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Glomerella leaf spot (GLS) of apple (Malus×domesticaBorkh.), caused by Glomerella cingulata, is an emerging fungal epidemic threatening the apple industry. Little is known about the molecular mechanism underlying resistance to this devastating fungus. In this study, high-throughput sequencing technology was used to identify microRNAs (miRNAs) involved in GLS resistance in apple. We focused on miRNAs that target genes related to disease and found that expression of a novel miRNA, Md-miRln20, was higher in susceptible apple varieties than in resistant ones. Furthermore, its target gene Md-TN1-GLSexhibited the opposite expression pattern, which suggested that the expression levels of Md-miRln20and its target gene are closely related to apple resistance to GLS. Furthermore, downregulation of Md-miRln20in susceptible apple leaves resulted in upregulation of Md-TN1-GLSand reduced the disease incidence. Conversely, overexpression of Md-miRln20in resistant apple leaves suppressed Md-TN1-GLSexpression, with increased disease incidence. We demonstrated that Md-miRln20negatively regulates resistance to GLS by suppressing Md-TN1-GLSexpression and showed, for the first time, a crucial role for miRNA in response to GLS in apple.

Alternarialeaf spot in apple (Malusx domestica), caused by the fungal pathogen Alternaria alternataf. sp. mali(also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. malithat we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternarialeaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternarialeaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.