Identification of MicroRNA, Especially miR390 in Apple response to Alternaria atanata infection by RNA-seq and Agrobacterium co-infiltration Assay

MicroRNAs (miRNAs) are major regulatory functions on plant growth and development as well as in multiple stress responses. In recent years, multiple studies have shown that miRNAs participate in regulating diverse aspects of the plant-pathogen interactions that cause plant diseases, but little is known about the potential existence and functional roles of pathogen-responsive miRNAs in apple (Malus domestica Borkh.). Here, we found pathogen-responsive miRNA classified into 41 families via deep-sequencing-based miRNA seq profiling from an infection time course of apple leaves challenged by Alternaria alternata apple pathotype (AAAP). These miRNAs were predicted to target transcripts for 673 genes, including multiple R genes and transcription factors, such as LRR, RPK2, LACCASE, and MYB, among others. A follow-up analysis revealed that many AAAP responsive miRNAs and their predicted targets exhibited differential expression patterns in susceptible (Starking Delicious) vs. resistant (Jonathon) apple cultivars, further indicating that these miRNAs were involved in the regulation of AAAP infection in apples after transcription. Then, we focused specifically on a potential negative regulatory impact of mdm miR390a on disease resistance and predicted the impact may result from targeting and inhibiting the expression of the MdRPK2 and MdLRR8 genes, which was confirmed experimentally in both Agrobacterium co-infiltration assays in tobacco (Nicotiana benthamiana) and apple (Malus domestica Gala). The result showed that overexpression of mdm miR390a enhanced the sensitivity to AAAP infection, and overexpression of MdLRR8 or MdRPK2 improved the resistance to AAAP while co-expression with mdm miR390a counteracted the resistance. Above all, we provide a large amount of data resource for the study of miRNA species and miRNA based regulatory mechanisms in tree fruit. In addition, our study uncovered a fascinating case in which the plant-genome-encoded miRNA induced by fungus apparently increases the susceptibility of the plant host to fungal infection through post-transcriptional R gene knockdown mechanism.