The Venturia inaequalis effector repertoire is dominated by expanded families with predicted structural similarity, but unrelated sequence, to avirulence proteins from other plant-pathogenic fungi

Abstract

Background Scab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth by V. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required. Results We generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector from Leptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi. Conclusions Our study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis and provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

Prediction of effector protein structures from fungal phytopathogens enables evolutionary analyses

“…We believe that ancestral origins of many species-specific effector families can be revealed through finer sampling of fungal species. Although MAX effectors were nearly exclusive to M. oryzae in our study, V. inaequalis in Ascomycota was suggested to encode many MAX effector-like proteins 21 . Similarly, Sporisorium reilianum related to U. maydis encodes Tin2-like effectors found exclusively in U. maydis in our study 46 .…”

“…lycopersici could be grouped into a few structural families, including Fol dual-domain (FOLD) effector family 8 . In Venturia inaequalis, MAX effectors represented one of the most expanded families 21 . Such structural analyses have reinforced the divergent evolution hypothesis in that pathogen virulence factors may have evolved through frequent duplications and rapid divergence of common folds.…”

The structural landscape and diversity ofPyricularia oryzaeMAX effectors revisited

“…V. inaequalis is an ascomycete fungus, in the Venturiaceae family, responsible for apple scab disease. Assessing models of V. inaequalis MAX-like effectors [45] against the criteria we defined here to assign MAX effectors to subfamilies, revealed that none of the models fitted into any of the MAX subfamilies. Indeed, V. inaequalis MAX effectors present three remarkable disulfide bonds.…”

“…Moreover, MAX-like effectors of V. inaequalis usually possess a C-terminal helical extension connected to the MAX core domain via a specific disulfide bond that was not observed in any MAX from P. oryzae . This makes these newly discovered MAX-like effectors from V. inaequalis another subfamily with unique sequence/structure features [45]. As V. inaequalis exclusively colonizes and releases effectors in the subcuticular host environment without penetrating the underlying epidermal cells, their function, and thus targets, are probably fundamentally different from the other studied MAX effectors from P. oryzae .…”

Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungusMagnaporthe oryzae

“…Studies focused on the biology of MAX effectors have demonstrated how these MAX effectors target host proteins (Bentham et al, 2021;Maidment et al, 2021;Oikawa et al, 2020), how they are detected by the plant immune system (De la De la Concepcion et al, 2021a;Guo et al, 2018;Maqbool et al, 2015;Ortiz et al, 2017), and how this interaction has shaped the evolution of host immunity (Bialas et al, 2021;De la Concepcion et al, 2021b). These studies have led to the first steps in the development of synthetic intracellular immune receptors with bespoke pathogen recognition (Bentham et al, 2022;Cesari et al, 2022;De la Concepcion et al, 2019;Kourelis et al, 2023;Liu et al, 2021;Maidment et al, 2023), a goal long pursued in plant biotechnology (Cadiou et al, 2023;Rodriguez-Moreno et al, 2017;Zdrzałek et al, 2023). Despite this progress, studies addressing functional characterization and experimental determination of non-MAX effectors from M. oryzae have remained somewhat behind in comparison to MAX effectors.…”