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Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

ABSTRACT: The fungus Zymoseptoria tritici causes Septoria tritici leaf blotch, which poses a serious threat to temperate-grown wheat. Recently, we described a raft of molecular tools to study the biology of this fungus in vitro. Amongst these are 5 conditional promoters (Pnar1, Pex1A, Picl1, Pgal7, PlaraB), which allow controlled over-expression or repression of target genes in cells grown in liquid culture. However, their use in the host-pathogen interaction in planta was not tested. Here, we investigate the behaviour of these promoters by quantitative live cell imaging of green-fluorescent protein-expressing cells during 6 stages of the plant infection process. We show that Pnar1 and Picl1 are repressed in planta and demonstrate their suitability for studying essential gene expression and function in plant colonisation. The promoters Pgal7 and Pex1A are not fully-repressed in planta, but are induced during pycnidiation. This indicates the presence of inducing galactose or xylose and/or arabinose, released from the plant cell wall by the activity of fungal hydrolases. In contrast, the PlaraB promoter, which normally controls expression of an ?-l-arabinofuranosidase B, is strongly induced inside the leaf. This suggests that the fungus is exposed to L-arabinose in the mesophyll apoplast. Taken together, this study establishes 2 repressible promoters (Pnar1 and Picl1) and three inducible promoters (Pgal7, Pex1A, PlaraB) for molecular studies in planta. Moreover, we provide circumstantial evidence for plant cell wall degradation during the biotrophic phase of Z. tritici infection.

SUBMITTER: Fantozzi E

PROVIDER: S-EPMC7812376 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

Fantozzi Elena E Kilaru Sreedhar S Cannon Stuart S Schuster Martin M Gurr Sarah J SJ Steinberg Gero G

Fungal genetics and biology : FG & B 20201209

The fungus Zymoseptoria tritici causes Septoria tritici leaf blotch, which poses a serious threat to temperate-grown wheat. Recently, we described a raft of molecular tools to study the biology of this fungus in vitro. Amongst these are 5 conditional promoters (Pnar1, Pex1A, Picl1, Pgal7, PlaraB), which allow controlled over-expression or repression of target genes in cells grown in liquid culture. However, their use in the host-pathogen interaction in planta was not tested. Here, we investigate ...[more]

PMID: 33309991

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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.

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Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

Publications

Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

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