Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Chitin‐binding lysin motif (LysM) effectors contribute to the virulence of various plant‐pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil‐borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage‐specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage‐specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin‐induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage‐specific LysM effector of strain VdLs17 contributes to virulence in planta.     

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up‐regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici–wheat pathosystem.     

Two stripe rust effectors impair wheat resistance by suppressing import of host Fe–S protein into chloroplasts

Several effectors from phytopathogens usually target various cell organelles to interfere with plant defenses, and they generally contain sequences that direct their translocation into organelles, such as chloroplasts. In this study, we characterized a different mechanism for effectors to attack chloroplasts in wheat (Triticum aestivum). Two effectors from Puccinia striiformis f. sp. tritici (Pst), Pst_4, and Pst_5, inhibit Bax-mediated cell death and plant immune responses, such as callose deposition and reactive oxygen species (ROS) accumulation. Gene silencing of the two effectors induced significant resistance to Pst, demonstrating that both effectors function as virulence factors of Pst. Although these two effectors have low sequence similarities and lack chloroplast transit peptides, they both interact with TaISP (wheat cytochrome b6–f complex iron–sulfur subunit, a chloroplast protein encoded by nuclear gene) in the cytoplasm. Silencing of TaISP impaired wheat resistance to avirulent Pst and resulted in less accumulation of ROS. Heterogeneous expression of TaISP enhanced chloroplast-derived ROS accumulation in Nicotiana benthamiana. Co-localization in N. benthamiana and western blot assay of TaISP content in wheat chloroplasts show that both effectors suppressed TaISP from entering chloroplasts. We conclude that these biotrophic fungal effectors suppress plant defenses by disrupting the sorting of chloroplast protein, thereby limiting host ROS accumulation and promoting fungal pathogenicity.

Despite the lack of chloroplast transit peptide, rust effectors affect chloroplast-mediated defenses by suppressing import of host Fe–S protein to chloroplast to promote pathogenicity of stripe rust.     

A Cu-only superoxide dismutase from stripe rust fungi functions as a virulence factor deployed for counter defense against host-derived oxidative stress

Plants quickly accumulate reactive oxygen species (ROS) to resist against pathogen invasion, while pathogens strive to escape host immune surveillance by degrading ROS. However, the nature of the strategies that fungal pathogens adopt to counteract host-derived oxidative stress is manifold and requires deep investigation. In this study, a superoxide dismutase (SOD) from Puccinia striiformis f. sp. tritici (Pst) PsSOD2 with a signal peptide (SP) and the glycophosphatidyl inositol (GPI) anchor, strongly induced during infection, was analysed for its biological characteristics and potential role in wheat–Pst interactions. The results showed that PsSOD2 encodes a Cu-only SOD and responded to ROS treatment. Heterologous complementation assays in Saccharomyces cerevisiae suggest that the SP of PsSOD2 is functional for its secretion. Transient expression in Nicotiana benthamiana leaves revealed that PsSOD2 is localized to the plasma membrane. In addition, knockdown of PsSOD2 by host-induced gene silencing reduced Pst virulence and resulted in restricted hyphal development and increased ROS accumulation. In contrast, heterologous transient assays of PsSOD2 suppressed flg22-elicited ROS production. Taken together, our data indicate that PsSOD2, as a virulence factor, was induced and localized to the plasma membrane where it may function to scavenge host-derived ROS for promoting fungal infection.     

Tracking costs of virulence in natural populations of the wheat pathogen, Puccinia striiformis f.sp. tritici

Background  

Costs of adaptation play an important role in host-parasite coevolution. For parasites, evolving the ability to circumvent host resistance may trade off with subsequent growth or transmission. Such costs of virulence (sensu plant pathology) limit the spread of all-infectious genotypes and thus facilitate the maintenance of genetic polymorphism in both host and parasite. We investigated costs of three virulence factors in Puccinia striiformis f.sp. tritici, a fungal pathogen of wheat (Triticum aestivum).     

Remarkable recent changes in the genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 haplotypes present in earlier Z. tritici isolate collections, with up to c.18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of haplotypes allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted resequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world in 2013–2017. However, in contrast to the data from previous AvrStb6 population studies, we report a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of haplotypes, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence haplotypes tend to be eliminated in subsequent populations.     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

Identification of New Sources of Resistance to Septoria Tritici Blotch Caused by Zymoseptoria tritici

Twenty‐nine synthetic hexaploid wheats (SHWs) were evaluated for resistance to five isolates of Zymoseptoria tritici, a devastating wheat pathogen worldwide. The five Z. tritici isolates varied in their virulence spectra towards wheat genotypes, indicating that they have distinct set of avirulence genes. New isolate‐specific resistances were identified that could be used in wheat breeding programmes. Comparing with the previous studies, the number of specific resistances identified in this study is considerable. Among 150 interactions, 78 isolate‐specific resistances were identified. Interestingly, 21 wheat genotypes showed specific responses to one or more isolates tested. Of these, 12 genotypes were highly resistant to all isolates, indicating that they possess known or novel effective resistance genes. The Stb15 and Stb16/Stb17 are effective resistance genes towards isolates used in this study, indicating that the conferred resistance in these genotypes is due to the presence of either of these genes in combination or individually. Alternatively, they may carry novel broad‐spectrum resistance gene(s) that their identification is of interest. Our data suggest that the presence of complete resistance to various Z. tritici isolates in SHWs justifies the need for more in‐depth research to characterize the likely novel genes.     

Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host–pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z. tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild‐type phenotypes. In planta expression analyses showed that ZtWor1 is up‐regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z. tritici wild‐type strain and the ZtWor1 mutant, which was particularly expressed in planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the β‐subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock‐out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.     

The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species

During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum –tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology-based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.     

Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.     

Evolutionary analyses of the avirulence effector AvrStb6 in global populations of Zymoseptoria tritici identify candidate amino acids involved in recognition

We analysed the population genetic diversity of AvrStb6, the first avirulence gene cloned from the wheat pathogen Zymoseptoria tritici, using 142 Z. tritici strains sampled from four wheat fields growing on three continents. Although AvrStb6 was located in a recombination hotspot, it was found in every strain, with 71 polymorphic sites that produced 41 distinct DNA haplotypes encoding 30 AvrStb6 protein isoforms. An AvrStb6 homologue was found in the closest known relative, Z. pseudotritici, but not in three other closely related Zymoseptoria species, indicating that this gene has emerged in Zymoseptoria quite recently. Two AvrStb6 homologues with nucleotide similarities greater than 70% were identified on chromosome 10 in all Z. tritici isolates, suggesting that AvrStb6 belongs to a multigene family of candidate effectors that has expanded recently through gene duplication. The AvrStb6 sequences exhibited strong evidence for non‐neutral evolution, including a large number of non‐synonymous mutations, with significant positive diversifying selection operating on nine of the 82 codons. It appears that balancing selection is operating across the entire gene in natural field populations. There was also evidence for co‐evolving codons within the gene that may reflect compensatory mutations associated with the evasion of recognition by Stb6. Intragenic recombination also appears to have affected the diversity of AvrStb6.     

The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine-rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity-associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern-triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine-proline-alanine-arginine-serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full-length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.