Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici.
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ABSTRACT:
SUBMITTER: Mirzadi Gohari A
PROVIDER: S-EPMC6638687 | biostudies-literature | 2014 May
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC6638447 | biostudies-literature
Project description:The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.
| S-EPMC5724701 | biostudies-literature
Project description:BackgroundThe fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)-secreted, membrane-bound nanoparticles containing molecular cargo-being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.ResultsZymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs.ConclusionWe have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.
| S-EPMC7501697 | biostudies-literature
Project description:BACKGROUND:The gene content of a species largely governs its ecological interactions and adaptive potential. A species is therefore defined by both core genes shared between all individuals and accessory genes segregating presence-absence variation. There is growing evidence that eukaryotes, similar to bacteria, show intra-specific variability in gene content. However, it remains largely unknown how functionally relevant such a pangenome structure is for eukaryotes and what mechanisms underlie the emergence of highly polymorphic genome structures. RESULTS:Here, we establish a reference-quality pangenome of a fungal pathogen of wheat based on 19 complete genomes from isolates sampled across six continents. Zymoseptoria tritici causes substantial worldwide losses to wheat production due to rapidly evolved tolerance to fungicides and evasion of host resistance. We performed transcriptome-assisted annotations of each genome to construct a global pangenome. Major chromosomal rearrangements are segregating within the species and underlie extensive gene presence-absence variation. Conserved orthogroups account for only ~?60% of the species pangenome. Investigating gene functions, we find that the accessory genome is enriched for pathogenesis-related functions and encodes genes involved in metabolite production, host tissue degradation and manipulation of the immune system. De novo transposon annotation of the 19 complete genomes shows that the highly diverse chromosomal structure is tightly associated with transposable element content. Furthermore, transposable element expansions likely underlie recent genome expansions within the species. CONCLUSIONS:Taken together, our work establishes a highly complex eukaryotic pangenome providing an unprecedented toolbox to study how pangenome structure impacts crop-pathogen interactions.
| S-EPMC7014611 | biostudies-literature
Project description:Osmotic stress is a ubiquitous and potent stress for all living organisms, but few studies have investigated the genetic basis of salt tolerance in filamentous fungi. The main aim of this study was to identify regions of the genome associated with tolerance to potassium chloride (KCl) in the wheat pathogen Zymoseptoria tritici. A secondary aim was to identify candidate genes affecting salt tolerance within the most promising chromosomal regions. We achieved these aims with a quantitative trait locus (QTL) mapping study using offspring from 2 crosses grown in vitro in the presence or absence of osmotic stress imposed by 0.75 M KCl. We identified significant QTL for most of the traits in both crosses. Several QTLs overlapped with QTL identified in earlier studies for other traits, and some QTL explained trait variation in both the control and salt stress environments. A significant QTL on chromosome 3 explained variation in colony radius at 8-day postinoculation (dpi) in the KCl environment as well as colony radius KCl tolerance at 8 dpi. The QTL peak had a high logarithm of the odds ratio (LOD) and encompassed an interval containing only 36 genes. Six of these genes present promising candidates for functional analyses. A gene ontology (GO) enrichment analysis of QTL unique to the KCl environment found evidence for the enrichment of functions involved in osmotic stress responses.
| S-EPMC10700024 | biostudies-literature
Project description:In host-pathogen interactions RNA interference (RNAi) has emerged as a pivotal mechanism to modify both, the immune responses of the host as well as the pathogenicity and virulence of the pathogen. In addition, in some fungi RNAi is also known to affect chromosome biology via its effect on chromatin conformation. Previous studies reported no effect of the RNAi machinery on the virulence of the fungal plant pathogen Zymoseptoria tritici however the role of RNAi is still poorly understood in this species. Herein, we elucidate whether the RNAi machinery is conserved within the genus Zymoseptoria. Moreover, we conduct functional analyses of Argonaute and Dicer-like proteins and test if the RNAi machinery affects chromosome stability. We show that the RNAi machinery is conserved among closely related Zymoseptoria species while an exceptional pattern of allelic diversity was possibly caused by introgression. The deletion of Ago1 reduced the ability of the fungus to produce asexual propagules in planta in a quantitative matter. Chromosome stability of the accessory chromosome of Z. tritici was not prominently affected by the RNAi machinery. These results indicate, in contrast to previous finding, a role of the RNAi pathway during host infection, but not in the stability of accessory chromosomes in Z. tritici.
| S-EPMC8303167 | biostudies-literature
Project description:Development of novel strategies to control fungal plant pathogens requires understanding of their cellular organisation and biology. Live cell imaging of fluorescent organelle markers has provided valuable insight into various aspects of their cell biology, including invasion strategies in plant pathogenic fungi. Here, we introduce a set of 17 vectors that encode fluorescent markers to visualize the plasma membrane, endoplasmic reticulum (ER), chromosomes, the actin cytoskeleton, peroxisomes and autophagosomes in the wheat pathogen Zymoseptoria tritici. We fused either enhanced green-fluorescent protein (eGFP) or a codon-optimised version of GFP (ZtGFP) to homologues of a plasma membrane-located Sso1-like syntaxin, an ER signalling and retention peptide, a histone H1 homologue, the LifeAct actin-binding peptide, a mitochondrial acetyl-CoA dehydrogenase, a peroxisomal import signal and a homologue of the ubiquitin-like autophagosomal protein Atg8. We expressed these markers in wildtype strain IPO323 and confirmed the specificity of these markers by counterstaining or physiological experiments. This new set of molecular tools will help understanding the cell biology of the wheat pathogen Z. tritici.
| S-EPMC5536155 | biostudies-literature
Project description:The fungal wheat pathogen Zymoseptoria tritici possesses a large complement of accessory chromosomes showing presence/absence polymorphism among isolates. These chromosomes encode hundreds of genes; however, their functional role and why the chromosomes have been maintained over long evolutionary times are so far not known. In this study, we addressed the functional relevance of eight accessory chromosomes in reference isolate IPO323. We induced chromosome losses by inhibiting the ?-tubulin assembly during mitosis using carbendazim and generated several independent isogenic strains, each lacking one of the accessory chromosomes. We confirmed chromosome losses by electrophoretic karyotyping and whole-genome sequencing. To assess the importance of the individual chromosomes during host infection, we performed in planta assays comparing disease development results in wild-type and chromosome mutant strains. Loss of the accessory chromosomes 14, 16, 18, 19, and 21 resulted in increased virulence on wheat cultivar Runal but not on cultivars Obelisk, Titlis, and Riband. Moreover, some accessory chromosomes affected the switch from biotrophy to necrotrophy as strains lacking accessory chromosomes 14, 18, 19, and 21 showed a significantly earlier onset of necrosis than the wild type on the Runal cultivar. In general, we observed that the timing of the lifestyle switch affects the fitness of Z. tritici Taking the results together, this study was the first to use a forward-genetics approach to demonstrate a cultivar-dependent functional relevance of the accessory chromosomes of Z. tritici during host infection.IMPORTANCEZymoseptoria tritici is a prominent fungal pathogen of wheat of worldwide distribution. This fungus shows a remarkable genome organization, with a large number of chromosomes that are present in only some isolates and therefore considered to be "accessory" chromosomes. To date, the function of these accessory chromosomes in Z. tritici has been unknown, although their maintenance in the species over evolutionary times suggests a functional relevance. Here we deleted whole accessory chromosomes to test the effect of these chromosomes on host specificity and virulence of the fungus. We show for the first time that some accessory chromosomes of Z. tritici affect the fitness of the fungus during host infection in a cultivar-dependent manner. These results show that the accessory chromosomes encode host-specific virulence determinants having a negative effect on fitness. Understanding the population dynamic of the accessory chromosomes and the molecular interaction of pathogen and plant traits is crucial to improve wheat-breeding strategies.
| S-EPMC5705923 | biostudies-literature
Project description:Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non-host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II)2 Cys6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non-host infections and that lower expression corresponds to an incompatible infection of non-hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection.
| S-EPMC6913241 | biostudies-literature
Project description:BACKGROUND:Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence. RESULTS:We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes. CONCLUSIONS:The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.
| S-EPMC5765654 | biostudies-literature