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

Reviewer #1 (Public Review):

Yu et al. investigated Fusarium oxysporum f. sp. lycopersici SIX effectors structure using experimental and computational approaches, and while doing so, the authors identified several SIX effectors as member of the FOLD family, and expanded the known diversity of the SIX effectors. A very interesting and novel finding is the presence of FOLD putative effectors in other Ascomycetes secretome, sharing structural similarities with SIX effectors Avr1, Avr3 and SIX6.

By performing technically sound predictions and experimental confirmation, the authors also confirmed co-operative interactions between Fol effectors, something that was previously known for different pairs of proteins, expanding this observation for new SIX effectors. In addition, the authors contributed to the understanding of the interaction Fol effectors, specifically FOLD and LARS effectors, - I receptors to suppress immunity by structurally similar effectors.

The conclusions of this paper are supported by data and I think it is a pioneer study analyzing the correspondence between AlphaFold predictions and experimentally derived structures, highlighting that models can answer the scientific questions in some cases but could not be enough in others.

Reviewer #2 (Public Review):

Yu et al. investigated the structural landscape of 'secreted in xylem' (SIX) effector (virulence and avirulence) proteins from the plant-pathogenic fungus, Fusarium oxysporum f. sp. lycopersici (Fol), with the goal of better understanding effector function and recognition by host (tomato) immune receptors. In recent years, several experimental and computational studies have shown that many effector proteins of plant-associated fungi can be assigned to one of a few major structural families. In the study by Yu et al., X-ray crystallography was used to show that two avirulence effectors of Fol, Avr1 (SIX4) and Avr3 (SIX1), which are recognized by the tomato immune receptors I and I-3, respectively, form part of a new structural family, the Fol dual-domain (FOLD) family, found across three fungal divisions. Using AlphaFold2, an ab initio structural prediction tool, the authors then predicted the structures of all proteins within the Fol SIX effector repertoire (and other effector candidates) and provided evidence that two other effectors, SIX6 and SIX13, also belong to this family.

In addition to identifying members of the FOLD family, structural prediction revealed that proteins of the Fol effector repertoire can largely be classified into a reduced set of structural families. Examples included four members of the ToxA-like family (including Avr2 (SIX3) and SIX8), as well as four members of a new family, Family 4 (including SIX5 and PSL1). Given previous studies had demonstrated that Avr2 (ToxA-like) and SIX5 (Family 4) interact and function together and that the genes encoding these proteins are divergently transcribed, and because homologues of SIX8 (ToxA-like) and PSL1 (Family 4) from another Fusarium pathogen are functionally dependent on each other and, in the case of Fol, are encoded by genes that are next to each other in the genome, the authors hypothesized that SIX8 and PSL1 may also physically interact. In line with this, co-incubation of the SIX8 and PSL1 proteins, followed by size exclusion chromatography (SEC), gave elution and gel migration profiles consistent with interaction in the form of a heterodimer. AlphaFold2-Multimer modelling then suggested that this interaction was mediated through an intermolecular disulfide bond. Such a prediction was subsequently confirmed through mutational analysis of the relevant cysteine residue in each protein in conjunction with SEC.

Finally, using a variant (homologue) of Avr1 from another Fusarium pathogen, as well as chimeric forms of this protein that integrated regions of Avr1 from Fol, Yu et al. determined through co-expression assays in Nicotiana benthamiana with the I immune receptor, as well as subsequent ion leakage assays, that the C-domain of Avr1 is recognized by the I immune receptor. Furthermore, through these assays, the authors were also able to show that surface-exposed residues in the C-domain enable Avr1 to evade recognition by a variant of the I receptor in Moneymaker tomato that does not provide resistance to Fol.

Overall, the manuscript presents a large body of work that is well supported by the data. A key strength of the manuscript is the validation (benchmarking) of protein structures predicted using AlphaFold2, which is a first for largescale effector structure prediction papers published to date. Another key strength is the use of largescale effector structure predictions to make hypotheses about functional relationships or interactions that are then tested (i.e. the SIX8-PSL1 protein interaction and recognition of Avr1 by the I immune receptor). This testing again goes above and beyond the large scale effector structure prediction papers published to date. Taken together, this showcases how experimental and computational experiments can be effectively combined to provide biologically relevant data for the plant protection and molecular plant-microbe interactions fields.

In terms of weaknesses, the manuscript could have validated the SIX8-PSL1 protein interaction with in planta experiments, such as co-immunoprecipitation assays or co-localization experiments in conjunction with confocal microscopy, to provide support for the interaction in a plant setting. However, given what is already known about the Avr2-SIX5 interaction, these additional experiments are not crucial and could instead form part of a follow-up study. With regards to the Agrobacterium tumefaciens-mediated transient expression assays involving co-expression of the Avr1 effector and I immune receptor, the authors need to make clear how many biological replicates were performed as this information is only provided for the ion leakage assay.

Reviewer #3 (Public Review):

In this work, the authors shed light onto the structures of Fusarium oxysporum f.sp. lycopersici proteins involved in the infection of tomato. They unravelled several new secreted effector protein structures and additionally used computational approaches to structurally classify the remaining effectors known from this pathogen. Through this they uncovered a new and unique structural class of proteins which they found to be present and widely distributed in fungal plant pathogens and plant symbiotic fungi. The authors further predicted structural models for the full SIX effector set revealing that genome-proximal effector pairs share similar structural classes. Building on their Avr1 structure, the authors also define the C-terminal domain and specific amino acid residues that are essential to Avr1 detection by its cognate immune receptor.

A major strength of this work is a portfolio of several (Avr1, Avr3, SIX6, SIX8) new structurally resolved proteins which led to the discovery that several of them fall into the same structural class. These findings are supported by strong results.

The experiments addressing the structure-function relationship of Avr1's avirulence activity are highly relevant to our understanding of disease resistance mechanisms against Fusarium, but will require additional controls to allow for solid conclusions to be drawn. In particular, it needs to be demonstrated that specific I gene alleles are at all required for FonSIX4's cell death activity in N.benthamiana leaves or whether FonSIX4 and those of some chimeric proteins is independent of the tomato I receptor. Complementary work in Fusarium mutants lacking Avr1 and expressing chimeric versions would document that the observations from transient expressions in Nicotiana benthamiana are relevant in the biological context of a Fusarium/tomato interaction.

The discovered solvent-exposed residues conditioning Avr1 recognition by the I receptor seem to be positioned in an area of the protein which had previously been highlighted as being highly variable in FOLD proteins of symbiotic fungi but it is not clear from the work whether this is indeed the case or whether Avr1 differs significantly in its structure from FOLD proteins found in other fungi.
It also remains to be addressed whether the residues conditioning avirulence activity is also crucial for virulence activity in Fusarium?

This work uncovered a new structural class of proteins with critical roles in plant-pathogen interactions. Structure-based predictions and genome-wide comparisons have emerged as a new approach enabling the identification of similar proteins with divergent sequences. The work undertaken by the authors adds to a growing body of work in plant-microbe research, predominantly from pathogenic organisms, and more recently in symbiotic fungi.