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

Abstract

BackgroundScab, 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. However, in the presence of corresponding host resistance proteins, these effectors are recognized as avirulence determinants to activate plant defences. To develop durable control strategies against scab, a better understanding of the molecular mechanisms underpinning subcuticular growth by V. inaequalis is required. Likewise, more information is needed on the role that effectors play in activating, suppressing or circumventing resistance protein-mediated defences.ResultsWe generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five in planta gene expression clusters or waves corresponding to three specific infection stages: early, mid and mid-late infection. Early infection was characterized by genes encoding plant cell wall-degrading enzymes (PCWDEs) and proteins associated with oxidative stress responses. Mid infection was characterized by genes encoding transporter proteins. Finally, mid-late infection was characterized by genes encoding PCWDEs and effector candidates (ECs), with most ECs belonging to expanded protein families. To gain insights into function, AlphaFold2 was used to predict the tertiary structures of proteinaceous ECs. Strikingly, 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 structural effector families. In addition, several other ECs, including an EC family with amino acid similarity to the AvrLm6 effector from Leptosphaeria maculans, were predicted to adopt a KP6/ferredoxin-like fold. Thus, proteins with a KP6/ferredoxin-like fold may represent yet another structural family of effectors shared among plant-pathogenic fungi.ConclusionsOur study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis, and reinforces the idea that fungal effectors share a limited number of structural folds. Importantly, our study also provides an enriched list of V. inaequalis ECs that can be investigated for roles in virulence and avirulence, and raises the possibility that apple resistance proteins can be engineered to recognize EC family folds or host components targeted by multiple EC family members.

Comparative computational structural genomics highlights divergent evolution of fungal effectors

“…We believe that ancestral origins of many nearly or entirely 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 the order Pleosporales of Ascomycota encodes many MAX effectors (Rocafort et al, 2022). Similarly, Sporisorium reilianum related to U. maydis encodes Tin2-like effectors found to be exclusive in U. maydis in our study (Tanaka et al, 2019).…”

“…lycopersici could be grouped into a few structural families, including Fol dual-domain (FOLD) effector family (Yu et al, 2021). In Venturia inaequalis, MAX effectors represent one of the most expanded families (Rocafort et al, 2022). Such structural analyses have reinforced the divergent evolution hypothesis in that pathogen virulence factors may have evolved through frequent duplications and functional diversification of ancient folds.…”

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

“…oryzae effectors and 25% of the cloned AVR effectors, and most of them are expressed during early biotrophic stages of rice infection. Such an expansion has also been observed in Venturia inaequalis , where a family of 75 in planta -upregulated MAX effectors has been identified [ 37 ]. De Guillen et al [ 32 ] hypothesized that, in the case of Magnaporthe , the expansion of the MAX family occurred in a common ancestor of M .…”

“…Three V . inaequalis effectors were also recently predicted to belong to the FOLD family [ 37 ]. Finally, ToxA, which was first described in P .…”

“…lycopersici and V . inaequalis [ 33 , 34 , 37 – 39 ]. The scenario observed for the LARS, RALPH, MAX, FOLD and ToxA-like examples suggests that a wide variety of effectors, without any apparent sequence relationship, could in fact constitute a limited set of structurally conserved effector families and that they have expanded in some fungal lineages or even in several fungal classes.…”