Exploring the infection strategy of Colletotrichum fructicola in pecan and two effectors Cf-ID1 and Cf-ID2 were characterized using unique molecular identifier-RNA sequencing technology

The anthracnose disease caused by <i>Colletotrichum fructicola</i> has widely occurred in pecan (<i>Carya illinoinensis</i>) in China, seriously affecting its fruit yield and quality. However, the details of the infection strategy of <i>C. fructicola</i> remain to be elucidated. In this study, unique molecular identifier-RNA sequencing (UMI RNA-seq) was used to analyze differentially expressed genes (DEGs) of <i>C. fructicola</i> and candidate effectors were predicted. Two candidate effectors were identified during the early infection stages of <i>C. fructicola</i>. There were 6,822 DEGs at three infection timepoints (6, 24, and 36 h post-inoculation), and these genes were involved in spore germination, nutrient uptake, detoxification, secretion of toxic substances (such as effectors and toxins), inhibition of the host's immune response, and protein post-translational modification, which participated in the pathogenic process of <i>C. fructicola</i>. Moreover, 191 candidate effectors were predicted and their expression trends were divided into five clusters. Two candidate effectors Cf-ID1 and Cf-ID2 were selected for functional validation, and they were demonstrated to trigger cell death and immune response in <i>Nicotiana benthamiana</i>. Cf-ID1 and Cf-ID2 are located in both cytoplasm and nucleus and could suppress the infection of <i>C. fructicola</i> by eliciting defense responses in <i>N. benthamiana</i>. This study provided valuable information for in-depth research on the pathogenesis of <i>C. fructicola</i>.