Open Access

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

  • Peng Ling1, 2,
  • Meinan Wang2, 3,
  • Xianming Chen1, 2Email author and
  • Kimberly Garland Campbell1, 4
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 sfi I 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 sfi I-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 sfi I 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 Hin dIII/Eco RI. 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.
https://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 of Puccinia striiformis f. sp. tritici. The insert sizes of 320 randomly picked cDNA clones were determined by Hin dIII/Eco RI 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 sfi I A site (5'AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3'), and 20 pmol of CDSIII/3' end polyT PCR primers containing sfi I 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 sfi I digestion, 100 μL sfi I 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 sfi I 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 sfi I-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 sfi I A and sfi I 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 Hin dIII and Eco RI 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 Hin dIII and Eco RI. 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.Google Scholar
  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. 10.1006/geno.1996.0567.PubMedView ArticleGoogle Scholar
  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. 10.1101/gr.145100.PubMed CentralPubMedView ArticleGoogle Scholar
  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. 10.1105/tpc.105.035980.View ArticleGoogle Scholar
  5. Chen XM: Epidemiology of barley stripe rust and races of Puccinia striiformis f. sp. hordei: the firstdecade in the United States. Cereal Rusts and Powdery Mildews Bulletin. 2004, 2004/1029chen., [http://www.crpmb.org/]Google Scholar
  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 ArticleGoogle Scholar
  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 ArticleGoogle Scholar
  8. Chen XM, Line RF, Leung H: Relationship between virulence variation and DNA polymorphism in Puccinia striiformis. Phytopathology. 1993, 83: 1489-1497. 10.1094/Phyto-83-1489.View ArticleGoogle Scholar
  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. 10.1094/Phyto-85-1335.View ArticleGoogle Scholar
  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. 2004, August . Abstracts A2.10, Cereal Rusts and Powdery Mildews BulletinGoogle Scholar
  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. 10.1094/PDIS.2002.86.1.39.View ArticleGoogle Scholar
  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. 10.1105/tpc.020040.PubMed CentralPubMedView ArticleGoogle Scholar
  14. Dubin HJ, Stubbs RW: Epidemic spread of barley stripe rust in South America. Plant Dis. 1986, 70: 141-144.View ArticleGoogle Scholar
  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 CentralPubMedView ArticleGoogle Scholar
  16. Ewing B, Green P: Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998, 8: 186-194.PubMedView ArticleGoogle Scholar
  17. Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene. 1983, 25: 263-269. 10.1016/0378-1119(83)90230-5.PubMedView ArticleGoogle Scholar
  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. 2004, August . Abstracts A1.47, Cereal Rusts and Powdery Mildews BulletinGoogle Scholar
  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.Google Scholar
  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. 10.1093/dnares/12.1.53.PubMedView ArticleGoogle Scholar
  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. 10.1016/0378-1119(94)90433-2.PubMedView ArticleGoogle Scholar
  22. Kozak M: Interpreting cDNA sequences: some insights from studies on translation. Mammalian Genome. 1996, 7: 563-574. 10.1007/s003359900171.PubMedView ArticleGoogle Scholar
  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.PubMedView ArticleGoogle Scholar
  24. Line RF: Stripe rust of wheat and barley in North America: a retrospective historical review. Ann Rev Phytopathol. 2002, 40: 75-118. 10.1146/annurev.phyto.40.020102.111645.View ArticleGoogle Scholar
  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-Google Scholar
  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.PubMedView ArticleGoogle Scholar
  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. 10.1016/0378-1119(94)90802-8.PubMedView ArticleGoogle Scholar
  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 ArticleGoogle Scholar
  30. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  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. 10.1126/science.1071006.PubMedView ArticleGoogle Scholar
  32. Stubbs RW: Stripe rust. The Cereal Rusts: Diseases, distribution, epidemiology and control. Edited by: Roelfs AP, Bushnell WR. 1985, Academic Press, Orlando, FL, II: 61-101.View ArticleGoogle Scholar
  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. 10.1016/S0378-1119(97)00411-3.PubMedView ArticleGoogle Scholar
  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: 36-10.1186/1471-2164-5-36.PubMed CentralPubMedView ArticleGoogle Scholar
  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. 10.1016/j.crvi.2003.09.036.PubMedView ArticleGoogle Scholar
  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.PubMedGoogle Scholar

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© Ling et al; licensee BioMed Central Ltd. 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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