Identification of microRNA transcriptome in apple response to Alternaria alternata infection and evidence that miR390 is negative regulator of defense response

MicroRNAs (miRNAs) play an important regulatory role in plant growth and development, as well as in response to multiple stresses. In recent years, multiple studies have shown that miRNAs participate in regulating diverse aspects of the plant-pathogen interactions, but little is known about the potential existence and functional roles of pathogen-responsive miRNAs in apple (Males x domestica Borkh.). Here, we identified pathogen-responsive miRNAs in apple leaves challenged by Alternaria alternata apple pathotype (AAAP). These AAAP-responsive miRNAs were classified into 41 families and predicted to target 673 genes, including multiple R genes and transcription factors, such as LRR, RPK2, LACCASE, and MTh, among others. A follow-up analysis revealed that some of the AAAP-responsive miRNAs and their predicted targets exhibited differential expression patterns in the disease-susceptible vs. disease-resistant apple cultivars, i.e. Starking Delicious' vs. 'Jonathon', indicating that these miRNAs are involved in the regulation of apple resistance to AAAP infection. 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 (Mattis x domestica 'Gala'). The results 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, beyond presenting a large data resource for studies of miRNA species and miRNA-based regulation mechanisms in tree fruit, our study uncovered a fascinating case wherein a fungus-induced plant-genome-encoded miRNA apparently increases the susceptibility of the plant host to fungal infection via a post-transcriptional R-gene-knockdown mechanism.

Automated summary

miRNAs play a significant regulatory role in plant responses to pathogens, with some miRNAs in apple leaves targeting genes involved in disease resistance. Specifically, the study found that certain miRNAs and their target genes exhibit differential expressions in different apple cultivars, shedding light on their roles in regulating resistance mechanisms. Additionally, a fungus-induced plant-genome-encoded miRNA was found to increase susceptibility to fungal infection via a post-transcriptional R-gene-knockdown mechanism.