title: Identifying secreted proteins of Marssonina brunnea by degenerate PCR.
abstract: Marssonina brunnea is an important fungal pathogen of the Populus genus. To further our understanding of the pathogenesis of M. brunnea, we initiated a proteome-level study of the fungal secretome. Using de novo peptide sequencing by MS/MS, we obtained peptide sequences for 32 protein spots. Four proteins were identified by sequence homology to conserved proteins in public databases using MS-driven BLAST. To identify additional protein spots, we combined a degenerate PCR method, based on the Consensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) method, and a rapid amplification of cDNA ends method to clone the full-length cDNA fragments encoding the proteins identified in the gel. Using this method, we cloned the full-length cDNA fragments encoding 11 M. brunnea-specific proteins. This method provides an efficient approach to identification of species-specific proteins of non-sequenced organisms. Furthermore, we analyzed the expression patterns of these genes during infection. We found that most of the identified secreted proteins could be induced in artificial medium after hyphae entered poplar apoplast spaces. We propose that for the host-specialized M. brunnea, the elongation of hyphae has evolved closely with the secretion of apoplastic proteins.
authors.affiliation: Nanjing Forestry University, Nanjing, People's Republic of China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Cheng
authors.forename: Youzhi
authors.initials: Y
authors.lastname: Cao
authors.forename: Cong
authors.initials: C
authors.lastname: Jiang
authors.forename: Li'an
authors.initials: L
authors.lastname: Xu
authors.forename: Mingxiu
authors.initials: M
authors.lastname: Wang
authors.forename: Shougong
authors.initials: S
authors.lastname: Zhang
authors.forename: Minren
authors.initials: M
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D005656:Fungal Proteins