Abstract
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.
Highlights
The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world
extracellular vesicles (EVs) can be isolated from Z. tritici cultures To determine if Z. tritici secretes EVs under in vitro culture conditions, as described for other fungi, we isolated EVs from Z. tritici broth cultures using a differential ultracentrifugation (DUC) method adapted from Rodrigues et al [59] and Thery et al [60]
The apparent lack of smaller particles represented in the nanoparticle tracking analysis (NTA) size distribution when compared to the transmission electron microscopy (TEM) measurements, may be artefactual of NTA systems’ known underreporting of particles < 50 nm and/or the dehydration and shrinkage caused by negatively staining EVs for TEM [21, 33]
Summary
The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Significant genomic, transcriptomic and functional work has aimed to identify and characterise these Z. tritici pathogenicity factors, with many studies focusing on proteins with canonical N-terminal secretion signals (or signal peptides (SPs)) that are putatively exported from the cell via the ER-Golgi pathway [5, 7,8,9,10,11] Despite these efforts, few secreted effector proteins have been shown to contribute to the disease phenotype, likely partly due to the functional redundancy in effector proteins and the quantitative nature of host resistance/pathogen virulence in the wheat—Z. tritici interaction [5, 12,13,14]. The role of these non-canonical secretory pathways in the Z. tritici—wheat interaction is unexplored
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