Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

Abstract

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.

Advances in Understanding Fusarium graminearum: Genes Involved in the Regulation of Sexual Development, Pathogenesis, and Deoxynivalenol Biosynthesis

“…Host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) are emerging biotechnological approaches that use double-stranded RNA (dsRNA) to target essential fungal genes and suppress their expression. Several studies have demonstrated that HIGS and SIGS can effectively reduce FHB symptoms and mycotoxin accumulation by targeting genes involved in fungal growth, virulence, and toxin biosynthesis [159,160]. HIGS and SIGS offer several advantages over other control methods, such as specificity, durability, safety, and compatibility with existing breeding programs.…”

Extracellular RNA: mechanisms of secretion and potential functions

“…Recently, the group of Aline Koch has suggested that plants EVs may only play a minor or indirect role in the delivery and uptake of host‐induced gene silencing (HIGS)- and spray‐induced gene silencing (SIGS)-associated RNAs. They showed that co-cultivation of Fusarium graminearum ( Fg ) with EVs (P100) isolated from CYP3RNA-expressing A. thaliana plants as well as from CYP3RNA-sprayed barley plants did not have any effect on the expression of CYP51 in Fg ( Schlemmer et al , 2022 ). Interestingly, they had previously shown that >70% of HIGS-derived siRNAs in Arabidopsis were found to be extravesicular ( Schlemmer et al , 2021 ).…”

RNA-Based Control of Fungal Pathogens in Plants

“…Recently, EVs isolated from barley (Hordeum vulgare) plants sprayed with a dsRNA construct that targeted the three F. graminearum CYP51 genes for silencing were found to harbor siRNAs derived from the topically applied RNA [119]. However, when these CYP51 siRNA-containing EVs were used to treat F. graminearum infection, no significant inhibition of the degree of infection or target gene silencing was observed [120].…”