Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BackgroundStagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained.ResultsIn this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome.ConclusionWe conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

[1]  K. Rybak,et al.  Structural Characterisation of the Interaction between Triticum aestivum and the Dothideomycete Pathogen Stagonospora nodorum , 2006, European Journal of Plant Pathology.

[2]  K. Malcolm,et al.  A Genomic and Proteomic Analysis of Activation of the Human Neutrophil by Lipopolysaccharide and Its Mediation by p38 Mitogen-activated Protein Kinase* , 2002, The Journal of Biological Chemistry.

[3]  Ling Li,et al.  Assessment and improvement of the Plasmodium yoelii yoelii genome annotation through comparative analysis , 2008, ISMB.

[4]  Steven Salzberg,et al.  GlimmerM, Exonomy and Unveil: three ab initio eukaryotic genefinders , 2003, Nucleic Acids Res..

[5]  K. Rybak,et al.  Investigating the role of calcium/calmodulin‐dependent protein kinases in Stagonospora nodorum , 2006, Molecular microbiology.

[6]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[7]  A. Millar,et al.  Proteomic identification of extracellular proteins regulated by the Gna1 Galpha subunit in Stagonospora nodorum. , 2009, Mycological research.

[8]  James K. Hane,et al.  Dothideomycete–Plant Interactions Illuminated by Genome Sequencing and EST Analysis of the Wheat Pathogen Stagonospora nodorum[W][OA] , 2007, The Plant Cell Online.

[9]  Akhilesh Pandey,et al.  Genome annotation of Anopheles gambiae using mass spectrometry-derived data , 2005, BMC Genomics.

[10]  James C. Wright,et al.  Exploiting proteomic data for genome annotation and gene model validation in Aspergillus niger , 2009, BMC Genomics.

[11]  C. Yanofsky.  Using Studies on Tryptophan Metabolism to Answer Basic Biological Questions , 2003, The Journal of Biological Chemistry.

[12]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.

[13]  Jacob D. Jaffe,et al.  Proteogenomic mapping as a complementary method to perform genome annotation , 2004, Proteomics.

[14]  P. Solomon,et al.  Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat. , 2006, Molecular plant pathology.

[15]  A Signaling-Regulated, Short-Chain Dehydrogenase of Stagonospora nodorum Regulates Asexual Development , 2008, Eukaryotic Cell.

[16]  Paul Horton,et al.  Nucleic Acids Research Advance Access published May 21, 2007 WoLF PSORT: protein localization predictor , 2007 .

[17]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.