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In silico prediction and characterization of secondary metabolite biosynthetic gene clusters in the wheat pathogen Zymoseptoria tritici.

ABSTRACT: BACKGROUND:Fungal pathogens of plants produce diverse repertoires of secondary metabolites, which have functions ranging from iron acquisition, defense against immune perturbation, to toxic assaults on the host. The wheat pathogen Zymoseptoria tritici causes Septoria tritici blotch, a foliar disease which is a significant threat to global food security. Currently, there is limited knowledge of the secondary metabolite arsenal produced by Z. tritici, which significantly restricts mechanistic understanding of infection. In this study, we analyzed the genome of Z. tritici isolate IP0323 to identify putative secondary metabolite biosynthetic gene clusters, and used comparative genomics to predict their encoded products. RESULTS:We identified 32 putative secondary metabolite clusters. These were physically enriched at subtelomeric regions, which may facilitate diversification of cognate products by rapid gene rearrangement or mutations. Comparative genomics revealed a four gene cluster with significant similarity to the ferrichrome-A biosynthetic locus of the maize pathogen Ustilago maydis, suggesting this siderophore is deployed by Z. tritici to acquire iron. The Z. tritici genome also contains several isoprenoid biosynthetic gene clusters, including one with high similarity to a carotenoid/opsin producing locus in several fungi. Furthermore, we identify putative phytotoxin biosynthetic clusters, suggesting Z. tritici can produce an epipolythiodioxopiperazine, and a polyketide and non-ribosomal peptide with predicted structural similarities to fumonisin and the Alternaria alternata AM-toxin, respectively. Interrogation of an existing transcriptional dataset suggests stage specific deployment of numerous predicted loci during infection, indicating an important role of these secondary metabolites in Z. tritici disease. CONCLUSIONS:We were able to assign putative biosynthetic products to numerous clusters based on conservation amongst other fungi. However, analysis of the majority of secondary metabolite loci did not enable prediction of a cluster product, and consequently the capacity of these loci to play as yet undetermined roles in disease or other stages of the Z. tritici lifecycle is significant. These data will drive future experimentation for determining the role of these clusters and cognate secondary metabolite products in Z. tritici virulence, and may lead to discovery of novel bioactive molecules.

SUBMITTER: Cairns T

PROVIDER: S-EPMC5561558 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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In silico prediction and characterization of secondary metabolite biosynthetic gene clusters in the wheat pathogen Zymoseptoria tritici.

Cairns Timothy T Meyer Vera V

BMC genomics 20170817 1

<h4>Background</h4>Fungal pathogens of plants produce diverse repertoires of secondary metabolites, which have functions ranging from iron acquisition, defense against immune perturbation, to toxic assaults on the host. The wheat pathogen Zymoseptoria tritici causes Septoria tritici blotch, a foliar disease which is a significant threat to global food security. Currently, there is limited knowledge of the secondary metabolite arsenal produced by Z. tritici, which significantly restricts mechanis ...[more]

PMID: 28818040

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Project description:Background:The 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. Results:Zymoseptoria 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. Conclusion:We 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.

Project description:BACKGROUND:The 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. RESULTS:There 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. CONCLUSIONS:This 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.

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.

Dataset Information

In silico prediction and characterization of secondary metabolite biosynthetic gene clusters in the wheat pathogen Zymoseptoria tritici.

Publications

In silico prediction and characterization of secondary metabolite biosynthetic gene clusters in the wheat pathogen Zymoseptoria tritici.

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