Abstract:

Objective: This study conducts a functional analysis of the SNARE protein Nyv1, which regulates vesicle transport in Cytospora chrysosperma, to explore its mechanism of action in the growth, development, and pathogenic processes of the fungus. It provides an important reference for understanding the regulatory mechanism of SNARE proteins in the pathogenic process of C. chrysosperma and for developing effective prevention and control strategies for this disease. Method: 1) Utilizing the amino acid sequence of Nyv1 from Saccharomyces cerevisiae for Blastp analysis to identify its homologous CcNyv1 gene. 2) Using bioinformatics software to perform multiple sequence alignments of the amino acid sequence encoded by CcNyv1 and homologous Nyv1 sequences in other fungi, and to construct a phylogenetic tree. 3) Employing the split-marker method and PEG-mediated genetic transformation system to obtain CcNyv1 gene deletion mutants and complemented strains. 4) Observing mycelial growth rate through PDA plate growth experiments, and analyzing whether the CcNyv1 gene, along with Longin and SNARE domains, affect the nutritional growth of mycelia and their response to salt stress. 5) Observing the expansion of disease spots on scalded branches of one-year-old poplars to assess the impact of the CcNyv1 gene, along with Longin and SNARE domains, on the pathogenicity of the poplar rot fungus. 6) Employing FM4-64 dye staining to investigate whether the CcNyv1 gene and the Longin, SNARE domains affect the endocytosis of the fungus. Result: 1) Through a Blast comparison using the amino acid sequence of Saccharomyces cerevisiae Nyv1, its homologous gene CcNyv1 was identified in C. chrysosperma. This gene is 780 bp long, contains one intron, and encodes a 233 amino acid vesicle transport-related R-SNARE protein, which includes an N-terminal Longin domain and a C-terminal SNARE domain. 2) Multiple sequence comparisons and phylogenetic analyses show that CcNyv1 and its homologous Nyv1 genes in ascomycetes have a high degree of sequence similarity, are evolutionarily conserved, and the CcNyv1 in C. chrysosperma is most closely related to the homologous gene in Cryphonectria parasitica. 3) Using the split-marker method, CcNyv1 gene deletion mutants, Longin domain deletion mutants, SNARE domain deletion mutants, and CcNyv1 gene complemented strains were obtained. 4) Analysis of mycelial growth phenotype and pathogenic phenotype on poplar branches shows that the CcNyv1 gene deletion mutants and Longin, SNARE domain deletion mutants exhibit significant defects in mycelial nutritional growth, response to salt stress, and pathogenicity. 5) Comparison of vesicle distribution in wild-type and deletion mutant strains stained with FM4-64 reveals that the CcNyv1 gene and Longin, SNARE domains affect the endocytosis of C. chrysosperma. Conclusion: The SNARE protein CcNyv1 of C. chrysosperma plays a significant regulatory role in aspects such as nutritional growth, stress response, pathogenicity, and endocytosis.

Key words: poplar, canker disease, Cytospora chrysosperma, vesicle transport, SNARE, CcNyv1, pathogenicity