A Single-Nucleotide Polymorphism in the Promoter of a Hairpin RNA Contributes to Alternaria alternata Leaf Spot Resistance in Apple (Malus × domestica)

Apple leaf spot caused by the Alternaria alternata f. sp mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA) named MdhpRNA277 that produces small RNAs and is induced by ALT1 infection in ‘NGR196’ apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in the resistant apple variety ‘Hanfu’ and at low levels in the susceptible variety ‘NGR196’ following ALT1 infection. MdhpRNA277 was strongly induced in ‘NGR196’ but not ‘Hanfu’ following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated ‘NGR196’ versus ‘Hanfu’. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between ‘NGR196’ (pMdhpRNA277-NGR196) and ‘Hanfu’ (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-NGR196, but not to pMdhpRNA277-HF. Transgenic ‘GL-3’ apple expressing pMdhpRNA277-NGR196:MdhpRNA277 was more susceptible to ALT1 infection than plants expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and reduced expression of the five target R genes. We confirmed that the SNP in pMdhpRNA277 is associated with A. alternata leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to A. alternata leaf spot.