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

Kunz, L.; Jigisha, J.; Menardo, F.; Sotiropoulos, A. G.; Zbinden, H.; Zou, S.; Tang, D.; Hueckelhoven, R.; Keller, B.; Mueller, M. C.

doi:10.1101/2024.07.10.602895

bioRxiv · 10.1101/2024.07.10.602895

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

Kunz, L.·Jigisha, J.·Menardo, F.·Sotiropoulos, A. G.·Zbinden, H.·Zou, S.·Tang, D.·Hueckelhoven, R.·Keller, B.·Mueller, M. C.

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Abstract

Wheat production is threatened by multiple fungal pathogens, such as the wheat powdery mildew fungus (Blumeria graminis f. 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. While R gene cloning has accelerated recently, AVR identification in many pathogens including Bgt lags behind, preventing pathogen-informed deployment of resistance sources. Here we describe a new "avirulence depletion (AD) assay" for rapid identification of AVR genes in Bgt. 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 the AVR component of the wheat immune receptor Pm3a to a 25kb genomic interval in Bgt harboring a single effector, the previously described AvrPm3a2/f2. Subsequently, we applied the AD assay to map the unknown AVR effector recognized by the Pm60 immune receptor. We show that AvrPm60 is encoded by three tandemly arrayed, nearly identical effector genes that trigger an immune response upon co- expression with Pm60 and its alleles Pm60a and Pm60b. We furthermore provide evidence that Pm60 outperforms Pm60a and Pm60b through more efficient recognition of AvrPm60 effectors, suggesting it should be prioritized for wheat breeding. Finally, we show that virulence towards Pm60 is caused by simultaneous deletion of all AvrPm60 gene paralogs and that isolates lacking AvrPm60 are especially prevalent in the US thereby limiting the potential of Pm60 in this region. The AD assay is a powerful new tool for rapid and inexpensive AVR identification in Bgt with the potential to contribute to pathogen-informed breeding decisions for the use of novel R genes and regionally tailored gene deployment.

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Kunz, L., Jigisha, J., Menardo, F., Sotiropoulos, A. G., Zbinden, H., Zou, S., Tang, D., Hueckelhoven, R., Keller, B., Mueller, M. C.. 2024-07-16. Avirulence depletion assay: combining R gene-mediated selection with bulk sequencing for rapid avirulence gene identification in wheat powdery mildew. https://doi.org/10.1101/2024.07.10.602895

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