The structural repertoire of Fusarium oxysporum f. sp. lycopersici effectors revealed by experimental and computational studies.

Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. 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, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant-fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens.

A rust-fungus Nudix hydrolase effector decaps mRNA in vitro and interferes with plant immune pathways.

To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.

Molecular plant immunity against biotrophic, hemibiotrophic, and necrotrophic fungi.

Pathogenic fungi use diverse infection strategies to obtain nutrients from plants. Biotrophic fungi feed only on living plant tissue, whereas necrotrophic fungi kill host cells to extract nutrients. To prevent disease, plants need to distinguish between pathogens with different life cycles, as a successful defense against a biotroph, which often involves programmed cell-death around the site of infection, is not an appropriate response to some necrotrophs. Plants utilize a vast collection of extracellular and intracellular receptors to detect the signatures of pathogen attack. In turn, pathogens are under strong selection to mask or avoid certain receptor responses while enhancing or manipulating other receptor responses to promote virulence. In this review, we focus on the plant receptors involved in resistance responses to fungal pathogens and highlight, with examples, how the infection strategy of fungal pathogens can determine if recognition responses are effective at preventing disease.