Marion Claudia Müller

Proteins with a tandem kinase structure have recently emerged as players in race-specific resistance in cereal crops. However, the molecular understanding of these immune receptors’ resistance mechanisms is limited by the lack of knowledge about the pathogen effectors that they recognise. In this work, we identify AvrWTK4, the wheat powdery mildew RNase-like effector recognised by the wheat tandem kinase protein WTK4, through a combination of bi-parental genetic mapping and mutagenesis. We demonstrate that mutations in the AvrWTK4 gene or a reduction of its expression lead to virulence on WTK4 . Transfection of AvrWTK4 specifically induced cell death in WTK4 -expressing Aegilops tauschii protoplasts. The avirulent AvrWTK4 variant interacts more strongly than the virulent variant with the N-terminal heavy metal-associated (HMA)-like domain of WTK4. We found that the integrated HMA-like domain possibly serves as a direct effector-binding decoy for WTK4, providing a mechanistic paradigm for effector recognition by tandem kinase proteins.

Summary: The receptor kinase FERONIA (FER) is a susceptibility factor for biotrophic powdery mildew fungal pathogens in Arabidopsis thaliana , but the underlying molecular mechanisms remain largely unknown. FER is required for the perception of endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides to control various aspects of plant growth, development and immunity. RALFs are either perceived by FER/LORELEI‐LIKE GPI‐ANCHORED PROTEIN (LLG) heterocomplexes to induce cellular responses or bind to LEUCINE‐RICH REPEAT EXTENSIN (LRX) proteins as cell wall structural components. Combining genetics, cell biology and biochemistry, we found that FER's endogenous RALF ligands are necessary for full colonization success of the powdery mildew‐species Erysiphe cruciferarum . We reveal that LLGs and LRXs are also powdery mildew susceptibility factors. We show that cell wall remodeling and apoplastic pH homeostasis, hallmark features of RALF function, support powdery mildew reproductive success. We provide data that RALF‐dependent powdery mildew pathogenesis is partially independent of FER. Powdery mildew fungi likely do not produce RALF peptide mimics, suggesting their reliance on endogenous RALFs for successful host colonization. We propose that powdery mildew fungi require RALF‐mediated modulation of apoplastic pH and pectin remodeling for successful host colonization, highlighting a new susceptibility mechanism by obligate biotrophic fungi.

Some filamentous plant‑pathogenic fungi have comparably large genome sizes within the fungal kingdom due to the proliferation of transposable elements (TEs). Blumeria hordei ( Bh ), the causal agent of the powdery mildew disease on barley, is a filamentous obligate biotrophic fungus. Compared to other ascomycetes, it contains a low number of genes but a high genomic TE content of approximately 75%. Yet, a comprehensive understanding of the contribution of TEs to the RNA and protein landscape of Bh is lacking. Here, we use Bh as a model to study transcripts and proteins derived from genes and individual TEs. Therefore , we created two high‑quality genome assemblies of the German Bh isolate TUM1 and the Australian Bh isolate AUS1. We applied deep proteomics with mass spectrometry, long‑read and short‑read sequencing on both DNA and RNA. Based on these multi‑omic resources, we completed nearly gapless genome assemblies, new gene and TE annotations, and effector predictions. Using long-read RNA sequencing, we detected extensive co-transcription of TEs and genes as TE-gene chimeric transcripts. We identified previously unpredicted splice variants or genes, partially supported by proteomics. The intergenic and TE genomic space of Bh TUM1 gives rise to thousands of transcripts and several novel TE-derived proteins that lack from previous TE protein predictions. Together, this supports an existing potential for expression of novel transcripts and proteins from highly abundant TEs in the Bh genome.

The fungal crop pathogen Blumeria hordei , causal agent of powdery mildew on barley, presents life-history and epidemiological characteristics, as well as and selective pressures due to modern agriculture leading to expected sweepstakes reproduction, that is highly skewed offspring distributions. Using genome-wide polymorphism data and population genomics inferences, we aim to 1) infer the past demographic history and the strength of sweepstakes reproduction in B. hordei , and 2) quantify the contributions of these selective and neutral processes in the genome. An new inference method based on Neural Posterior Estimation and diversity and linkage disequilibrium statistics was developed and tested on simulated and B. hordei genomic data. We confirm that B. hordei exhibits a moderate sweepstakes reproduction ( α -parameter of 1.6). We highlight that the Site Frequency Spectrum (SFS) appears sensitive to the joint occurrence of sweepstakes and recent demographic changes, which may caution on the reliability of the SFS to infer sweepstakes reproduction. We then scan the genome for selective sweeps, adjusting the significance thresholds of the methods for demographic history and sweepstakes reproduction, thereby yielding a counterintuitive result. When conditioning the significance threshold for sweep detection on simulations under sweepstakes and demography, a very large number of putatively selected regions is found (11.6% of the genome). We suggest that sweepstakes reproduction in B. hordei is due to 1) neutrality (clonal/sexual phases and Boom-and-Bust cycles) generating a genome-wide level of background noise in the coalescent genealogies, and 2) selective sweepstakes due to pervasive positive selection. Our findings have important implications for both population genomic methodology and our understanding of pathogen evolution.

Broad-spectrum resistance genes are highly valuable for sustainable crop protection, yet the molecular basis of their activity is often unknown. The Pm3 allelic series in wheat encodes NLR receptors that recognize avirulence (AVR) effectors of wheat powdery mildew. Here, we show that near-identical Pm3 alleles vary greatly in resistance efficacy and broadness against a global mildew isolate collection and subsequently use this model system to study the mechanisms underlying broad-spectrum resistance. We demonstrate that two alleles, Pm3d and Pm3e , provide resistance against most isolates worldwide, by each recognizing two AVR genes, thereby lowering the risk of resistance breakdown. Pm3d recognizes two highly similar RNase-like AVRs, encoded by gene paralogs. In contrast, Pm3e detects two structurally diverse AVRs: one shared with Pm3d, the other originating from a large, uncharacterized protein family with a discrete structural fold. Using chimeric Pm3 NLRs, we identify specificity-defining polymorphisms of Pm3d and Pm3e against their diverse effector targets. Lastly, we demonstrate that Pm3d and Pm3e activities can be combined in engineered Pm3 NLRs, thereby further extending their recognition spectrum. Our findings highlight the potential of Pm3 immune receptors for long-lasting wheat protection by demonstrating their versatility in recognizing structurally diverse effectors and their amenability to NLR engineering.

Durable resistance to soil-borne pathogens remains elusive in cereals, partly because susceptibility (S) genes that facilitate root infection have not been identified in monocots. In the model legume Medicago truncatula , the SCAR/WAVE complex member MtAPI functions as a root S-gene for microbial invasion. Whether SCAR gene associated susceptibility function is conserved in monocots, and whether SCAR gene inactivation can enhance root resistance in cereals, remains unknown. Here, we identify and characterize three SCAR genes in barley: HvSCAR-A , HvSCAR-B , and HvSCAR-C . Cross-species complementation assays indicate that HvSCAR-B and HvSCAR-C are functionally similar to MtAPI . While hscar-b and hvscar-c single mutants exhibited no major growth defects, hvscar-a mutants showed strongly reduced seed production, and a hvscar-b/c double mutant displayed shorter root hairs. Notably, the hvscar-b/c double mutant exhibited increased resistance to the hemibiotrophic pathogen Phytophthora palmivora but greater colonization by the symbiotic arbuscular mycorrhizal fungus Funneliformis mosseae , underscoring a complex role in plant root - microbe interactions. Our findings reveal a conserved susceptibility function of SCAR genes in monocots and identify api monocot homologs as promising targets for engineering disease resistance in cereals. This study offers new insights into SCAR protein functional diversification and its potential for improving root health in crop plants.

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.

Broad-spectrum resistance genes are highly valuable for sustainable crop protection, yet the molecular basis of their activity is often unknown. The Pm3 allelic series in wheat encodes NLR receptors that recognize avirulence (AVR) effectors of wheat powdery mildew. Here, we show that near-identical Pm3 alleles vary greatly in resistance efficacy and broadness against a global mildew isolate collection and subsequently use this model system to study the mechanisms underlying broad-spectrum resistance. We demonstrate that two alleles, Pm3d and Pm3e , provide resistance against the majority of isolates worldwide, by each recognizing two AVR effectors from powdery mildew, thereby lowering the risk of resistance breakdown. While Pm3d recognizes two closely related RNase-like AVR effectors, Pm3e detects two structurally diverse AVRs, including an effector belonging to a large, uncharacterized protein family with a novel structural fold. Using chimeric Pm3 NLRs, we identify specificity-defining polymorphisms of Pm3d and Pm3e against their diverse effector targets. Lastly, we demonstrate that Pm3d and Pm3e activities can be combined in engineered Pm3 NLRs, thereby further extending their recognition spectrum. Our findings highlight the potential of Pm3 immune receptors for long-lasting wheat protection by demonstrating their versatility in recognizing structurally diverse effectors and their amenability to NLR engineering.

Proteins with a tandem kinase structure have recently emerged as new players in race-specific resistance in cereal crops. However, the molecular understanding of these novel immune receptors’ resistance mechanisms is limited by the lack of knowledge about the pathogen effectors that they recognise. In this work, we identify AvrWTK4, the wheat powdery mildew RNase-like effector recognised by the wheat tandem kinase immune receptor WTK4, through a combination of bi-parental genetic mapping and mutagenesis. We demonstrate that mutations in the AvrWTK4 gene or a reduction of its expression lead to virulence on WTK4 . Transfection of AvrWTK4 specifically induced cell death in WTK4 -containing Aegilops tauschii protoplasts. The avirulent AvrWTK4 variant interacts more strongly than the virulent variant with the N-terminal heavy metal-associated (HMA)-like domain of WTK4. These findings further highlight that integrated domains in tandem kinase proteins serve as decoys for pathogen effectors, which could be leveraged to design novel recognition specificities.