Artificial nanovesicles for dsRNA delivery in spray‐induced gene silencing for crop protection

Abstract

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre-and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.

Spray-induced gene silencing (SIGS) as a tool for the management of Pine Pitch Canker forest disease

“…This study shows promising results for SIGS. However, the stability and duration of the silencing are limited due to RNA instability/degradation under environmental conditions (Hoang et al, 2022; Niño-Sánchez et al, 2022; Qiao et al, 2023). Re-applying dsRNAs regularly would be feasible in the field and might help to overcome the issues related to the loss of efficacy over time.…”

“…Although we demonstrated that recurring dsRNAs applications improve the efficiency and durability of SIGS, we did not achieve complete disease protection or control. The use of various types of nanocarriers such as artificial vesicles (Qiao et al, 2023), clay nanoparticles (BioClay TM ) (Mitter et al, 2017; Niño-Sánchez et al, 2022), or carbon-based (Wang et al, 2023) among others, can enhance RNA stability and uptake efficiency by the pathogens. Furthermore, it has also been shown to increase the protection window against fungal pathogens, viruses and insects (Jain et al, 2022; Mitter et al, 2017; Mosa and Youssef, 2021).…”

Engineered dsRNA-protein nanoparticles for effective long-distance transport, delivery and gene silencing in plants

“…However, the silencing effects of these complexes are somewhat limited to the local areas of application (Jiang et al ., 2014; Zhang et al ., 2019; Demirer et al ., 2020; Zhang et al ., 2021; Zhang et al ., 2022). Also, some nanoparticles such as LDH nanosheets and artificial nanovesicles have been shown to facilitate the long-distance transport of dsRNA molecules (Li et al ., 2015; Mitter et al ., 2017; Qiao et al ., 2023). However, it is unknown whether these dsRNA-nanocomplexes can penetrate plant cell membrane, as these studies have primarily focused on investigating their impact on gene expression in insects, fungi, or viruses that infected the experimental plants (Li et al ., 2015; Mitter et al ., 2017; Qiao et al ., 2023).…”

“…Also, some nanoparticles such as LDH nanosheets and artificial nanovesicles have been shown to facilitate the long-distance transport of dsRNA molecules (Li et al ., 2015; Mitter et al ., 2017; Qiao et al ., 2023). However, it is unknown whether these dsRNA-nanocomplexes can penetrate plant cell membrane, as these studies have primarily focused on investigating their impact on gene expression in insects, fungi, or viruses that infected the experimental plants (Li et al ., 2015; Mitter et al ., 2017; Qiao et al ., 2023). As shown here, our cBSA/dsRNA nanocomplexes may present a highly useful and more convenient tool that not only enables the delivery of RNA molecules into plant cells but also facilitates their long-distance transport within the plant and systemic silencing effects of target genes.…”

“…While there have been reports on the long-distance transport of nanoparticle-RNA in plants, these studies document the silencing effects on genes in fungal pathogens, insect pests, or viruses that infect plants, rather than on endogenous plant genes. (Li et al ., 2015; Mitter et al ., 2017; Qiao et al ., 2023). Therefore, achieving long-distance transport of nanoparticle-RNA complexes is highly desirable, as it would enable systemic gene silencing effects to be obtained, even when double-stranded RNA molecules are applied locally.…”