Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Abstract

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 – 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.

Size‐exclusion chromatography allows the isolation of EVs from the filamentous fungal plant pathogen Fusarium oxysporum f. sp. vasinfectum (Fov)

“…These domains include LysM [66], NPP1 [67], and AltA‐1 [68]. Our results support the recent discovery of potential effectors in EVs from the wheat pathogen Zymoseptoria tritici [29]. The presence of effectors with and without signal peptides inside fungal EVs could offer answers to unknown infection mechanisms where fungi rely on unconventional secretion to colonize the host.…”

“…Of the 48 potential markers identified in C. albicans , 11 were detected in both CD and SDB EVs. One of the main candidates is Sur7 [18], and the presence of this protein was recently confirmed in EVs from C. neoformans and Z. tritici [29, 73], but its homologue was not detected in Fov EVs. This may be explained by the lack of homology between the C. albicans and Fov versions of Sur7, which only share 32% identity.…”

“…Aspergillus fumigatus produces EVs that can alter immune responses from the host [26] and Aspergillus flavus EVs induce M1 polarization of macrophages and may favor fungal clearance in larval models [27]. The only proteomic characterizations of EVs from plant pathogens are from Alternaria infectoria [28], Zymoseptoria tritici [29] and a previous Fov analysis [30]. EVs from A. infectoria contain proteins involved in cell wall remodeling, synthesis of polyketides and metabolism [28], while Z. tritici EVs transport the putative fungal EV marker Sur7 [16, 29].…”

“…The only proteomic characterizations of EVs from plant pathogens are from Alternaria infectoria [28], Zymoseptoria tritici [29] and a previous Fov analysis [30]. EVs from A. infectoria contain proteins involved in cell wall remodeling, synthesis of polyketides and metabolism [28], while Z. tritici EVs transport the putative fungal EV marker Sur7 [16, 29]. Fov EVs contained substantially more proteins than EVs from other filamentous fungi [30].…”

RevisitingCryptococcusextracellular vesicles properties and their use as vaccine platforms

“…Sur7 proteins have been recently identified as EV-protein markers in C. albicans and in the wheat pathogen Zymoseptoria tritici (Dawson et al 2020, Hill and Solomon 2020). This latter hypothesis, together with whether or not the smaller undecorated EVs are a result of the endosomal secretory pathways, thought to be exosomes being released by multivesicular bodies (MVB), still needs to be further explored.…”