Cloning and Characterization of miRNAs and Their Targets, Including a Novel miRNA-Targeted NBS–LRR Protein Class Gene in Apple (Golden Delicious)

ABSTRACTMicroRNA (miRNA) has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple (Malus domestica cv. Golden Delicious) by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 (Malus domestica microRNA Ln11), targeted an apple nucleotide-binding site (NBS)–leucine-rich repeat (LRR) class protein coding gene (Md-NBS). 5′ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11–12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ‘JiGuan’ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ‘FuJi’ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS–LRR protein class genes in woody plants.SUMMARY146 microRNA (miRNA) precursors were cloned from apple, with 11 found to be novel. The genomic locations of the miRNAs were determined and their expression levels analyzed in five different tissues. The novel miRNA, Md-miRLn11, was shown to suppress the expression of the Md-NBS gene and decrease resistance to apple Alternaria leaf spot.