Identification and functional analysis of miRNAs in N. benthamiana in response to A. longipes infection

Alternaria longipes (A. longipes) is the main pathogen causing tobacco brown spot, one of the most severe diseases of tobacco. Numerous studies have indicated that miRNAs are widely involved in interactions between pathogens and host plants. To characterize the roles of miRNAs in A. longipes infection of Nicotiana benthamiana (N. benthamiana), specific infection methods to isolate infected and sampled regions of N. benthamiana leaves were used. The process of A. longipes infection of N. benthamiana leaves was characterized by determining a series of critical physiological indices. The results revealed that 3 days after infection by A. longipes was the critical stage of A. longipes infection of N. benthamiana leaves. miRNAs expressed in response to A. longipes infection were identified through sRNA high-throughput sequencing. A total of 175 novel miRNAs were identified in the N. benthamiana genome through 6 sRNA libraries. Of these, 37 miRNAs were differentially expressed between control and infected leaves. The abundance of 15 miRNAs, including nbe-miRN2031*, nbe-miR482b, and nbe-miR398, was found to be especially upregulated in leaves infected with A. longipes. These miRNAs mainly targeted resistance proteins of N. benthamiana. Further expression analysis of the miRNAs and their targets indicated that miRNAs substantially affected the expression of resistance genes during infection by A. longipes. Moreover, nbe-miR482b and nbe-miRN2031* transiently silenced via STTM technology decreased injury levels caused by A. longipes, suggesting that this fungus might regulate the immune response of host plants by regulating several key miRNAs. This study primarily clarified the potential roles of miRNAs during A. longipes infection and provided critical information for an in-depth understanding of the infection mechanism used by A. longipes on plants.