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

Author
item Ling, Peng
item MEINAN, WANG - NW A&F U,YANGLING CHINA
item Chen, Xianming
item Garland-Campbell, Kimberly

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2007
Publication Date: 6/4/2007
Citation: Ling, P., Meinan, W., Chen, X., Garland Campbell, K.A. 2007. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici). BMC Genomics 8:145.

Interpretive Summary: The stripe rust fungus is an important pathogen that causes destructive diseases of cereal crops and grasses including wheat and barley. Despite of its economic importance, little is know about the genome and genes of the pathogen because its difficulty to be cultured on artificial media and lifecycle of the fungus limit the application of classic genetic and conventional molecular techniques in studying the fungus. To study the functional genomics and identify genes involving in the biology of the pathogen, we constructed a full-length cDNA library with the spores of the wheat stripe rust pathogen. The cDNA library consists of 42,240 clones. We sequenced 196 randomly picked cDNA clones and compared their sequences with those of genes from other fungi in the GenBank databases. From the 196 clones, we identified 51 genes encoding 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. Phylogenetic analyses by sequence comparison of five of the genes revealed evolutionary relationships of the wheat stripe rust pathogen to other fungi. The results demonstrated the usefulness of the full-length cDNA library in identifying functional genes of the pathogen.

Technical Abstract: A full-length cDNA library with complete genomic coverage is a powerful tool for functional genomic studies. We have constructed a full-length cDNA library from urediniospores of race PST-78 of Puccinia striiformis f. sp. tritici, the fungal pathogen causing wheat stripe rust. The full-length cDNA library consisted of 42,240 clones with an average cDNA insert of 1.9 kb. Of 196 randomly picked full-length cDNA clones that were sequenced, complete sequences were obtained for 149 clones and 47 clones had two partial sequences. The sequences of the 196 clones were compared to the fungal and all-organism sequences in the NCBI databases through BLAST searching and comparison. Of the 196 clones, 74 clones (37.8%) had significant homology at an e-value of 10-5 or less to the sequences of genes characterized in other fungi, 33 clones (16.8%) had significant homology to hypothetical proteins identified in other fungi, 38 clones (19.4%) had some homology at an e-value of higher than 1.0E-05, and the remaining 51 clones (26.0%) were unique and did not have significant homology with any previously reported sequences in the fungal database. We have identified 51 genes encoding 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, '-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. Phylogenetic analyses were conducted by sequence comparison of five genes (elongation factor, TATA-box binding protein, beta tubulin, nucleoside diphosphate kinase, and mitogen-activated protein kinase), which revealed evolutionary relationships of the wheat stripe rust pathogen to other fungi. The results demonstrated the usefulness of the full-length cDNA library in identifying functional genes of the pathogen.