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Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogenColletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, includingArabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14‐3‐3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection onArabidopsisleaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.

 

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 distinguishbona fideEV cargo from merely co‐purifying contaminants.

 

Abstract Extracellular RNA (exRNA) has long been considered as cellular waste that plants can degrade and utilize to recycle nutrients. However, recent findings highlight the need to reconsider the biological significance of RNAs found outside of plant cells. A handful of studies suggest that the exRNA repertoire, which turns out to be an extremely heterogenous group of non-coding RNAs, comprises species as small as a dozen nucleotides to hundreds of nucleotides long. They are found mostly in free form or associated with RNA-binding proteins, while very few are found inside extracellular vesicles (EVs). Despite their low abundance, small RNAs associated with EVs have been a focus of exRNA research due to their putative role in mediating trans-kingdom RNAi. Therefore, non-vesicular exRNAs have remained completely under the radar until very recently. Here we summarize our current knowledge of the RNA species that constitute the extracellular RNAome and discuss mechanisms that could explain the diversity of exRNAs, focusing not only on the potential mechanisms involved in RNA secretion but also on post-release processing of exRNAs. We will also share our thoughts on the putative roles of vesicular and extravesicular exRNAs in plant–pathogen interactions, intercellular communication, and other physiological processes in plants. 

Extracellular RNA (exRNA) has long been considered as cellular waste that plants can degrade and utilize to recycle nutrients. However, recent findings highlight the need to reconsider the biological significance of RNAs found outside of plant cells. A handful of studies suggest that the exRNA repertoire, which turns out to be an extremely heterogenous group of non-coding RNAs, comprises species as small as a dozen nucleotides to hundreds of nucleotides long. They are found mostly in free form or associated with RNA-binding proteins, while very few are found inside extracellular vesicles (EVs). Despite their low abundance, small RNAs associated with EVs have been a focus of exRNA research due to their putative role in mediating transkingdom RNA interference. Therefore, non-vesicular exRNAs have remained completely under the radar until very recently. Here we summarize our current knowledge of the RNA species that constitute the extracellular RNAome and discuss mechanisms that could explain the diversity of exRNAs, focusing not only on the potential mechanisms involved in RNA secretion but also on post-release processing of exRNAs. We will also share our thoughts on the putative roles of vesicular and extravesicular exRNAs in plant-pathogen interactions, intercellular communication, and other physiological processes in plants. 

Extracellular vesicles (EVs) are nano-sized, lipid compartments that mediate the intercellular transport of lipids, proteins, nucleic acids and metabolites. During infectious diseases, EVs released by host cells promote immune responses, while those released by pathogens attempt to subvert host immunity. There is a growing body of research investigating the role of fungal EVs in plant pathosystems. It is becoming clear that EVs released by fungal phytopathogens play a role during infection through the transport of protein effectors, toxic metabolites and RNA. Here, we discuss recent findings on EVs in fungal phytopathogens, including the methods employed in their isolation, their characterization, contents and functionality, as well as the key questions remaining to be addressed. 

Abstract Host-induced gene silencing (HIGS) refers to the silencing of genes in pathogens and pests by expressing homologous double-stranded RNAs (dsRNA) or artificial microRNAs (amiRNAs) in the host plant. The discovery of such trans-kingdom RNA silencing has enabled the development of RNA interference-based approaches for controlling diverse crop pathogens and pests. Although HIGS is a promising strategy, the mechanisms by which these regulatory RNAs translocate from plants to pathogens, and how they induce gene silencing in pathogens, are poorly understood. This lack of understanding has led to large variability in the efficacy of various HIGS treatments. This variability is likely due to multiple factors, such as the ability of the target pathogen or pest to take up and/or process RNA from the host, the specific genes and target sequences selected in the pathogen or pest for silencing, and where, when, and how the dsRNAs or amiRNAs are produced and translocated. In this review, we summarize what is currently known about the molecular mechanisms underlying HIGS, identify key unanswered questions, and explore strategies for improving the efficacy and reproducibility of HIGS treatments in the control of crop diseases. 

Abstract Previously, we have shown that apoplastic wash fluid (AWF) 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 AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, 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.