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.

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 ß-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.

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

The effector SnTox3 from Parastagonospora nodorum elicits a strong necrotic response in susceptible wheat and also interacts with wheat pathogenesis-related protein 1 (TaPR-1), although the function of this interaction in disease is unclear. Here, we dissect TaPR1 function by studying SnTox3-TaPR1 interaction and demonstrate the dual functionality of SnTox3. We utilized site-directed mutagenesis to identify an SnTox3 variant, SnTox3P173S , that was unable to interact with TaPR1 in yeast-two-hybrid assays. Additionally, using recombinant proteins we established a novel protein-mediated phenotyping assay allowing functional studies to be undertaken in wheat. Wheat leaves infiltrated with TaPR1 proteins showed significantly less disease compared to control leaves, correlating with a strong increase in defence gene expression. This activity was dependent on release of the TaCAPE1 peptide embedded within TaPR1 by an unidentified serine protease. The priming activity of TaPR1 was compromised by SnTox3 but not the noninteracting variant SnTox3P173S , and we demonstrate that SnTox3 prevents TaCAPE1 release from TaPR1 in vitro. SnTox3 independently functions to induce necrosis through recognition by Snn3 and also suppresses host defence through a direct interaction with TaPR1 proteins. Importantly, this study also advances our understanding of the role of PR1 proteins in host-microbe interactions as inducers of host defence signalling.

Parastagonospora nodorum and Related Species in Western Canada: Genetic Variability and Effector Genes.

Parastagonospora nodorum is an important fungal pathogen that causes Septoria nodorum blotch (SNB) in wheat. This pathogen produces several necrotrophic effectors that act as virulence factors; three have been cloned, SnToxA, SnTox1, and SnTox3. In this study, P. nodorum and its sister species P. avenaria f. tritici (Pat1) were isolated from wheat node and grain samples collected from distanced sites in western Canada during 2018. The presence of effector genes and associated haplotypes were determined by PCR and sequence analysis. An internal transcribed spacer-restriction fragment length polymorphism test was developed to distinguish between leaf spotting pathogens (P. nodorum, Pat1, Pyrenophora tritici-repentis, and Bipolaris sorokiniana). P. nodorum was mainly recovered from wheat nodes and to a lesser extent from the grains, while Pat1 was exclusively isolated from grain samples. The effector genes were present in almost all P. nodorum isolates, with the ToxA haplotype 5 (H5) being most prevalent, while a novel ToxA haplotype (denoted here H21) is reported for the first time. In Pat1, only combinations of SnTox1 and SnTox3 genes were present. A ToxA haplotype network was also constructed to assess the evolutionary relationship among globally found haplotypes to date. Finally, cultivars representing wheat development in Canada for the last century were tested for sensitivity to Sn-effectors and to the presence of Tsn1, the ToxA sensitivity gene. Of tested cultivars, 32.9 and 56.9% were sensitive to SnTox1 and SnTox3, respectively, and Tsn1 was present in 59% of the cultivars. In conclusion, P. nodorum and Pat1 were prevalent wheat pathogens in Canada with a potential tissue-specific colonization capacity, while producing necrotrophic effectors to which wheat is sensitive.

Differential effector gene expression underpins epistasis in a plant fungal disease.

Fungal effector-host sensitivity gene interactions play a key role in determining the outcome of septoria nodorum blotch disease (SNB) caused by Parastagonospora nodorum on wheat. The pathosystem is complex and mediated by interaction of multiple fungal necrotrophic effector-host sensitivity gene systems. Three effector sensitivity gene systems are well characterized in this pathosystem; SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. We tested a wheat mapping population that segregated for Snn1 and Snn3 with SN15, an aggressive P. nodorum isolate that produces SnToxA, SnTox1 and SnTox3, to study the inheritance of sensitivity to SnTox1 and SnTox3 and disease susceptibility. Interval quantitative trait locus (QTL) mapping showed that the SnTox1-Snn1 interaction was paramount in SNB development on both seedlings and adult plants. No effect of the SnTox3-Snn3 interaction was observed under SN15 infection. The SnTox3-Snn3 interaction was however, detected in a strain of SN15 in which SnTox1 had been deleted (tox1-6). Gene expression analysis indicates increased SnTox3 expression in tox1-6 compared with SN15. This indicates that the failure to detect the SnTox3-Snn3 interaction in SN15 is due - at least in part - to suppressed expression of SnTox3 mediated by SnTox1. Furthermore, infection of the mapping population with a strain deleted in SnToxA, SnTox1 and SnTox3 (toxa13) unmasked a significant SNB QTL on 2DS where the SnTox2 effector sensitivity gene, Snn2, is located. This QTL was not observed in SN15 and tox1-6 infections and thus suggesting that SnToxA and/or SnTox3 were epistatic. Additional QTLs responding to SNB and effectors sensitivity were detected on 2AS1 and 3AL.

Ethylene-Cytokinin Interaction Determines Early Defense Response of Wheat against Stagonospora nodorum Berk.

Ethylene, salicylic acid (SA), and jasmonic acid are the key phytohormones involved in plant immunity, and other plant hormones have been demonstrated to interact with them. The classic phytohormone cytokinins are important participants of plant defense signaling. Crosstalk between ethylene and cytokinins has not been sufficiently studied as an aspect of plant immunity and is addressed in the present research. We compared expression of the genes responsible for hormonal metabolism and signaling in wheat cultivars differing in resistance to Stagonospora nodorum in response to their infection with fungal isolates, whose virulence depends on the presence of the necrotrophic effector SnTox3. Furthermore, we studied the action of the exogenous cytokinins, ethephon (2-chloroethylphosphonic acid, ethylene-releasing agent) and 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) on infected plants. Wheat susceptibility was shown to develop due to suppression of reactive oxygen species production and decreased content of active cytokinins brought about by SnTox3-mediated activation of the ethylene signaling pathway. SnTox3 decreased cytokinin content most quickly by its activated glucosylation in an ethylene-dependent manner and, furthermore, by oxidative degradation and inhibition of biosynthesis in ethylene-dependent and ethylene-independent manners. Exogenous zeatin application enhanced wheat resistance against S. nodorum through inhibition of the ethylene signaling pathway and upregulation of SA-dependent genes. Thus, ethylene inhibited triggering of SA-dependent resistance mechanism, at least in part, by suppression of the cytokinin signaling pathway.

Reactive Oxygen Species in Host Plant Are Required for an Early Defense Response against Attack of Stagonospora nodorum Berk. Necrotrophic Effectors SnTox.

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of the Stagonospora nodorum Berk. are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox-status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation at the early stages of infection has not been studied. We studied the early stage of infection of various wheat genotypes with S nodorum isolates -Sn4VD, SnB, and Sn9MN, carrying a different set of NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, SnTox3 significantly contributed to cause disease, and the virulence of the isolates depended on their differential expression in plants (Triticum aestivum L.). The Tsn1-SnToxA, Snn1-SnTox1and Snn3-SnTox3 interactions played an important role in inhibition ROS production at the initial stage of infection. The Snn3-SnTox3 inhibited ROS production in wheat by affecting NADPH-oxidases, peroxidases, superoxide dismutase and catalase. The Tsn1-SnToxA inhibited ROS production in wheat by affecting peroxidases and catalase. The Snn1-SnTox1 inhibited the production of ROS in wheat by mainly affecting a peroxidase. Collectively, these results show that the inverse gene-for gene interactions between effector of pathogen and product of host sensitivity gene suppress the host's own PAMP-triggered immunity pathway, resulting in NE-triggered susceptibility (NETS). These results are fundamentally changing our understanding of the development of this economical important wheat disease.

Assessing European Wheat Sensitivities to Parastagonospora nodorum Necrotrophic Effectors and Fine-Mapping the Snn3-B1 Locus Conferring Sensitivity to the Effector SnTox3.

Parastagonospora nodorum is a necrotrophic fungal pathogen of wheat (Triticum aestivum L.), one of the world's most important crops. P. nodorum mediates host cell death using proteinaceous necrotrophic effectors, presumably liberating nutrients that allow the infection process to continue. The identification of pathogen effectors has allowed host genetic resistance mechanisms to be separated into their constituent parts. In P. nodorum, three proteinaceous effectors have been cloned: SnToxA, SnTox1, and SnTox3. Here, we survey sensitivity to all three effectors in a panel of 480 European wheat varieties, and fine-map the wheat SnTox3 sensitivity locus Snn3-B1 using genome-wide association scans (GWAS) and an eight-founder wheat multi-parent advanced generation inter-cross (MAGIC) population. Using a Bonferroni corrected P ≤ 0.05 significance threshold, GWAS identified 10 significant markers defining a single locus, Snn3-B1, located on the short arm of chromosome 5B explaining 32% of the phenotypic variation [peak single nucleotide polymorphisms (SNPs), Excalibur_c47452_183 and GENE-3324_338, -log10P = 20.44]. Single marker analysis of SnTox3 sensitivity in the MAGIC population located Snn3-B1 via five significant SNPs, defining a 6.2-kb region that included the two peak SNPs identified in the association mapping panel. Accordingly, SNP Excalibur_c47452_183 was converted to the KASP genotyping system, and validated by screening a subset of 95 wheat varieties, providing a valuable resource for marker assisted breeding and for further genetic investigation. In addition, composite interval mapping in the MAGIC population identified six minor SnTox3 sensitivity quantitative trait loci, on chromosomes 2A (QTox3.niab-2A.1, P-value = 9.17-7), 2B (QTox3.niab-2B.1, P = 0.018), 3B (QTox3.niab-3B.1, P = 48.51-4), 4D (QTox3.niab-4D.1, P = 0.028), 6A (QTox3.niab-6A.1, P = 8.51-4), and 7B (QTox3.niab-7B.1, P = 0.020), each accounting for between 3.1 and 6.0 % of the phenotypic variance. Collectively, the outcomes of this study provides breeders with knowledge and resources regarding the sensitivity of European wheat germplasm to P. nodorum effectors, as well as simple diagnostic markers for determining allelic state at Snn3-B1.

A functionally conserved Zn2 Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat.

The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.