Abstract:

Objective: Colletotrichum fructicola is the pathogen of anthracnose in Camellia oleifera. In this study, bioinformatics research methods were used to screen and analyze the effectors of Colletotrichum fructicola, and their functions were verified, so as to provide a theoretical basis for the research of disease-related genes of Colletotrichum fructicola, and to provide a reference for disease management in Camellia oleifera cultivation. Method: RNA-seq sequencing was performed at the non-infection stage (conidia) and infection stage (lesion tissue of Camellia oleifera leaves) of C. fruiticola. A series of bioinformatics software, such as BUSCA, target P, big PI predictor, and databases of GO, KEGG and PHI, were used to screen out the candidate effectors and then the effectors were quantitatively analyzed with qRT-PCR. The full-length gene of the candidate effector was cloned, and the corresponding gene function was verified by tobacco transient expression system and DAB staining. Result: The transcriptome data showed that there were 7 850 differentially expressed genes at the infection stage compared to the conidia stage. Predictive analysis of the encoded proteins of differentially expressed genes revealed that 345 proteins were classical secretory proteins, accounting for 4.39% of the total. The amino acid length of the classical secretory protein was evenly distributed in each segment, and the protein that meets the effector screening conditions (< 300 aa) accounted for about 36% of the total protein, which was relatively small. The length of the signal peptide was concentrated in 18-20 aa (48.7%), and the signal peptide cleavage site was SPase I type(88.41%). A total of 164 pieces of pathway information were gained by KEGG functional enrichment analysis, most of which were fungal metabolic pathways, secondary metabolites, and pathways related to starch and sucrose metabolism. The homology comparison result of PHI pathogen database showed that there were 80 genes related to fungal pathogenicity in the classical secretory protein, including a known effector. With the length of amino acid < 300 and cysteine residue number ≥ 4 as screening conditions, 17 candidate effector were screened out in C. fructicola after filtering the proteins with annotated functions. Nine candidate effectors were randomly selected for qRT-PCR analysis, and the results showed that the expression of all nine genes were up-regulated, which was in accordance with the results of transcriptome data. Four candidate effectors were cloned and verified by tobacco transient expression system and DAB staining, indicating that all candidate effectors was able to lead to plant cell necrosis. Conclusion: Multiple effectors are expressed during the infection period of Camellia oleifera by C. fructicola, while most of these proteins are not expressed or slightly expressed during the conidia period. This proves that the effectors is the key factor in the interaction between plant and pathogen.

Key words: Camellia oleifera anthracnose, Colletotrichum fructicola, transcriptome, bioinformatics, effector, Tobacco transient expression, secretory protein