Fusarium graminearum (Fg) is a necrotrophic fungal pathogen that causes devastating diseases on its crop hosts barley and wheat. Recently, small RNAs (sRNAs) were identified as mobile communication signals between eukaryotes and their pathogens, symbionts or parasites. It has been shown that pathogens secrete sRNAs as effectors to suppress plant immunity and plants use endogenous sRNAs to resist infection, a phenomenon termed cross-kingdom RNAi; ckRNAi. However, little is known about the transport of fungus- or plant produced sRNAs to silence genes that contribute to immunity. Extracellular vesicles (EVs) are predicted playing a key role in the bidirectional transfer of sRNAs that mediate ckRNAi. To address this knowledge gap, we investigated the effects of EVs isolated from barley and Fg on their counterparts during plant-fungal interaction. Towards this, we developed a protocol for the isolation of EVs from Fg liquid cultures and assessed how Fg EVs contribute to fungal pathogenesis in barley using infiltration assays. To test the interdependence of EVs during Barley-Fg interaction, we treated Fg cultures with barley EVs. We found that infiltration of Fg EVs caused host specific phytotoxic effects in barley and barley EVs impaired Fg growth. Of note, Fg cultures showed an increase in purple pigmentation upon inoculation with barley EVs, suggesting a stress-induced premature formation of fruiting bodies. Together, our results demonstrate that EVs contribute to the Barley-Fg interaction, however, further studies are needed to unravel the nature of EV cargoes (e.g. protein and/or sRNA) responsible for affecting its plant/fungus counterpart.

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

Incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto plant leaves successfully protects plants against invading pathogens with RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their interacting pathogens. Recently, we found that EVs isolated from HIGS or SIGS plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from RNA-sprayed barley ( Hordeum vulgare ) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum ( Fg ). Encouraged by our previous finding that dropping barley-derived EVs on Fg cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated Fg with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of Fg macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect Fg growth and showed no gene silencing activity on FgCYP51 genes. We conclude that either the amount of spray-derived sRNA was insufficient to induce target gene silencing (SIGS) in Fg or Fg uptake of plant EVs from liquid cultures was inefficient or impossible.