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Summary The effector SnTox3 from Parastagonospora nodorum elicits a strong necrotic response in susceptible wheat and also interacts with wheat pathogenesis‐related protein 1 (TaPR‐1), although the function of this interaction in disease is unclear. Here, we dissect TaPR1 function by studying SnTox3–TaPR1 interaction and demonstrate the dual functionality of SnTox3. We utilized site‐directed mutagenesis to identify an SnTox3 variant, SnTox3P173S, that was unable to interact with TaPR1 in yeast‐two‐hybrid assays. Additionally, using recombinant proteins we established a novel protein‐mediated phenotyping assay allowing functional studies to be undertaken in wheat. Wheat leaves infiltrated with TaPR1 proteins showed significantly less disease compared to control leaves, correlating with a strong increase in defence gene expression. This activity was dependent on release of the TaCAPE1 peptide embedded within TaPR1 by an unidentified serine protease. The priming activity of TaPR1 was compromised by SnTox3 but not the noninteracting variant SnTox3P173S, and we demonstrate that SnTox3 prevents TaCAPE1 release from TaPR1 in vitro. SnTox3 independently functions to induce necrosis through recognition by Snn3 and also suppresses host defence through a direct interaction with TaPR1 proteins. Importantly, this study also advances our understanding of the role of PR1 proteins in host–microbe interactions as inducers of host defence signalling.

Plant pathogens secrete proteins, known as effectors, that function in the apoplast and inside plant cells to promote virulence. Effectors can also be detected by cell-surface and cytosolic receptors, resulting in the activation of defence pathways and plant immunity. Our understanding of fungal effector function and detection by immunity receptors is limited largely due to high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. Recent studies have demonstrated that fungal effectors can be grouped into structural classes despite significant sequence variation. Using protein x-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class. Using AlphaFold ab initio protein structure prediction, benchmarked against the experimentally determined structures, we demonstrate SIX6 and SIX13 are FOLD effectors. We show that the conserved N-domain of Avr1 and Avr3 is sufficient for recognition by their corresponding, but structurally-distinct, immunity receptors. Additional structural prediction and comparison indicate that 11 of the 14 SIX effectors group into four structural families. This revealed that genetically linked effectors are related structurally, and we provide direct evidence for a physical association between one divergently-transcribed effector pair. Collectively, these data indicate that Fol secretes groups of structurally-related molecules during plant infection, an observation that has broad implications for our understanding of pathogen virulence and the engineering of plant immunity receptors.

Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment.Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence.We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties.This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations.