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

Abstract

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