Identifying secreted proteins of Marssonina brunnea by degenerate PCR
Qiang Cheng1, Youzhi Cao, Cong Jiang
1Nanjing Forestry University, Nanjing, People's Republic of China.
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.
Insights
Marssonina brunnea, a poplar pathogen, was studied at the proteome level. Researchers identified novel secreted proteins, revealing insights into fungal pathogenesis and host-specific evolution.
Area of Science:
- Mycology
- Plant Pathology
- Proteomics
Background:
- Marssonina brunnea is a significant fungal pathogen impacting Populus trees.
- Understanding its pathogenesis is crucial for developing effective disease management strategies.
Purpose of the Study:
- To investigate the proteome of Marssonina brunnea, focusing on secreted proteins.
- To identify novel M. brunnea-specific proteins and analyze their expression during host infection.
Main Methods:
- Proteomic analysis using MS/MS for de novo peptide sequencing.
- Combined degenerate PCR (CODEHOP) and rapid amplification of cDNA ends (RACE) for full-length cDNA cloning.
- Gene expression analysis during host-pathogen interaction.
Main Results:
- Identified 32 protein spots, with 4 confirmed via homology searches.
- Successfully cloned 11 M. brunnea-specific proteins using CODEHOP and RACE.
- Most identified secreted proteins were induced after hyphal entry into the poplar apoplast.
Conclusions:
- The developed method efficiently identifies species-specific proteins in non-sequenced organisms.
- Hyphal elongation in host-specialized M. brunnea is closely linked to the secretion of apoplastic proteins.
- This study provides a foundation for understanding M. brunnea pathogenesis and host interactions.
