The structural repertoire ofFusarium oxysporumf. sp.lycopersicieffectors revealed by experimental and computational studies

Abstract

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.

TheVenturia inaequaliseffector repertoire is expressed in waves and is dominated by expanded families with predicted structural similarity to avirulence proteins from other plant-pathogenic fungi

“…Taken together, our study on V. inaequalis , along with previous studies on effector proteins from M. oryzae , F. oxysporum and other fungi [43, 53, 62, 117, 120], reinforce the idea that fungal effectors are often sequence-diverse, but share a limited number of structural folds. The presence of common structural folds in effectors without obvious sequence similarity could be the result of diversifying selection, where the effectors have evolved rapidly to a point where almost all amino acid sequence similarity, with the exception of residues involved in the maintenance of the overall structural fold, is lost [53].…”

“…To gain insights into the putative function of proteinaceous ECs, we predicted their tertiary structures using AlphaFold2 [42], which has been successfully benchmarked against pathogen effectors of known tertiary structure [43, 44], and then investigated these structures for similarity to proteins of characterized tertiary structure (and in some cases, function) using the Dali server [45]. This analysis was specifically performed on the most highly expressed member from each EC family (which we referred to as the main family member) as well as each singleton, expressed during the host infection-specific waves.…”

“…without its predicted N-terminal peptide) was used as input. For the Avr1/Six4-like family, the published pro-domain [43] was also removed. For those ECs that had <30 proteins with amino acid sequence similarity in the NCBI database, as identified by a BLASTp analysis in conjunction with an e-value threshold of ≤0.05, a custom MSA was generated and used as input.…”

“…To gain insights into the putative function of proteinaceous ECs, we predicted their tertiary structures using AlphaFold2 [42], which has been successfully benchmarked against pathogen effectors of known tertiary structure [43,44], and then investigated these structures for similarity to proteins of characterized tertiary structure (and in some cases, function) using the Dali server [45].…”

The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report

“…Several examples indicate that AlphaFold models exhibit r.m.s.d. values of lower than 1.5 A ˚with the final structures (Dowah et al, 2021;Gao et al, 2021;Kuttiyatveetil et al, 2021;Milla ´n et al, 2021;Yin et al, 2021;Yu et al, 2021;Fowler & Williamson, 2022;Paul et al, 2022), even for proteins without related structural templates deposited in the PDB. However, the impact of these high-quality models in structural biology goes far beyond the simple case of molecular replacement.…”