Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.

Background: Fungal extracellular vesicles (EVs) have been implicated in host-pathogen and pathogen-pathogen communication in some fungal diseases. In depth research into fungal EVs has been hindered by the lack of specific protein markers such as those found in mammalian EVs that have enabled sophisticated isolation and analysis techniques. Despite their role in fungal EV biogenesis, ESCRT proteins such as Vps23 (Tsg101) and Bro1 (ALIX) are not present as fungal EV cargo. Furthermore, tetraspanin homologs are yet to be identified in many fungi including the model yeast S. cerevisiae. Objective: We performed de novo identification of EV protein markers for the major human fungal pathogen Candida albicans with adherence to MISEV2018 guidelines. Materials and methods: EVs were isolated by differential ultracentrifugation from DAY286, ATCC90028 and ATCC10231 yeast cells, as well as DAY286 biofilms. Whole cell lysates (WCL) were also obtained from the EV-releasing cells. Label-free quantitative proteomics was performed to determine the set of proteins consistently enriched in EVs compared to WCL. Results: 47 proteins were consistently enriched in C. albicans EVs. We refined these to 22 putative C. albicans EV protein markers including the claudin-like Sur7 family (Pfam: PF06687) proteins Sur7 and Evp1 (orf19.6741). A complementary set of 62 EV depleted proteins was selected as potential negative markers. Conclusions: The marker proteins for C. albicans EVs identified in this study will be useful tools for studies on EV biogenesis and cargo loading in C. albicans and potentially other fungal species and will also assist in elucidating the role of EVs in C. albicans pathogenesis. Many of the proteins identified as putative markers are fungal specific proteins indicating that the pathways of EV biogenesis and cargo loading may be specific to fungi, and that assumptions made based on studies in mammalian cells could be misleading. Abbreviations: A1-ATCC10231; A9-ATCC90028; DAY B-DAY286 biofilm; DAY Y-DAY286 yeast; EVextracellular vesicle; Evp1extracellular vesicle protein 1 (orf19.6741); GOgene ontology; Log 2 (FC)log 2 (fold change); MCCmembrane compartment of Can1; MDSmultidimensional scaling; MISEVminimal information for studies of EVs; sEVssmall EVs; SPsignal peptide; TEMstetraspanin enriched microdomains; TMtransmembrane; VDMvesicle-depleted medium; WCLwhole cell lysate ARTICLE HISTORY

Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro . However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays , with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.

Extracellular vesicles (EVs) are nano-sized compartments involved in cell communication and macromolecule transport that are well characterized in mammalian organisms. Fungal EVs transport virulence-related cargo and modulate the host immune response, but most work has been focused on human yeast pathogens. Additionally, the study of EVs from filamentous fungi has been hindered by the lack of protein markers and efficient isolation methods. In this study we performed the isolation and proteomic characterization of EVs from the filamentous cotton pathogen Fusarium oxysporum f. sp. vasinfectum (Fov). EVs were recovered from two different growth media, Czapek Dox and Saboraud's dextrose broth, and purified by size-exclusion chromatography. Our results show that the EV proteome changes depending on the growth medium but EV production remains constant. EVs contained proteins involved in polyketide synthesis, cell wall modifications, proteases and potential effectors. These results support a role in modulation of host-pathogen interactions for Fov EVs.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and β-glucan with NaD1 and other plant defensins. We discovered that the β-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

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Fusarium graminearum (F. graminearum) is a filamentous fungus that infects cereals such as corn, wheat, and barley, with serious impact on yield as well as quality when the grain is contaminated with mycotoxins. Despite the huge impact of F. graminearum on food security and mammalian health, the mechanisms used by F. graminearum to export virulence factors during infection are not fully understood and may involve non-classical secretory pathways. Extracellular vesicles (EVs) are lipid-bound compartments produced by cells of all kingdoms that transport several classes of macromolecules and are implicated in cell–cell communication. EVs produced by human fungal pathogens carry cargo that facilitate infection, leading us to ask whether plant fungal pathogens also deliver molecules that increase virulence via EVs. We examined the metabolome of the EVs produced by F. graminearum to determine whether they carry small molecules that could modulate plant–pathogen interactions. We discovered that EVs from F. graminearum were produced in liquid medium-containing inducers of trichothecene production, but in lower quantities compared to other media. Nanoparticle tracking analysis and cryo-electron microscopy revealed that the EVs were morphologically similar to EVs from other organisms; hence, the EVs were metabolically profiled using LC-ESI-MS/MS. This analysis revealed that EVs carry 2,4-dihydroxybenzophenone (BP-1) and metabolites that have been suggested by others to have a role in host–pathogen interactions. BP-1 reduced the growth of F. graminearum in an in vitro assay, suggesting that F. graminearum might use EVs to limit metabolite self-toxicity.