Comparative computational structural genomics highlights divergent evolution of fungal effectors

Abstract

Elucidating the evolution of pathogen effector molecules is critical to understand infection mechanisms of fungal phytopathogens and ensure crop security. However, rapid diversification that diminishes sequence similarities between homologous effectors has largely concealed the roots of effector evolution. We predicted the folds of 26,653 secreted proteins from 21 species with AlphaFold and performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded effector families and common folds present across the fungal species. Extreme expansion of nearly lineage-specific effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified additional lineage-specific effector families that include known (a)virulence factors, such as AvrSr35, AvrSr50 and Tin2. These families represented new classes of sequence-unrelated structurally similar effectors. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Sub-and neo-functionalized structurally similar effectors reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds that have been conserved across fungi. Collectively, our study highlights diverse mechanisms of effector evolution and supports divergent evolution of effectors that leads to emergence of pathogen virulence from ancestral proteins.

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)

“…Such sequence diversification is anticipated to have roles in, for example, the diversification of effector functions, as well as the evasion of host immunity. In some cases, this diversification can result in an almost complete loss of sequence similarity between family members, with similarity only retained at the protein tertiary structure level, as has recently been shown for several effector families of plant-pathogenic fungi using the groundbreaking ab initio protein structure prediction tool, AlphaFold2 (Rocafort et al, 2022;Seong & Krasileva, 2022). However, in many cases, sequence identity between family members is maintained, even across different fungal species.…”

Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae

“…The complexity of the spatio-temporal regulation of effector gene expression– in which cytoplasmic effectors localise specifically at the BIC during invasive hyphal growth –suggests that sophisticated transcriptional regulation must be present to ensure that effectors are produced and deployed at the correct time and in the correct cells. But even though effectors have been studied intensively in M. oryzae , leading to new insight into their host targets and structure-function relationships (38, 39), very little is known about effector gene regulation. In contrast to bacterial pathogens, where effector gene regulation is well understood and associated with co-ordinate operon-based gene control (40), there have been very few regulators of fungal effector expression identified to date.…”

Doomed by popularity: The broad use of the Pm8 resistance gene in wheat resulted in hypermutation of the AvrPm8 gene in the powdery mildew pathogen

“…tritici and Blumeria graminis f. sp. hordei (barley powdery mildew) revealed that a high proportion of effector genes encode for small, secreted proteins with a predicted RNAse-like structure (Pedersen et al ., 2012; Menardo et al ., 2017; Seong & Krasileva, 2022). Several RNAse-like effectors have furthermore been implicated in virulence processes of Blumeria (Zhang et al ., 2012; Pliego et al ., 2013; Ahmed et al ., 2015; Ahmed et al ., 2016; Pennington et al ., 2019).…”