The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro‐domain protease processing of fungal effectors

Abstract

Summary Plant pathogens cause disease through secreted effector proteins, which act to promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilized a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat‐lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination and functional studies. We show this approach can be successfully applied to study effectors from other pathogenic fungi. The β‐barrel fold of SnTox3 is a novel fold among fungal effectors. Structure‐guided mutagenesis enabled the identification of residues required for Snn3 recognition. SnTox3 is a pre‐pro‐protein, and the pro‐domain of SnTox3 can be cleaved in vitro by the protease Kex2. Complementing this, an in silico study uncovered the prevalence of a conserved motif (LxxR) in an expanded set of putative pro‐domain‐containing fungal effectors, some of which can be cleaved by Kex2 in vitro. Our in vitro and in silico study suggests that Kex2‐processed pro‐domain (designated here as K2PP) effectors are common in fungi and this may have broad implications for the approaches used to study their functions.

pOPIN-GG: A resource for modular assembly in protein expression vectors

“…The different pOPIN-GG acceptors are crosscompatible for co-expression, allowing for ease of co-expression and purification of protein complexes. Finally, the utility of the pOPIN-GG vectors presented here have already been successfully tested by the community for the expression and purification of a fungal effector from Parastagonospora nodorum (Outram et al, 2021), as well as for detailing the structural mechanisms underpinning the evolution of Magnaporthe oryzae effectors for a specific host target (Bentham et al, 2021).…”

Simple and efficient heterologous expression of necrosis‐inducing effectors using the model plant Nicotiana benthamiana

“…Therefore, this lack of a visible phenotype precludes this coexpression approach from being used to study these interactions (as it can be with many biotrophic effectors). Consequently, as an alternative approach, NEs are often expressed using heterologous systems for characterization and subsequent phenotyping on host plants (Liu et al, 2009(Liu et al, , 2012Outram et al, 2021;Sung et al, 2021;Zhang et al, 2017). Almost exclusively, NEs have been expressed by using microbial expression systems such as Escherichia coli and Pichia pastoris.…”

“…It has been demonstrated that the cysteines in many of these effectors form disulfide bonds providing stability for these proteins in the apoplast (Saunders et al, 2012). For instance, the Tox3 and Tox1 effectors from P. nodorum contain six and 16 cysteines respectively, and at least one disulfide bond is required for their necrosis-inducing activity (Liu et al, 2009(Liu et al, , 2012Outram et al, 2021). Similarly, Cladosporium fulvum effector Avr2 has four disulfide bonds that are essential for its function of inhibiting cysteine protease Rcr3 in tomato plants (Rooney et al, 2005).…”

“…Similarly, Cladosporium fulvum effector Avr2 has four disulfide bonds that are essential for its function of inhibiting cysteine protease Rcr3 in tomato plants (Rooney et al, 2005). Moreover, some effectors such as ToxA and Tox3 appear to contain pro-domains subsequent to their secretion signals that are thought to be important for folding and are processed prior to secretion from the fungi (Liu et al, 2009;Outram et al, 2021;Tuori et al, 2000). Recently, Outram et al (2021) reported that the prodomain of Tox3 was cleaved by KEX2 protease that lead to a processed protein with demonstrably higher activity indicating that some NEs require posttranslational modification and processing to become fully active.…”

“…Moreover, some effectors such as ToxA and Tox3 appear to contain pro-domains subsequent to their secretion signals that are thought to be important for folding and are processed prior to secretion from the fungi (Liu et al, 2009;Outram et al, 2021;Tuori et al, 2000). Recently, Outram et al (2021) reported that the prodomain of Tox3 was cleaved by KEX2 protease that lead to a processed protein with demonstrably higher activity indicating that some NEs require posttranslational modification and processing to become fully active. However, because of these requirements, such proteins are difficult to express using a bacterial system due to the absence of eukaryotic cell organelles necessary for disulfide bond formation and posttranslational modifications.…”