Remarkable recent changes in the genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici.
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SUBMITTER: Stephens C
PROVIDER: S-EPMC8358995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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: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:Zymoseptoria tritici is a globally distributed fungal pathogen which causes Septoria tritici blotch on wheat. Management of the disease is attempted through the deployment of resistant wheat cultivars and the application of fungicides. However, fungicide resistance is commonly observed in Z. tritici populations, and continuous monitoring is required to detect breakdowns in fungicide efficacy. We recently reported azole-resistant isolates in Australia; however, it remained unknown whether resistance was brought into the continent through gene flow or whether resistance emerged independently. To address this question, we screened 43 isolates across five Australian locations for azole sensitivity and performed whole-genome sequencing on 58 isolates from seven locations to determine the genetic basis of resistance. Population genomic analyses showed extremely strong differentiation between the Australian population recovered after azoles began to be used and both Australian populations recovered before azoles began to be used and populations on different continents. The apparent absence of recent gene flow between Australia and other continents suggests that azole fungicide resistance has evolved de novo and subsequently spread within Tasmania. Despite the isolates being distinct at the whole-genome level, we observed combinations of nonsynonymous substitutions at the CYP51 locus identical to those observed elsewhere in the world. We observed nine previously reported nonsynonymous mutations as well as isolates that carried a combination of the previously reported L50S, S188N, A379G, I381V, Y459DEL, G460DEL, and N513K substitutions. Assays for the 50% effective concentration against a subset of isolates exposed to the tebuconazole and epoxiconazole fungicides showed high levels of azole resistance. The rapid, parallel evolution of a complex CYP51 haplotype that matches a dominant European haplotype demonstrates the enormous potential for de novo resistance emergence in pathogenic fungi.IMPORTANCE Fungicides are essential to control diseases in agriculture because many crops are highly susceptible to pathogens. However, many pathogens rapidly evolve resistance to fungicides. A large body of studies have described specific mutations conferring resistance and have often made inferences about the origins of resistance based on sequencing data from the target gene alone. Here, we show the de novo acquisition of resistance to the ubiquitously used azole fungicides in genetically isolated populations of the wheat pathogen Zymoseptoria tritici in Tasmania, Australia. We confirm evidence for parallel evolution through genome-scale analyses of representative worldwide populations. The emergence of complex resistance haplotypes following a well-documented recent introduction of azoles into Australian farming practices demonstrates how rapidly chemical resistance evolves in agricultural ecosystems.
| S-EPMC6365823 | biostudies-literature
Project description: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.
| S-EPMC6637991 | 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: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:BackgroundLaccases are multicopper oxidases, which are assigned into auxiliary activity family 1 (AA1) in the CAZy database. These enzymes, catalyzing the oxidation of phenolic and nonphenolic substrates coupled to reduction of O2 to H2O, are increasingly attractive as eco-friendly oxidation biocatalysts. Basidiomycota laccases are well characterized due to their potential in de-lignification of lignocellulose. By contrast, insight into the biochemical diversity of Ascomycota counterparts from saprophytes and plant pathogens is scarce.ResultsHere, we report the properties of the laccase from the major wheat pathogen Zymoseptoria tritici (ZtrLac1A), distinguished from common plant fungal pathogens by an apoplastic infection strategy. We demonstrate that ZtrLac1A is appended to a functional starch-binding module and displays an activity signature disfavoring relatively apolar phenolic redox mediators as compared to the related biochemically characterized laccases. By contrast, the redox potential of ZtrLac1A (370 mV vs. SHE) is similar to ascomycetes counterparts. The atypical specificity is consistent with distinctive sequence substitutions and insertions in loops flanking the T1 site and the enzyme C-terminus compared to characterized laccases.ConclusionsZtrLac1A is the first reported modular laccase appended to a functional starch-specific carbohydrate binding module of family 20 (CBM20). The distinct specificity profile of ZtrLac1A correlates to structural differences in the active site region compared to previously described ascomycetes homologues. These differences are also highlighted by the clustering of the sequence of ZtrLac1A in a distinct clade populated predominantly by plant pathogens in the phylogenetic tree of AA1 laccases. The possible role of these laccases in vivo merits further investigations. These findings expand our toolbox of laccases for green oxidation and highlight the binding functionality of CBM-appended laccases as versatile immobilization tags.
| S-EPMC6700816 | biostudies-literature
Project description:BackgroundThe ascomycete fungus Zymoseptoria tritici (synonyms: Mycosphaerella graminicola, Septoria tritici) is a major pathogen of wheat that causes the economically important foliar disease Septoria tritici blotch. Despite its importance as a pathogen, little is known about the reaction of this fungus to light. To test for light responses, cultures of Z. tritici were grown in vitro for 16-h days under white, blue or red light, and their transcriptomes were compared with each other and to those obtained from control cultures grown in darkness.ResultsThere were major differences in gene expression with over 3400 genes upregulated in one or more of the light conditions compared to dark, and from 1909 to 2573 genes specifically upregulated in the dark compared to the individual light treatments. Differences between light treatments were lower, ranging from only 79 differentially expressed genes in the red versus blue comparison to 585 between white light and red. Many of the differentially expressed genes had no functional annotations. For those that did, analysis of the Gene Ontology (GO) terms showed that those related to metabolism were enriched in all three light treatments, while those related to growth and communication were more prevalent in the dark. Interestingly, genes for effectors that have been shown previously to be involved in pathogenicity also were upregulated in one or more of the light treatments, suggesting a possible role of light for infection.ConclusionsThis analysis shows that Z. tritici can sense and respond to light with a huge effect on transcript abundance. High proportions of differentially expressed genes with no functional annotations illuminates the huge gap in our understanding of light responses in this fungus. Differential expression of genes for effectors indicates that light could be important for pathogenicity; unknown effectors may show a similar pattern of transcription. A better understanding of the effects of light on pathogenicity and other biological processes of Z. tritici could help to manage Septoria tritici blotch in the future.
| S-EPMC7382159 | biostudies-literature
Project description:The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential. IMPORTANCE Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici, have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides' efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici. The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici.
| S-EPMC5656749 | biostudies-literature
Project description:Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a fungal wheat pathogen that causes significant global yield losses. Within Z. tritici populations, quantitative differences in virulence among different isolates are commonly observed; however, the genetic components that underpin these differences remain elusive. In this study, intraspecific comparative transcriptomic analysis was used to identify candidate genes that contribute to differences in virulence on the wheat cultivar WW2449. This led to the identification of a multicopy gene that was not expressed in the high-virulence isolate when compared to the medium- and low-virulence isolates. Further investigation suggested this gene resides in a 7.9-kb transposon. Subsequent long-read sequencing of the isolates used in the transcriptomic analysis confirmed that this gene did reside in an active Class II transposon, which is composed of four genes named REP9-1 to -4. Silencing and overexpression of REP9-1 in two distinct genetic backgrounds demonstrated that its expression alone reduces the number of pycnidia produced by Z. tritici during infection. The REP9-1 gene identified within a Class II transposon is the first discovery of a gene in a transposable element that influences the virulence of Z. tritici. This discovery adds further complexity to genetic loci that contribute to quantitative virulence in this important pathogen.
| S-EPMC8232023 | biostudies-literature