Isolation and characterization of barley (<i>Hordeum vulgare</i>) extracellular vesicles and their role in RNAi-based crop protection

Schlemmer, Timo; Weipert, Lisa; Preußer, Christian; Hardt, Martin; Moebus, Anna; Barth, Patrick; Busche, Tobias; Koch, Aline

doi:10.1101/2021.04.26.441409

Cited by 3 publications

References 21 publications

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Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

Karimi

Baldrich

Rutter

et al. 2021

Preprint

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Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside EVs and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they comprise both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m(6)A). Consistent with this, we identified a putative m(6)A-binding protein in apoplastic wash fluid, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that these extravesicular RNAs mediate host-induced gene silencing, rather than RNA inside EVs.

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Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

Karimi

Baldrich

Rutter

et al. 2021

Preprint

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RNA-Based Control of Fungal Pathogens in Plants

Mann

Sawyer

Gardiner

et al. 2023

IJMS

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Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20–40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

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Beyond Loading: Functions of Plant ARGONAUTE Proteins

Liang,

Wang,

et al. 2023

IJMS

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ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.

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Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles and their role in RNAi-based crop protection

Cited by 3 publications

References 21 publications

Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

RNA-Based Control of Fungal Pathogens in Plants

Beyond Loading: Functions of Plant ARGONAUTE Proteins

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