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Highlights d Phytophthora infection increases production of a pool of secondary siRNAs in Arabidopsis d Secondary siRNAs from a PPR gene cluster contribute to defense against Phytophthora d PPR-siRNAs potentially silence Phytophthora transcripts to confer resistance d Phytophthora effector PSR2 suppresses the biogenesis of PPR-siRNAs to promote infection

Small RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile membrane-bound compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that Arabidopsis (Arabidopsis thaliana) EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of "tiny RNAs" (10 to 17 nt) that are highly enriched in EVs. This RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

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 and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-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 coimmunoprecipitated 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 exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.

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24Small RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most 25 eukaryotic organisms and regulate numerous biological functions, including transposon 26 silencing, development, reproduction, and stress responses, typically via control of the 27 stability and/or translation of target mRNAs. Major classes of sRNAs in plants include 28 microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel 29 as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. 30In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile 31 lipid compartments that participate in intercellular communication. In addition to sRNAs, 32EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here 33 we report that plant EVs also contain diverse species of sRNA. We found that specific 34 miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously 35 overlooked class of "tiny RNAs" (10 to 17 nt) that are highly enriched in EVs. This new 36RNA category of unknown function has a broad and very diverse genome origin and might 37 correspond to degradation products. 39Small RNAs (sRNAs) are important 21 to 24 nucleotide (nt) non-coding signaling 40 molecules involved in a wide variety of processes, including plant development, 41 reproduction and defense (Samad et al., 2017). sRNAs can be divided into two 42 categories, microRNAs (miRNAs) and small interfering RNAs (siRNAs), based on the 43 differences in their biogenesis and mode of action. miRNAs originate from a single-44 stranded, self-complementary, non-coding RNA that forms a hairpin structure. In contrast, 45 siRNAs originate from a double-stranded RNA molecule synthesized by RNA-46 DEPENDENT RNA POLYMERASES (RDRs). siRNAs can further be divided into two 47 main categories, heterochromatic siRNAs (hc-siRNAs) and phased siRNAs, including 48 trans-acting siRNAs (tasi-RNAs). Both of these double-stranded RNA structures are 49 recognized by Dicer-like proteins (DCL), which cleave these RNAs into defined length 50 products. One strand of these products is then selectively loaded onto an ARGONAUTE 51 (AGO) protein and incorporated into the RNA-induced Silencing Complex (RISC). The 52 RISC uses the sRNA in a sequence-homology-dependent manner to negatively regulate 53 targets, typically mRNAs (Borges and Martienssen, 2015). 54 sRNAs are often mobile and function in non-cell autonomous silencing, which can 55 be either local or systemic. Local RNA silencing occurs among groups of adjacent cells 56 and can gradually spread from cell to cell (Marín-González and Suárez-López, 2012; 57 Dunoyer et al., 2013). Systemic silencing occurs over long distances and can spread 58 throughout an entire plant. While there are several documented cases of mobile small 59RNAs in plants, the mechanisms by which these RNAs move has yet to be clearly 60 established. Local RNA silencing is thought to involve the transport of RNAs through 61 plasmodesmata (PD) wi...

Summary Extracellular vesicles (EVs) are small, membrane‐enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long‐sought‐after mechanism for host‐induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co‐purifying contaminants.

Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves.The isolated EVs can be quantified using a fluorometric dye to assess total membrane content.