

          65I3

          EG374375

          1870

          F

          Hypothetical protein

          gb|EAK86140.1

          Ustilago maydis

          1.00E-129

          65O15

          EG374381

          1893

          F

          Hypothetical protein

          gi|71006255

          Ustilago maydis

          1.10E+00

          66B6

          EG374316

          1263

          F

          Hypothetical protein

          gb|EAK81690.1

          Ustilago maydis

          1.00E-03

          66B11a

          EG374437

          1145

          P

          Hypothetical protein

          AN2903.3b

          Aspergillus nidulans

          3.00E-57

          66B11b

          EG374438

          1200

          P

          Hypothetical protein

          FG10782.1b

          Fusarium graminearum

          5.00E-49

          66C18

          EG374327

          2043

          F

          Hypothetical protein

          gb|EAA59593.1

          Aspergillus nidulans

          2.00E-12

          70A3

          EG374360

          1835

          F

          Hypothetical protein

          SS1G_14513.1b

          Sclerotinia sclerotiorum

          8.00E-18

          70C17b

          EG374442

          1191

          P

          Hypothetical protein

          AN0768.3b

          Aspergillus nidulans

          1.00E-07

          70H16

          EG374426

          1121

          F

          Hypothetical protein

          gi|38100779

          Magnaporthe grisea

          2.60E+00

          70I19b

          EG374443

          1190

          P

          Hypothetical protein

          NCU02808.2b

          Neurospora crassa

          2.00E-08

          70K15b

          EG374320

          933

          P

          Hypothetical protein

          gi|58261561

          Cryptococcus neoformans

          1.00E-07

          70L24b

          EG374446

          1168

          P

          Hypothetical protein

          gb|EAA28928.1

          Neurospora crassa

          3.00E-23

          80I9

          EG374394

          1060

          F

          Hypothetical protein

          gi|58259618

          Cryptococcus neoformans

          1.50E+00

          90O3

          EG374410

          1725

          F

          Hypothetical protein

          gi|85119288

          Neurospora crassa

          1.20E-02

          90O18

          EG374411

          1973

          F

          Hypothetical protein

          CHG04543.1b

          Chaetomium globosum

          4.00E-07

          66C24b

          EG374440

          1271

          P

          Macrofage activating glycoprotein

          gi|15722495

          Cryptococcus neoformans

          3.00E-08

          30E3

          EG374335

          1406

          F

          Probable gEgh 16 protein

          emb|CAE85538.1

          Neurospora crassa

          8.00E-07

          60I8

          EG374355

          1039

          F

          Related to ars binding protein 2

          gi|18376044

          Neurospora crassa

          6.60E+00

          55J15b

          EG374285

          896

          P

          Telomeric sequence DNA

          gi|173051

          Saccharomyces cerevisiae

          2.00E-05

          55E7

          EG374477

          1253

          F

          Unknown protein in chromosome E

          gi|49654999

          Debaryomyces hansenii

          3.00E-06

          55F15a

          EG374281

          461

          P

          Unknown protein in chromosome G

          gi|50427978

          Debaryomyces hansenii

          2.00E-03

          60L20

          EG374361

          1646

          F

          Unknown protein in chromosome VI

          gi|39975020

          Magnaporthe grisea

          3.00E-18

          60N1

          EG374362

          2024

          F

          Unknown protein in chromosome 1

          gi|46110618

          Gibberella zeae

          2.00E-09

          70F20

          EG374415

          1818

          F

          Unknown protein in chromosome III

          gi|58270250

          Magnaporthe grisea

          1.60E+00

          80M4

          EG374396

          1985

          F

          Unknown protein in chromosome G

          gi|49657202

          Debaryomyces hansenii

          1.00E-03

          80N10

          EG374430

          563

          P

          Phytochrome

          gi|57337632

          Emericella nidulans

          4.30E-00

          90B8

          EG374400

          2011

          F

          Unknown protein in chromosome G

          gi|49657202

          Debaryomyces hansenii

          4.90E-02

          90L21

          EG374408

          2002

          F

          Unknown protein in chromosome A

          gi|49524079

          Candida glabrata

          1.20E+00

          a F = full-length sequence and P = partial sequence.

          b Data generated from Blastx search of the fungal database of the Broad Institute [34].

          Table 3

          cDNA clones that produced no hit in the Blastx search of the NCBI fungal databases

          Category & Clone no.

          GenBank accession

          Size (bp)

          Full length or partiala

          Category & clone no.

          GenBank accession

          Size (bp)

          Full length or partiala

          04A1

          EG374448

          1188

          F

          55N9

          EG374482

          1171

          F

          04C13

          EG374459

          1423

          F

          55B9a

          EG374292

          585

          P

          04P11

          EG374434

          1133

          F

          55B9b

          EG374293

          930

          P

          100B17

          EG374489

          1137

          F

          58E11a

          EG374301

          542

          P

          10B5

          EG374492

          1161

          F

          58G18a

          EG374303

          791

          P

          10C11

          EG374503

          1235

          F

          58J11a

          EG374308

          672

          P

          10I7

          EG374324

          1112

          F

          58J15a

          EG374310

          921

          P

          10K3

          EG374328

          1687

          F

          58L3

          EG374487

          959

          F

          10L3

          EG374272

          1099

          F

          58M15a

          EG374314

          719

          P

          10N5

          EG374329

          1090

          F

          58M15b

          EG374315

          718

          P

          10O19

          EG374332

          1359

          F

          58M7a

          EG374312

          788

          P

          30I15a

          EG374417

          1032

          P

          58M7b

          EG374313

          934

          P

          32B15

          EG374294

          1296

          F

          58N10a

          EG374317

          287

          P

          32H21b

          EG374436

          1249

          P

          58N10b

          EG374318

          837

          P

          35C19a

          EG374493

          739

          P

          60F10

          EG374353

          1131

          F

          35D23a

          EG374495

          775

          P

          60L12

          EG374358

          1239

          F

          35F14

          EG374453

          971

          F

          60O23

          EG374365

          1084

          F

          35F7

          EG374452

          1086

          F

          65C23

          EG374369

          2047

          F

          35G11b

          EG374498

          757

          P

          65G1

          EG374373

          1631

          F

          35I10a

          EG374501

          807

          P

          65G15b

          EG374514

          1158

          P

          35P11b

          EG374507

          682

          P

          65I10

          EG374376

          1010

          F

          40B2b

          EG374509

          860

          P

          65K18

          EG374377

          1230

          F

          40C12b

          EG374511

          921

          P

          65P1

          EG374384

          1814

          F

          40C19b

          EG374513

          857

          P

          66M21

          EG374349

          1437

          F

          40E10

          EG374467

          713

          F

          70C4

          EG374382

          1518

          F

          40E23

          EG374468

          734

          F

          70D12

          EG374393

          1285

          F

          40G6a

          EG374273

          779

          P

          70K15a

          EG374319

          722

          P

          40H8b

          EG374276

          811

          P

          70L24a

          EG374445

          1104

          P

          50M2

          EG374305

          1182

          F

          80D10

          EG374386

          1147

          F

          55C20

          EG374474

          868

          F

          80E22

          EG374388

          2064

          F

          55E2

          EG374476

          1272

          F

          80E4

          EG374387

          1173

          F

          55F12

          EG374478

          935

          F

          80F15

          EG374390

          2129

          F

          55F15b

          EG374282

          865

          P

          80G19

          EG374392

          1124

          F

          55J15a

          EG374284

          660

          P

          80N10a

          EG374429

          1091

          P

          55L18a

          EG374288

          930

          P

          80O12

          EG374398

          1517

          F

          55M5

          EG374480

          942

          F

          80O24

          EG374399

          2098

          F

          55N22a

          EG374290

          813

          P

          90H10

          EG374403

          1748

          F

          55N22a

          EG374291

          282

          P

          90K17

          EG374407

          1896

          F

          a F = full-length sequence and P = partial sequence.

          Identification of open reading frames

          Various lengths of open reading frames (ORFs) were identified from 167 cDNA clones using the Lasergene sequence analysis software (DNASTAR package, WI. USA). The quality of the cDNA libraries with respect to the full-length (intactness) of cDNA was evaluated using three parameters: 1) identification of the 5'-end sequence structures of the insert, 2) ATG start site at their 5'-end for complete ORF contents and 3) Blastx evaluation of pre-determined ORF with corresponding amino acid sequences in the GenBank. Multiple ORFs with different length were frequently identified in a given cDNA sequence. When methionine was found aligned (including gaps) with first amino acid of a completed sequence (within the longest ORFs) with the first ATG start codon at the 5' end, a cDNA sequence was determined as a full-length transcript. Most of the cDNA sequences retained the specific 5'-end priming sequences (5'-CGGCCGGG-3'). A total of 128 complete ORFs were identified with first translation initiation codon ATG. The longest ORF was 951 bp, and the shortest ORF was 93 bp. The longest ORF sequence was selected from each analyzed cDNA and validated with the corresponding amino acid sequences to determine the genuine ORF. Four cDNA sequences were identified which contain incomplete ORF sequences, indicating incomplete transcripts for those cDNA clones. Nearly 86% of the cDNA sequences were found containing completed ORFs with a translation initiation codon (ATG). Each of the validated ORFs was able to translate into a continuous protein sequence with a translation initiation codon. This finding indicated high percentage of cDNA clones containing full-length transcripts with various sizes of ORFs in the cDNA library.

          Discussion

          A cDNA library can provide molecular resources for analysis of genes involved in the biology of a plant pathogenic fungus, such as genes responsible for the development, survival, pathogeniCity and virulence. In order to initiate studies on the basic genome structure and gene expression of P. striiformis with infective State, we constructed a full-length cDNA library and a BAC library from urediniospores of a predominant race of P. striiformis f. sp. tritici [10]. The full-length cDNA library can be used to study the normal transcription profiles for the uredinial State, the biologically and epidemiologically essential stage of the fungus. The current cDNA library will serve as a major genetic resource for identifying and isolating full-length genes and functional units from the P. striiformis genome. Because this cDNA library was constructed from urediniospores of the pathogen, it should include expressed genes unique to this spore stage. Therefore, the cDNA library should have avoided EST limitations that are commonly generated by automatic assemblies of transcripts from different tissues. Controlled greenhouse conditions and careful handling of the plants and spores minimized possibility of contaminations by other fungal spores. Powdery mildew or leaf rust, which sometimes contaminates stripe rust spores, were not observed on the stripe rust - sporulating plants. Therefore, genes or cDNA sequences identified in this study should be from urediniospores of P. striiformis f. sp. tritici. This also was confirmed in a separate study, in which primers of all 12 randomly picked cDNA clones were successfully amplified clones in the BAC library constructed with the same race of the pathogen (data not shown).

          A urediniospore of P. striiformis is an infectious structure that is critical for the rust to initiate the infection process. Although the fungus produces other spores, teliospores and basidiospores, they do not result in infection of host plants because the fungus does not have alternate hosts for basidiospores to infect. Compared to mycelium, a urediniospore is relatively more resistant to adverse environmental conditions. Therefore, the urediniospore stage should contain most of the pathogen genes involved in the pathogen development, survival and pathogeniCity. Thus, our first full-length cDNA library for P. striiformis was constructed using urediniospores. Such transcript (gene) collection should include the genes that are important for the unique physical properties and characters of the urediniospores of P. striiformis. These genes are essential to maintain their germination and infective abilities. Therefore, the current full-length cDNA library would be one of the useful genomic resources for the functional genomic study of this important agricultural pathogen. Our full-length cDNA library reported here is the first large scale transcript collection for P. striiformis. As expression of certain genes are stage-specific and genes involved in plant-pathogen interactions express in haustoria [4, 13], currently, we are working together with Scot Hulbert's lab to construct a full-length cDNA library from haustoria of the same stripe rust race used in this study.

          The technology used in this study for full-length cDNA enrichment is robust and only requires less than 1 μg of starting total RNA. By using the MMLV reverse transcriptase, only the 5'-end tagged cDNAs are not prematurely terminated and can be amplified into full-length by an RNA oligo-specific primer [35, 37]. The size fractionation process was modified in this study to generate large directional full-length cDNA inserts, which enriched full-length cDNA clones to have an insert size up to 9 kb. The enrichment of the full-length cDNA was achieved by PCR amplification following the cDNA synthesis. Because selection bias could favor the smaller cDNA, we used fewer PCR cycles to minimize such bias as previously suggested [35]. The conventionally constructed cDNA libraries rarely carry cDNA inserts over 2 kb, because the longer transcripts are often easily truncated during cDNA synthesis process, causing size bias against the larger cDNA fragments in cloning process. In our study, up to 22 PCR amplification cycles were used to generate adequate amount cDNA for cloning. The evaluation of cDNA insert size and its distribution showed a low level of insert size bias in the final cDNA library. Most of the cDNA inserts ranged from 500 bp to 1,500 bp, and there were high number of cDNA clones harboring inserts over 3,000 bp. Such results indicate that the size fraction is an effective selection approach to ensure the full-length cDNA content level in the cDNA library. The high quality of the initial total RNA and the optimal LD PCR conditions also resulted in low size bias level for the insert size distribution in this library. High quality and adequate amount of the initial mRNA is the key for yielding sufficient amount of the first strand full-length cDNA by reverse transcription. To reduce the redundancy and to avoid underrepresentation of different transcript species, cDNA fragments with different fractionated sizes were balanced and subjected to library construction. A considerable number of clones with an insert over 3 kb were found in our cDNA library, such big insert size is rarely found in conventional cDNA libraries.

          The sequences of 5'-end transcripts are important for finding the signals for initiation of transcription. Irrespective of the length of cDNA, identification of the specific 5'-end nucleotide sequences in cDNA is commonly used to determine the full-length cDNA content and quality. In many cases, the 5'-end nucleotide sequences are referred to as a 5' cap structure [3, 15, 20, 27]. We also found that nearly 95% of the cDNA clones contained the known 5'-end sequence : 5'-CGGCCGGG-3' (DB Clontech. USA), where as (G)3 at 3'-end will bind to the intact reveres transcripts which has nucleotide priming site CCC at its 5'-end. Completed ORFs were identified in cDNA sequence having the 5'-end sequence structure (5'-CGGCCGGG-3'). Presence of the ATG initiation codon aligned with amino acid methionine also was used as an indicator for the quality of full-length cDNA.

          Blastx was used to search the entire NCBI GenBank with e-value of 10-5, which revealed 37% of the cDNA clones with high homologies to genes with known functions in the database. The relative low match rate to homologous genes from the blastx search might be due to the lack of gene information in the database for fungi. During the search process, the longest ORFs in each given cDNA sequence was also evaluated with amino acid alignments. The results showed that 86% of the cDNA clones contain ORFs with the translation initiation codon and stop codon. In addition, the existence of multi-exonic structure within some ORFs is additional evidence that supports their biological reality of genes or transcripts. The Kozak rules were found not totally applicable in determining ORFs in this study. Perhaps the Kozak rules are more suitable for analysis of mammalian genomes [22].

          So far, there have been no other reports on the genome of P. striiformis in relation to function and biology of this important pathogen. In this study, we have identified genes encoding 51 different protein products involved in eleven aspects of the pathogen cell biology and plant infection. These genes are the first group of genes reported for the stripe rust pathogen. The genes identified for virulence/infection can be used in transient expression to confirm their function in pathogeniCity. Although we sequenced only a small portion of the cDNA library, the study demonstrated the high efficiency of this procedure for the identification of putative genes of known function. As more and more genes with identified functions from other organisms are deposited into the databases, genes with important functions in P. striiformis should be more efficiently identified using our cDNA library. Even though sequences of only 196 clones were characterized in this study, we identified 19 cDNA clones encoding ribosomal RNA subunits, seven clones encoding deacetylase, and two clones encoding the glucose-repressible protein. The results may indicate the mRNA abundance of these genes. In this study, 10 cDNA clones had one of the two partial sequences with high homology (e-value ranging from 3E-06 to 5E-77) to genes identified in other fungi, but another partial sequence produced no hit. The results may indicate that these genes have very long sequences, and also may reflect that similar gene sequences in other fungi are mainly short EST sequences. When blastx search was conducted using other fungal genomic databases [34], seven cDNA clones, which produced no hit when blasted with the NCBI database, were identified to have some homology with unknown functions in various fungal species. In this study, we identified 37.2% of the clones with known genes, 18.4% encoding hypothetical proteins, and 25.5% no hit. These numbers are quite different from the 11%, 23%, and 66% of these categories, respectively, found in the urediniospore EST library of P. graminis f. sp. tritici, the wheat stem rust pathogen (L. Szabo, personal communication). The differences could be due to the clone sampling sizes of the studies and the different types of libraries (the full-length cDNA library for P. striiformis f. sp. tritici and conventional EST library for P. graminis f. sp. tritici). As more genes or ESTs from other Puccinia species infecting cereal crops become available, it will be more feasible to identify genes common to this group of the rust pathogens and also identify genes unique to particular species.

          Conclusion

          A full-length cDNA library was constructed using urediniospores of the wheat stripe rust pathogen. Using the library, we identified 51 genes involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. The results of function-identified genes demonstrated that the full-length library is useful in the study of functional genomics of the important plant pathogenic fungus. Research will be conducted to identify genes involved in the development, survival and pathogeniCity of the pathogen using the cDNA library.

          Methods

          Total RNA isolation from urediniospores of P. striiformis f. sp. tritici

          Urediniospores from race PST-78 of P. striiformis f. sp. tritici, a predominant race of the wheat stripe rust [11], were harvested from infected leaves 15 days after inoculation. The inoculation method and conditions for growing plants before and after inoculation were as described by Chen and Line [7]. For total RNA extraction, approximately 30 mg urediniospores were pre-chilled with liquid nitrogen in a glass vial. Spores were ground in liquid nitrogen with mortar and pestle, and then 10 mM Tris buffer (PH 8.0) was added. Ground frozen powder was transferred to an RNase-free microcentrifuge tube. The SV Total RNA Isolation kit (Pormega. Madison, WI. USA) was used to isolate total RNA from ground urediniospores. The extraction procedure recommended by the kit manufacturer was followed with slight modifications to adapt the use of fungal material. The quantity and purity of isolated total RNA was analyzed by 1% agarose gel electrophoresis and spectrophotometer.

          Full-length cDNA synthesis and size fractionation

          First-strand cDNA was synthesized from approximately 500 ng of total RNA using the Creator SMART cDNA Library Construction kit (DB Clontech. USA) following a slightly modified manufacturer's protocol. The first-strand cDNA mixture was used as template to synthesize double-stranded DNA with long distance (LD) PCR. PCR reactions were facilitated by 20 pmol of 5' end PCR primer containing sfiI A site (5'AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3'), and 20 pmol of CDSIII/3' end polyT PCR primers containing sfiI B site [5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3']. In a 100 μL PCR reaction, 2 μL first-stranded cDNA were used as the template. The PCR reaction mixture contained 20 pmol of 10× PCR buffer, dNTP mix and 5 units of Taq polymerase. The LD PCR was performed in a GeneAmp 9600 thermal cycler (ABI Biosystem, USA) with the following program: denature at 95°C for 20 s followed by 22 cycles of 95°C for 5 s, 68°C for 6 min and 4°C soaking. The double stranded cDNA was then treated with proteinase K at 45°C for 20 min to inactivate the remaining DNA polymerase. The double stranded cDNA was then phenol-extracted and precipitated with 10 μL of 3 M sodium acetate, 1.3 μL of glycogen (20 μg/μL) and 2.5 volumes of 100% ethanol. Double stranded cDNA pellet was washed with 80% ethanol, air dried and suspended in 20 μL of water.

          Double stranded cDNA was subjected to sfiI digestion, 100 μL sfiI digestion reaction containing 79 μL of cDNA, 10 μL 10× NE buffer 2 (New England Biolabs, USA) (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol), 1 μL of 100× BSA (100 μg/ml) and 10 units of sfiI restriction enzyme (New England Biolabs, USA). Digestion was performed under 50°C for 2 h. Digested cDNA was size-fractionated on 1% agarose gel with 6 V/cm electrophoresis and the size fraction of 500 bp to 10 kb was excised. The excised gel slice was further divided into 5 zones (5 smaller gel slices) corresponding to a cDNA size ranging from 500 bp to 10 kb. Then cDNA in each gel slice was extracted and purified using the MinElute Gel Extraction kit (Qiagen, USA). The final cDNA concentration was adjusted to 5 ng/μl.

          Construction of cDNA library

          Approximately 30 ng sfiI-digested cDNA fragments were ligated to 100 ng of the pDNR-LIB cloning vector (DB Clontech, USA) using T4 DNA ligase (New England Biolabs, USA) under 16°C for 16 h. The ligation product was directly transformed into competent cell DH10B (Epicentre Technologies, USA) by electroporation. After 1 h SOC recovery incubation, transformed bacterial strain were grown on LB agar plates containing chloramphenicol (12.5 μg/ml), incubated at 37°C for 20 h. Since only the cDNA fragments with both sfiI A and sfiI B ends were allowed to be ligated into vector pDNR-LIB, only the recombinant clones were able to grow and were clearly identified as white colonies. The cDNA clones were randomly sampled and mini-prepared for a quality check using HindIII and EcoRI double-digestion to release inserts. The ligations with insert size larger than 500 bp were selected for large scale transformation. These colonies were subsequently picked and arrayed with a Q-Bot (Genetix, UK) into 384-well micro-titer plates. Each well on the culture plate contained 75 μl of LB freezing storage medium [360 mM K2HPO4, 132 mM KH2PO4, 17 mM Na citrate, 4 mM MgSO4, 68 mM (NH4)2SO4, 44% (v/v) glycerol, 12.5 μg/ml of chloramphenicol, LB]. Colonies were incubated at 37°C overnight, and then stored at -70°C.

          Full-length cDNA library evaluation and cDNA clone sequence analysis

          To evaluate the quality of the current full-length cDNA library, 400 individual cDNA clones were randomly picked from 12 storage plates, and grown in 5 ml of LB with 12.5 μg/ml of chloramphenicol under 37°C with 200 rpm shaking for 16 h. Plasmid DNA was isolated using the alkaline-lysis method [30] and digested with HindIII and EcoRI. The cDNA inserts were analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. The average cDNA insert size and the cDNA length distribution profiles were obtained.

          Two hundred cDNA clones were randomly selected for sequencing analysis. Prior to sequencing, all plasmids were isolated from cDNA bacterial clones by cellular lysis and purified in 96-well plates. Single pass sequencing was performed from both directions using two "in-house" sequencing primers. Phred software [16] was used for base calling. Each sequence was edited manually by removing vector sequences and the ambiguous reads. The overlapping sequences (from both 3' and 5' ends) were evaluated and aligned into full consensus sequence contigs using the DNA analyzing software DNA for Windows 2.2.1 [12]. The non-overlapping sequences were formatted and treated as two separated sequence contigs. All aligned sequence contigs were analyzed with the Lasergene 5.0 software (DNA STAR, Madison, WI, USA) for identifying ORFs. Consensus sequences were searched against the National Center for Biotechnology Information (NCBI) [28] fungal database and the all-organism database under E-value of 10-3 and 10-6, respectively. The genuine ORF fragments were cross validated by these two different scales of NCBI blast analysis.

          Declarations

          Acknowledgements

          This research was supported in part by the US Department of Agriculture (USDA), Agricultural Research Service (ARS), USDA-ARS Postdoctoral Program, and Washington Wheat Commission. PPNS No. 0440, Department of Plant Pathology, College of Agricultural, Human, and Natural Resource Sciences Research Center, Project No. 13C-3061-3923, Washington State University, Pullman, WA 99164-6430, USA. We thank the Sequencing Core Facility of Washington State University for the support of automated cDNA clone array, Dr. Pat Okubara for the assistance on the NCBI database blast search, Mr. Dat Q. Le for his technical assistance. We also are grateful to Dr. Lee Hadwiger and Dr. Weidong Chen for their critical review of the manuscript.

          Authors’ Affiliations

          (1)
          US Department of Agriculture, Agricultural Research Service, Wheat Genetic, Quality, Physiology and Disease Research Unit
          (2)
          Department of Plant Pathology, Washington State University
          (3)
          College of Plant Protection, Northwest A&F University
          (4)
          Department of Soil and Crop Sciences, Washington State University

          References

          1. Calhoun DS, Arhana S, Vivar HE: Chemical control of barley stripe rust, a new disease for North America. Barley Newsl 1988, 32:109–112.
          2. Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, Muramatsu M, Hayashizaki Y, Schneider C: High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 1996, 37:327–336.View ArticlePubMed
          3. Carninci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, Cono H, Okazaki Y, Muramatsu M, Hayashizaki Y: Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapad discovery of new genes. Genome Res 2000, 10:1617–1630.View ArticlePubMed
          4. Catanzariti AM, Dodds PN, Lawreance GJ, Ayliffe MA, Ellis JG: Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors. Plant Cell 2000, 18:243–256.View Article
          5. Chen XM: Epidemiology of barley stripe rust and races of Puccinia striiformis f. sp. hordei : the firstdecade in the United States. [http://​www.​crpmb.​org/​]Cereal Rusts and Powdery Mildews Bulletin 2004. 2004/1029chen.
          6. Chen XM: Epidemiology and control of stripe rust [ Puccinia striiformis f. sp. tritici ] on wheat. Can J Plant Pathol 2005, 27:314–337.View Article
          7. Chen XM, Line RF: Inheritance of stripe rust resistance inwheat cultivars used to differentiate races of Puccinia striiformis in North America. Phytopathology 1992, 82:633–637.View Article
          8. Chen XM, Line RF, Leung H: Relationship between virulence variation and DNA polymorphism in Puccinia striiformis . Phytopathology 1993, 83:1489–1497.View Article
          9. Chen XM, Line RF, Leung H: Virulence and polymorphic DNA relationships of Puccinia striiformis f. sp. hordei to other rusts. Phytopathology 1995, 85:1335–1342.View Article
          10. Chen XM, Ling P: Towards cloning wheat genes for resistance to stripe rust and functional genomics of Puccinia striiformis f. sp. tritici . Proc of the 11th Intl Cereal Rusts and Powdery Mildew Conf., Norwich, England, 22–27 August 2004. Abstracts A2.10, Cereal Rusts and Powdery Mildews Bulletin
          11. Chen XM, Moore M, Milus EA, Long DL, Line RF, Marshall D, Jackson L: Wheat stripe rust epidemics and races of Puccinia striiformis f. sp. tritici in the United States in 2000. Plant Dis 2002, 86:39–46.View Article
          12. DNA for Windows[http://​www.​dna-software.​co.​uk]
          13. Dodds PN, Lawrence GJ, Catanzariti A, Ayliffe MA, Ellis JG: The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells. Plant Cell 2004, 16:755–768.View ArticlePubMed
          14. Dubin HJ, Stubbs RW: Epidemic spread of barley stripe rust in South America. Plant Dis 1986, 70:141–144.View Article
          15. Edery I, Chu LL, Sonenberg N, Pelletier J: An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture). Mol Cell Biol 1995, 15:3363–3371.PubMed
          16. Ewing B, Green P: Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998, 8:186–194.PubMed
          17. Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 1983, 25:263–269.View ArticlePubMed
          18. Hu GG, Linning R, Kamp A, Joseph C, McCallum B, Banks T, Cloutier S, Butterfield Y, Liu J, Kirkpatrick R, Stott J, Yang G, Smailus D, Jones S, Marra M, Schein J, Pei JM, Westwood T, Bakkeren G: Generation of a wheat leaf rust, Puccinia triticina , EST database and microarray from stage-specific cDNA libraries. Proc. of the 11th Int. Cereal Rusts and Powdery Mildew Conf., Norwich, England, 22–27 August 2004. Abstracts A1.47, Cereal Rusts and Powdery Mildews Bulletin
          19. Humphrey HB, Hungerford CW, Johnson AG: Stripe rust ( Puccinia glumarum ) of cereals and grasses in the United States. J Agric Res 1924, 29:209–227.
          20. Kato S, Ohtoko K, Ohtake H, Kimura T: Vector-capping: a simple method for preparing a high-quality full-length cDNA library. DNA Res 2005, 12:53–62.View ArticlePubMed
          21. Kato S, Sekine S, Oh SW, Kim NS, Umezawa Y, Abe N, Yokoyama KM, Aoki T: Construction of a human full-length cDNA bank. Gene 1994, 150:243–250.View ArticlePubMed
          22. Kozak M: Interpreting cDNA sequences: some insights from studies on translation. Mammalian Genome 1996, 7:563–574.View ArticlePubMed
          23. Lin KC, Bushnell WR, Szabo LJ, Smith AG: Isolation and expression of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions. Mol Plant-Microbe Interact 1996, 9:511–522.View ArticlePubMed
          24. Line RF: Stripe rust of wheat and barley in North America: a retrospective historical review. Ann Rev Phytopathol 2002, 40:75–118.View Article
          25. Line RF, Qayoum A: Virulence, aggressiveness, evolution, and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968–87. U.S. Department of Agriculture Technical Bulletin 1992, 1788:44.
          26. Liu Z, Szabo LJ, Bushnell WR: Molecular cloning andanalysis of abundant and stage-specific mRNAs from Pucciniagraminis . Mol Plant Microbe Interact 1993, 6:84–91.View ArticlePubMed
          27. Maruyama K, Sugano S: Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 1994, 138:171–174.View ArticlePubMed
          28. National Center for Biotechnology Information[http://​www.​ncbi.​nlm.​nih.​gov]
          29. Roelfs AP, Huerta-Espino J, Marshall D: Barley stripe rust in Texas. Plant Dis 1992, 76:538.View Article
          30. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1989.
          31. Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T, Nakajima M, Enju A, Akiyama K, Oono Y, Muramatsu M, Hayashizaki Y, Kawai J, Carninci P, Itoh M, Ishii Y, Arakawa T, Shibata K, Shinagawa A, Shinozaki K: Functional annotation of a full-length Arabidopsis cDNA collection. Science 2002,296(5565):141–145.View ArticlePubMed
          32. Stubbs RW: Stripe rust. The Cereal Rusts: Diseases, distribution, epidemiology and control (Edited by: Roelfs AP, Bushnell WR). Academic Press, Orlando, FL 1985, II:61–101.
          33. Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S: Construction and characterization of a full length-enriched and a 5'- end-enriched cDNA library. Gene 1997, 200:149–156.View ArticlePubMed
          34. The Broad Institute[http://​www.​broad.​mit.​edu]
          35. Wellenreuther R, Schupp I, Poustka A, Wiemann S: SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics 2004, 5:36.View ArticlePubMed
          36. Wiemann S, Mehrle A, Bechtel S, Wellenreuther R, Pepperkok R, Poustka A: cDNAs for functional genomics and proteomics: the German cDNA Consortium. C.R. Biol 2003, 326:1003–1009.View ArticlePubMed
          37. Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD: Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 2001, 30:892–897.PubMed

          Copyright

          © Ling et al. 2007

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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