The crystal structure of SnTox3 from the necrotrophic fungusParastagonospora nodorumreveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

Abstract

SummaryPlant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised 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. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi.The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici.We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence.

PR1‐mediated defence via C‐terminal peptide release is targeted by a fungal pathogen effector

“…SnTox3 was cloned, expressed and purified as described previously (Zhang et al ., 2017; Outram et al ., 2020). SnTox3 P173S was cloned from synthetic DNA (IDT gBlock), expressed and purified as described for SnTox3 (Outram et al ., 2020).…”

“…SnTox3 was cloned, expressed and purified as described previously (Zhang et al ., 2017; Outram et al ., 2020). SnTox3 P173S was cloned from synthetic DNA (IDT gBlock), expressed and purified as described for SnTox3 (Outram et al ., 2020). Five TaPR1 genes (TaPR1‐1, ‐5, ‐7, ‐16 and ‐18, codon‐optimized for S. cerevisiae ), lacking their N‐terminal signal peptides as determined by SignalP (Almagro Armenteros et al ., 2019), and TaPR1‐7ΔCAPE1 (TaPR1‐7 without the last 11 amino acids) were cloned into the expression vector pMCSG7 using ligation‐independent cloning (LIC) (Eschenfeldt et al ., 2009; Supporting Information Table S1).…”

“…The SnTox3 effector protein is a key determinant in the pathogen’s arsenal and contributes strongly to yield losses. Protein infiltration experiments with SnTox3 have shown that it can rapidly induce cell death and necrosis in the presence of the host susceptibility gene Snn3 (Liu et al ., 2009; Outram et al ., 2020). More recently, it has also been shown to directly interact with multiple members of the wheat pathogenesis‐related protein 1 (TaPR1) family, implying a potential role in manipulating host defence (Breen et al ., 2016).…”

TheParastagonospora nodorumnecrotrophic effector SnTox5 targets the wheat geneSnn5and facilitates entry into the leaf mesophyll

“…BLASTp search with the SnTox5 amino acid sequence showed that SnTox3 was the only hit in the NCBI non-redundant database and only showed 45% homology. Although this is not a high level of homology, it is interesting that SnTox3 is also a pre-pro protein with a Kex2 cleavage site (Liu et al 2009; Outram et al 2020) and structural homology is also observed based on similar placement of the cysteine residues, indicating a potentially similar mature protein structure. The Kex2 protease is unique to fungi and Outram et al (2020) showed that Kex2-processed pro-domain (K2PP) effectors were common in pathogenic fungi and included the P. nodorum effectors SnTox3 and SnToxA and now SnTox5.…”

“…The Kex2 protease is unique to fungi and Outram et al (2020) showed that Kex2-processed pro-domain (K2PP) effectors were common in pathogenic fungi and included the P. nodorum effectors SnTox3 and SnToxA and now SnTox5. Outram et al (2020) also presented the crystal structure of SnTox3 and experimentally demonstrated that the Kex2-processed pro-domain was critical for SnTox3 folding and activity. The validation of SnTox5 provides another K2PP effector interaction for further study of this class of effectors.…”