Virulence on Pm4 kinase-based resistance is determined by two divergent wheat powdery mildew effectors

nature plants Article https://doi.org/10.1038/s41477-025-02180-w Virulence on Pm4 kinase-based resistance is determined by two divergent wheat powdery mildew effectors Received: 28 July 2025 Accepted: 17 November 2025 Published online: 12 January 2026 Check for updates Zoe Bernasconi 1,6, Aline G. Herger 1,6, Maria Del Pilar Caro 1,6, Lukas Kunz 1,6, Marion C. Müller 1,5, Ursin Stirnemann 1, Megan A. Outram 2, Victoria Widrig 1,3, Matthias Neidhart1, Jonatan Isaksson 1, Seraina Schudel 1, Sebastian Rösli 1, Thomas Wicker Kyle W. Bender 1, Cyril Zipfel 1,4, Peter N. Dodds 2, Melania Figueroa 2, Javier Sánchez-Martín 1,3 & Beat Keller 1 , 1 The wheat resistance gene Pm4 encodes a kinase fusion protein and has gained particular attention as it confers race-specific resistance against two major wheat pathogens: powdery mildew and blast. Here we describe the identification of AvrPm4, the mildew avirulence effector recognized by Pm4, using UV mutagenesis, and its functional validation in wheat protoplasts. We show that AvrPm4 directly interacts with and is phosphorylated by Pm4. Using genetic association and quantitative trait locus mapping, we further demonstrate that the evasion of Pm4 resistance by virulent mildew isolates relies on a second fungal component, SvrPm4, which suppresses AvrPm4-induced cell death. Surprisingly, SvrPm4 was previously described as AvrPm1a. We show that SvrPm4, but not its inactive variant svrPm4, is recognized by the nucleotide-binding leucine-rich repeat immune receptor Pm1a. These multiple roles of a single effector provide a new perspective on fungal (a)virulence proteins and their combinatorial interactions with different types of immune receptors. Wheat yields are severely impacted by pests and pathogens, accounting for over 20% of global losses1. Resistance breeding is a key strategy for crop protection and reduction of pathogen-inflicted damage. It often relies on dominant resistance (R) genes encoding immune receptors that recognize pathogen-delivered avirulence (Avr) effectors and activate immune responses, usually culminating in cell death2,3. While most cloned R genes in wheat encode nucleotide-binding leucine-rich repeat (NLR) receptors, kinase fusion proteins (KFPs) are emerging as a distinct class of immune receptors specifically found in cereals4. Much of our current understanding of KFPs comes from tandem kinase proteins—a major KFP subclass—which consist of two kinase domains, sometimes with additional domains of unknown function5,6. Recent studies have demonstrated that some tandem kinase proteins rely on a helper NLR to trigger effector-induced cell death, as shown for Sr62 in Aegilops tauschii and RWT4 in wheat7,8. Beyond tandem kinase proteins, other KFPs, composed of at least one kinase domain and additional domains, have been described in cereals but remain poorly understood9,10. Among them, Pm4, a protein Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland. 2Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Canberra, Australian Capital Territory, Australia. 3Department of Microbiology and Genetics, Spanish-Portuguese Agricultural Research Centre, University of Salamanca, Salamanca, Spain. 4Sainsbury Laboratory, University of East Anglia, Norwich, UK. 5Present address: Phytopathology, TUM School of Life Sciences, Technical University of Munich, Freising, Germany. 6These authors contributed equally: Zoe Bernasconi, Aline G. Herger, Maria Del Pilar Caro, Lukas Kunz. e-mail: ; 1 Nature Plants | Volume 12 | January 2026 | 164–178 164 Article containing a serine/threonine kinase, multiple C2 domains and transmembrane regions, is particularly notable: the Pm4 gene, located on wheat chromosome 2A, encodes two alternative isoforms derived by alternative splicing, Pm4-V1 and Pm4-V2, both required for resistance to the biotrophic pathogen Blumeria graminis f. sp. tritici11. Pm4 occurs as several alleles (Pm4a–g), which encode highly similar proteins. Pm4a and Pm4b have been studied extensively in near-isogenic backgrounds, where they show partially overlapping race-specific resistance spectra against B. g. tritici11. Furthermore, recent work has shown that Pm4 also confers resistance to the hemibiotrophic wheat blast pathogen Magnaporthe oryzae12,13. In fact, the wheat blast resistance gene Rmg7 is identical to Pm4a, whereas the Rmg8 resistance gene on wheat chromosome 2B could be assigned to a Pm4 homoeologue with an identical sequence to that of Pm4f12. The corresponding wheat blast effector AVR-Rmg8 was cloned earlier14 and is recognized by multiple Pm4 alleles, including Pm4a, Pm4b and Pm4f12,13. The KFP encoded by Pm4 therefore represents a highly important resistance source in wheat with multiple alleles providing resistance to the obligate biotrophic B. g. tritici and the hemibiotrophic wheat blast pathogen simultaneously. Despite the progress made in describing novel KFPs, a mechanistic understanding of how such proteins induce immunity is yet to be established. Kinase activity in KFPs was suggested to be relevant for Avr effector recognition and/or downstream signalling, a fact further supported by the finding of loss-of-function mutants affected in the kinase domain of KFPs such as Pm4 (ref. 11). Moreover, the tandem kinase protein RWT4, encoded by an allele of the powdery mildew resistance gene Pm24 (refs. 15,16), phosphorylates the avirulence but not the virulence variant of the wheat blast effector PWT4, suggesting that the phosphorylation of the Avr effector plays a key role in the resistance mechanism17. For the barley stem rust resistance protein RPG1, two fungal proteins work synergistically to induce phosphorylation and trigger a hypersensitive response (HR)18. These examples also highlight the importance of direct interaction between KFPs and their recognized effectors. Understanding these mechanisms will require the identification and characterization of Avr effectors that interact with KFPs such as Pm4. Methods based on genetic association, such as biparental mapping or genome-wide association studies (GWAS), have been widely used to identify Avr loci in Blumeria and have been combined with cell death assays in heterologous systems such as Nicotiana benthamiana and host protoplasts19–21. More recently, AvrXpose, an approach based on UV mutagenesis and selection of gain-of-virulence mutants, has also proved effective in identifying genes controlling avirulence in B. g. tritici22. To date, all known Avr genes in B. g. tritici encode small (100–150 amino acid residues), secreted effector proteins with a predicted RNase-like structure and trigger a cell death response upon recognition by corresponding wheat NLR immune receptors (reviewed in ref. 23). In contrast, no B. g. tritici Avr effector recognized by a KFP has been identified so far. Suppressors of R-gene-mediated immunity have been reported across bacterial, oomycete and fungal (...truncated)