Structural analysis of Phytophthora suppressor of RNA silencing 2 (PSR2) reveals a conserved modular fold contributing to virulence

Abstract

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the “start” unit, various numbers of L-W-Y motifs as the “middle” units, and a degenerate L-W-Y as the “end” unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a “building block” to accelerate effector evolution in Phytophthora.

Effector prediction and characterization in the oomycete pathogen Bremia lactucae reveal host-recognized WY domain proteins that lack the canonical RXLR motif

“…Both tyrosine and phenylalanine are aromatic amino acids; however, the hydroxyl group on tyrosine makes it slightly bulkier, more polar, and introduces a potential phosphorylation site. Variation in this region is not uncommon in other oomycete species: the fifth of the seven domains in Psr2 of P. sojae has an F instead of a Y and is similar in structure to the single domain of ATR1 in H. arabidopsidis that has a cysteine at the Y position [31]. Further structural characterization is needed to reveal whether these substitutions alter tertiary structure and biological function.…”

“…Structural analysis on four different RXLR effectors from Phytophthora infestans (Avr3a and PexRD2), P. capsici (Avr3a11), and the downy mildew pathogen H. arabidopsidis (ATR1) revealed that the W and Y motifs form a~50 amino acid alpha-helical fold that may play a role in protein-protein interactions [27][28][29][30]. This effector-associated fold, termed the WY domain after its conserved tryptophan and tyrosine residues, is highly structurally conserved between effectors from multiple Peronosporales species, despite sharing less than 20% sequence similarity across the whole domain [27,31]. The WY domain appears to be specific to the Peronosporales and was predicted to be present in nearly half of the RXLR effectors of P. infestans and a quarter of the RXLR effectors in H. arabidopsidis [27].…”

“…WY domain containing proteins appear to interact with a variety of host targets, with Avr3a from P. infestans targeting the E3 ligase CMPG1 [34], P. infestans PexRD54 targeting potato autophagy-related protein ATG8 [36], PsAvh240 from P. sojae targeting an aspartic protease [37], and PSR1 and PSR2 from P. sojae suppressing host RNA silencing through interactions with the RNA helicase PINP1 and the dsRNA-binding protein DRB4, respectively [38][39][40][41]. Mutation analysis of the regions encoding the seven individual WY domains of PSR2 demonstrated differential contributions of each domain to virulence of P. sojae, suggesting that the WY domain may act as a module during effector evolution [31]. In addition to its roles in immune suppression, the WY domain has been shown to be important for immune recognition of the effector by nucleotide binding-leucine rich repeat (NB-LRR) resistance proteins [42].…”

Host-interactor screens ofPhytophthora infestansRXLR proteins reveal vesicle trafficking as a major effector-targeted process

“…The P. infestans genome harbors over 500 predicted RXLR effectors that are grouped into ~150 families and tend to exhibit sequence and expression polymorphisms between pathogen strains (Haas et al, 2009;Cooke et al, 2012;Yoshida et al, 2013;Pais et al, 2018). RXLR effectors are modular proteins with the N-terminal signal peptide and RXLR region mediating secretion and translocation into host cells, and the C-terminal end, often defined by the WY/LWY fold, carrying the effector biochemical activity (Whisson et al, 2007;Win et al, 2012a;He et al, 2019). To date, over a dozen P. infestans RXLR effectors have been functionally characterized for their virulence activities (Rovenich et al, 2014;Anderson et al, 2015;Du et al, 2015;Wang et al, 2015;Boevink et al, 2016;Dagdas et al, 2016;Yang et al, 2016;Turnbull et al, 2017;Wang et al, 2018).…”

RxLR and CRN effectors of the spinach downy mildew Peronospora effusa and their interacting plant proteins

“…The structure may llow the evolution of effectors to maintain their function while evading recognition by the plant immune system [77]. Effectors with WY-folds were thus far implicated in various processes such as RNA silencing suppression (Phytophthora suppressor of RNA silencing 2 (PSR2, P. sojae) and manipulation of vesicular trafficking (PexRD54, P. infestans) [78,79].…”