Molecular Mechanism of miR160d in Regulating Kiwifruit Resistance to <i>Botrytis cinerea</i>

Abstract

Gray mold caused by <i>Botrytis cinerea</i> leads to huge economic losses to the kiwifruit (<i>Actinidia chinensis</i>) industry. Elucidating the molecular mechanism responding to <i>B. cinerea</i> is the theoretical basis for the resistance to molecular breeding of kiwifruit. Previous studies have shown that miR160 regulates plant disease resistance through the indole-3-acetic acid (IAA) signaling pathway. In this study, kiwifruit "Hongyang" was used as the material, and Ac-miR160d and its target genes were identified and cloned. Overexpression and virus-induced gene silencing (VIGS) technology combined with RNA-seq were adopted to analyze the regulatory role of Ac-miR160d in kiwifruit resistance to <i>B. cinerea</i>. Silencing Ac-miR160d (<i>AcMIR160d</i>-KN) increased kiwifruit sensitivity to <i>B. cinerea</i>, whereas overexpression of Ac-miR160d (<i>AcMIR160d</i>-OE) increased kiwifruit resistance to <i>B. cinerea</i>, suggesting that Ac-miR160d positively regulates kiwifruit resistance to <i>B. cinerea</i>. In addition, overexpression of Ac-miR160d in kiwifruit increased antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD), and endogenous phytohormone IAA and salicylic acid (SA) content, in response to <i>B. cinerea</i>-induced stress. RNA-seq identified 480 and 858 unique differentially expressed genes in the <i>AcMIR160d</i>-KN vs CK and <i>AcMIR160d</i>-OE vs CK groups, respectively, with fold change ≥2 and false discovery rate <0.01. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that families associated with "biosynthesis of secondary metabolites" are possibly regulated by Ac-miR160d. "Phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "terpenoid backbone biosynthesis" were further activated in the two comparison groups upon <i>B. cinerea</i> infection. Our results may reveal the molecular mechanism by which miR160d regulates kiwifruit resistance to <i>B. cinerea</i> and may provide gene resources for molecular breeding in kiwifruit resistance.