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

Abstract

AbstractPlant 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.