In Silico Apple Genome-Encoded MicroRNA Target Binding Sites Targeting Apple Chlorotic Leaf Spot Virus

Apple chlorotic leaf spot virus (ACLSV) (genus, Trichovirus; family, Betaflexiviridae) is a widespread, deleterious, and the most damaging pathogen of pome and fruit trees including domesticated apple (Malus x domestica Borkh.), to which it is transmitted by grafting and pruning. The positive-sense, single-stranded RNA virus is 600-700 nm long and has a genome of 74.7-7.56 kbp in size, minus the poly-A tail and 3 & PRIME;- and 5 & PRIME;-untranslated regions. The genome has three overlapping open reading frames (ORFs) that encode a replication-associated protein (Rep), movement protein (MP), and coat protein (CP). RNA interference (RNAi)-mediated antiviral defense in eukaryotes has evolved to control infections in plant viruses. The objective of this study was to analyze locus-derived microRNAs (mdm-miRNAs) in the apple genome with potential for targeting ACLSV +ssRNA-encoded mRNAs, using a predictive approach that involves four algorithms. The goal is to mobilize the in silico-predicted endogenous mdm-miRNAs and trigger the RNAi pathway experimentally in apple trees to evaluate antiviral resistance to ACLSV. Experimentally validated apple (2n = 2X = 34) mdm-miRNAs (n = 322) were obtained from the miRBase database and aligned to the ACLSV genome (KU870525). Of the 322 targeting mature locus-derived mdm-miRNAs analyzed, nine apple mdm-miRNA homologs (mdm-miR395k, mdm-miR5225c, and mdm-miR7121 (a, b, c, d, e, f, g, h) were predicted by all "four algorithms", whereas fifty-eight mdm-miRNAs were identified as consensus binding sites by the combined results of two algorithms. The miRanda, RNA22, and TAPIR algorithms predicted binding of mdm-miR395k at nucleotide position 4691 and identified it as the most effective interacting mdm-miRNA targeting the virus ORF1 sequence. An integrated Circos plot was generated to validate the accuracy of target prediction and determine if apple mdm-miRNAs could bind to the predicted ACLSV mRNA target(s). A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The availability of validated locus-derived microRNAs (mdm-miRNAs) with predicted potential to target ACLSV in infected apple trees represents the first step toward development of ACLSV-resistant apple trees.