The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Motivation: RNA–protein interactions play essential roles in every living organism, with RNA transcription, processing, and translation being just a few examples. Therefore, determining the set of RNAs that are bound by individual RNA-binding proteins, as well as the precise location of the interaction, is crucial for biological understanding. CLIP (UV-cross-linking and immunoprecipitation) is a method developed to study these interactions. Several variations of the CLIP protocol have been developed, e.g. iCLIP (individual-nucleotide resolution CLIP), which offers nucleotide-precise resolution of the cross-linking event. Results: PARANOiD is a versatile software for fully automated analysis of iCLIP and iCLIP2 data. It contains all steps necessary for preprocessing, the determination of cross-link locations, and several additional steps, which can be used to detect specific characteristics, e.g. definite distances between cross-link events or identify binding motifs. Additionally, results are visualized as statistical plots for a quick overview and as standardized bioinformatics file formats, which can be used for further analysis steps. Availability and implementation: PARANOiD is published under the MIT license and is available from https://github.com/patrick-barth/PARANOiD. The documentation is available at https://paranoid.readthedocs.io/en/latest/index.html.

Summary: RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta‐analysis, we evaluated 89 studies that compare the efficiency of host‐induced gene silencing (HIGS) and spray‐induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly against biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. However, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3′ end and SIGS when targeting genes closer to the 5′ end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking, and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3’ end and SIGS when targeting genes closer to the 5’ end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.