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Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called 'cross-kingdom RNAi'. It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and annexins (ANNs), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not to non-EV-associated sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11 rh37 and ann1 ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RNA-binding proteins play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11 rh37 and ann1 ann2 showed increased susceptibility to Botrytis cinerea, suggesting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.

Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease is critical for global food security; however, most current disease management approaches rely on chemical fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important process through which RNA molecules target and silence complementary genes, regulating gene expression during both transcription and translation. Recently, it has been discovered that some species of fungi can efficiently take up RNAs originating from their host plant and the environment. If these RNAs are complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed "cross-kingdom RNAi," if the RNA originated from a plant host, or "environmental RNAi," if the RNA originated from the environment. These discoveries have inspired the development of spray-induced gene silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of SIGS is largely dependent on the ability of fungi to take up environmental RNAs. Here, we describe the protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy for that fungus.

Regulatory small RNAs are well known as antiviral agents, regulators of gene expression, and defenders of genome integrity in plants. Several studies over the last decade have also shown that some small RNAs are exchanged between plants and their pathogens and parasites. Naturally occurring trans -species small RNAs are used by host plants to silence mRNAs in pathogens. These gene-silencing events are thought to be detrimental to the pathogen and beneficial to the host. Conversely, trans- species small RNAs from pathogens and parasites are deployed to silence host mRNAs; these events are thought to be beneficial for the pests. The natural ability of plants to exchange small RNAs with invading eukaryotic organisms can be exploited to provide disease resistance. This review gives an overview of the current state of trans- species small RNA research in plants and discusses several outstanding questions for future research., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Some pathogens and pests deliver small RNAs (sRNAs) into host cells to suppress host immunity. Conversely, hosts also transfer sRNAs into pathogens and pests to inhibit their virulence. Although sRNA trafficking has been observed in a wide variety of interactions, how sRNAs are transferred, especially from hosts to pathogens and pests, is still unknown. Here, we show that host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen Botrytis cinerea These sRNA-containing vesicles accumulate at the infection sites and are taken up by the fungal cells. Transferred host sRNAs induce silencing of fungal genes critical for pathogenicity. Thus, Arabidopsis has adapted exosome-mediated cross-kingdom RNA interference as part of its immune responses during the evolutionary arms race with the pathogen., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Small RNAs have emerged as one of the most important regulators for gene expression in eukaryotes. Small RNA-mediated gene silencing has been shown to play an essential role in antiviral defense in both plant and animal systems (Li and Ding, 2005; Voinnet, 2005; Wang et al., 2006). These viral RNA-generated small interfering RNAs (siRNAs) are extragenomic in origin. Studies from our lab and others suggest that host-endogenous small RNAs also play an important role in plant defense in response to other pathogens besides viruses (Katiyar-Agarwal et al., 2006; Navarro et al., 2006). The methods described here provide an opportunity to identify many more novel pathogen-regulated small RNAs in plants, which will help in understanding the regulatory mechanism of plant immunity. Here, we introduce the approaches of powerful high-throughput parallel sequencing and hybridization-based technologies for the discovery and detection of pathogen-regulated small RNAs. We mainly compare and discuss the methods of low-molecular-weight (LMW) RNA extraction from pathogen-infected tissue and strategies for detecting endogenous small RNAs by Northern blot analysis.