Avirulence depletion assay: combiningRgene-mediated selection with bulk sequencing for rapid avirulence gene identification in wheat powdery mildew

Abstract

AbstractWheat production is threatened by multiple fungal pathogens, such as the wheat powdery mildew fungus (Blumeria graminisf. sp.tritici,Bgt). Wheat resistance breeding frequently relies on the use of resistance (R) genes that encode diverse immune receptors which detect specific avirulence (AVR) effectors and subsequently induce an immune response. WhileRgene cloning has accelerated recently,AVRidentification in many pathogens includingBgtlags behind, preventing pathogen-informed deployment of resistance sources. Here we describe a new “avirulence depletion (AD) assay” for rapid identification ofAVRgenes inBgt. This assay relies on the selection of a segregating, haploid F1 progeny population on a resistant host, followed by bulk sequencing, thereby allowing rapid avirulence candidate gene identification with high mapping resolution. In a proof-of- concept experiment we mapped theAVRcomponent of the wheat immune receptorPm3ato a 25kb genomic interval inBgtharboring a single effector, the previously describedAvrPm3a2/f2. Subsequently, we applied the AD assay to map the unknownAVReffector recognized by the Pm60 immune receptor. We show thatAvrPm60is encoded by three tandemly arrayed, nearly identical effector genes that trigger an immune response upon co- expression withPm60and its allelesPm60aandPm60b. We furthermore provide evidence thatPm60outperformsPm60aandPm60bthrough more efficient recognition ofAvrPm60effectors, suggesting it should be prioritized for wheat breeding. Finally, we show that virulence towardsPm60is caused by simultaneous deletion of allAvrPm60gene paralogs and that isolates lackingAvrPm60are especially prevalent in the US thereby limiting the potential ofPm60in this region. The AD assay is a powerful new tool for rapid and inexpensiveAVRidentification inBgtwith the potential to contribute to pathogen-informed breeding decisions for the use of novelRgenes and regionally tailored gene deployment.