Objective To explore and identify effector proteins in Colletotrichum higginsianum.

Method Based on the transcriptomic data from the interaction between Colletotrichum higginsianum and Arabidopsis thaliana, candidate effector proteins were first screened using approaches including signal peptide prediction, transmembrane domain analysis, and subcellular localization prediction. By analyzing the expression patterns of candidate effector proteins during infection, we further focused on those significantly up-regulated at specific infection stages. Functional validation was performed using a transient expression system in Nicotiana benthamiana to identify the effector proteins. Bioinformatic analyses, including subcellular localization, conserved domain, and phylogenetic analyses, were subsequently conducted on the identified effector proteins.

Result A total of 432 high-confidence candidate effector proteins were screened from 14 651 genes. We further focused on 21 proteins that were significantly up-regulated at specific infection stages and successfully identified three effector proteins: Ch3542, Ch13411 and Ch13653. Among them, Ch13411 and Ch13653 could individually induce cell death in N. benthamiana, whereas Ch3542 was able to suppress INF1-induced cell death, exhibiting an immune-suppressive function. Further analyses demonstrated that all three effector proteins possessed typical secretory signal peptides and showed membrane localization or cytosolic distribution in N. benthamiana cells. Conserved domain prediction revealed that Ch3542, Ch13411, and Ch13653 belonged to the glycosyl hydrolase family 16, DPBB domain-containing proteins, and M35 metallopeptidase family, respectively. Phylogenetic analysis indicated that these three effector proteins were relatively conserved within the genus Colletotrichum.

Conclusion This study established an integrated screening pipeline combining expression profiles and sequence characteristics, successfully identified key effector proteins of C. higginsianum, and provided important targets and a theoretical foundation for further elucidating its pathogenic mechanisms and plant immune interactions.