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In Anbetracht der hohen gesellschaftlichen Forderungen nach nachhaltigen und umweltschonenden Pflanzenschutzverfahren, stellt das Sprühen von RNA-basierten Wirkstoffen eine innovative und vielversprechende Alternative zu konventionellen chemisch-synthetischen Pflanzenschutzmitteln dar. Dabei vermitteln die sequenzspezifischen und somit maßgeschneiderten RNAs nicht nur eine hohe Selektivität, sondern lassen sich zudem sehr schnell adaptieren. Dadurch sind sie, verglichen mit der langwierigen, herkömmlichen Wirkstoff­entwicklung, schneller verfügbar. Trotz der vielfältigen Vorteile und des dringenden Bedarfs an Alternativen, stehen wir erst am Anfang des Transfers von RNA Sprays ins Freiland. Hier diskutieren wir die damit verknüpften wissenschaftlich-technischen, gesellschaftlichen und wirtschaftlichen Herausforderungen. Zudem zeigen wir die offenen Forschungsfragen auf, die es zu adressieren gilt, um RNA-basierte Pflanzenschutzmittel zeitnah zu etablieren. Zuletzt führen wir aktuelle Beispiele zu innovativen Lösungsansätzen aus der Forschung an, die die Weiterentwicklung von RNA Sprays für die Kontrolle von Schadinsekten in der Pflanzenproduktion zum Ziel haben. Teile dieses Artikels wurden bereits in einem breiter angelegten englischsprachigen Übersichtsartikel (Rank & Koch 2021) dargestellt und werden hier in einen detaillierteren Kontext eingebettet. Durch die Übersetzung ins Deutsche sollen sie zudem einem erweiterten Leserkreis zugänglich gemacht werden., Journal für Kulturpflanzen, Bd. 74 Nr. 03-04 (2022): Zukünftige Wege des Pflanzenschutzes gegen Schadinsekten

Additional file 6: Figure S6. The relative fungal growth in the co-culture was calculated using the EV-free cultivation conditions with enzymes and detergent only as a baseline. Control (wt): circle; CYP3RNA-expressing plants: triangle (a-f). Selected timepoints were chosen for statistical analysis. Differences between wt or CYP3RNA-expressing A. thaliana plants were calculated for each investigated volume and EV pre-treatment using a two-tailed Student’s t-test (p-value < 0.05). 5 µl EVs of wt plants: light green; 5 µl EVs of CYP3RNA-expressing A. thaliana plants: dark green; 10 µl EVs of wt plants: light brown; 10 µl EVs of CYP3RNA-expressing A. thaliana plants: dark brown (g).

Additional file 4: Figure S4. 5 µl (light blue cross) and 10 µl (gray triangle) of purified EVs from control (wt) and CYP3RNA-expressing A. thaliana plants were added to F. graminearum liquid culture. Growth was determined by optical density measurements between 23 and 42 h post-inoculation (hpi) for cultures treated with EVs out of all three fractions.

Additional file 2: Figure S2. EVs were mixed with PBS and PDB and quick frozen at the beginning [0 h (h)], 24 h and 48 h after incubation at 25 °C. Samples were thawed on ice and diluted with PBS (1:20). The final volume of 200 µl was then loaded into the Nanosight NS300 (Malvern Panalytical) and five measurements were performed at room temperature. Mean values are arranged as a black line, standard deviation is given as the red plot.

Additional file 3: Figure S3. EVs were mixed with supernatant of 24-h-old F. graminearum culture and incubated at 25 °C. Particle concentration was determined by NTA measurements.

Additional file 5: Figure S5. Purified A. thaliana EVs were treated with RNase A and proteinase K (yellow square) or Triton X-100, RNase A, and Proteinase K (green rhombus) after isolation and co-inoculated with F. graminearum. Additionally, untreated (orange circle) and EV-free PBS (brown cross) were co-inoculated as positive and negative controls.

Small (s)RNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through expression in transgenic plants and targeted gene silencing (host-induced gene silencing, HIGS). While HIGS strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unknown. Here we show that extracellular vesicles (EVs) purified fromArabidopsis thalianaplants expressing CYP3RNA, a dsRNA originally designed to target the threeCYP51genes of the fungal pathogenFusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. These transgene specific siRNAs had a length of 21 and 22 nucleotides with a bias towards 5’-uracil (U) and 5’-adenine (A). Notably, stringent protease and RNase treated EV fractions contained >70% less CYP3RNA-derived siRNAs, suggesting the presence of co-purified extravesicular nucleoprotein complexes stabilizing siRNAs outside of EVs. In addition, mutants of the ESCRT-III complex showed a loss of HIGS-mediated disease resistance and EVs isolated from these mutants were free of CYP3RNA-derived siRNAs. Together, these findings support the view that endosomal vesicle trafficking is required for HIGS mediating the transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells probably in an EV-independent manner.

In filamentous fungi, gene silencing through RNA interference (RNAi) shapes many biological processes, including pathogenicity. We explored the requirement of key components of fungal RNAi machineries, including DICER-like 1 and 2 (FgDCL1, FgDCL2), ARGONAUTE 1 and 2 (FgAGO1, FgAGO2), AGO-interacting protein FgQIP (QDE2-interacting protein), RecQ helicase (FgQDE3), and four RNA-dependent RNA polymerases (FgRdRP1, FgRdRP2, FgRdRP3, FgRdRP4), in the ascomycete mycotoxin-producing fungal pathogen Fusarium graminearum (Fg) for sexual and asexual multiplication, pathogenicity, and its sensitivity to double-stranded (ds)RNA. We corroborate and extend earlier findings that conidiation, ascosporogenesis, and Fusarium head blight (FHB) symptom development require an operable RNAi machinery. The involvement of RNAi in conidiation is dependent on environmental conditions as it is detectable only under low light (

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley—Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14a-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.