A novel miRNA negatively regulates resistance to Glomerella leaf spot by suppressing expression of an NBS gene in apple

Glomerella leaf spot(GLS)of apple(Malus×domestica Borkh.),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-GLS exhibited the opposite expression pattern,which suggested that the expression levels of Md-miRln20 and its target gene are closely related to apple resistance to GLS.Furthermore,downregulation of MdmiRln20 in susceptible apple leaves resulted in upregulation of Md-TN1-GLS and reduced the disease incidence.Conversely,overexpression of Md-miRln20 in resistant apple leaves suppressed Md-TN1-GLS expression,with increased disease incidence.We demonstrated that Md-miRln20 negatively regulates resistance to GLS by suppressing Md-TN1-GLS expression and showed,for the first time,a crucial role for miRNA in response to GLS in apple.

Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple

Alternaria leaf spot in apple(Malus x domestica),caused by the fungal pathogen Alternaria alternata f.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(CC R-NB-LRR)domain,interacts with another CC R-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.mali that we tested:ALT1,GBYB2,BXSB5,and BXSB7.In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf 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 Alternaria leaf spot resistance in apple.The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.