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Abstract Extracellular vesicles (EVs) produced by Arabidopsis (Arabidopsis thaliana) plants are highly heterogeneous in protein content. To understand the origins of plant EV heterogeneity, we used an unbiased proximity labeling approach to identify proteins and pathways involved in the secretion of distinct EV subpopulations. Proximity labeling, co-immunoprecipitation, and fluorescence microscopy in Nicotiana benthamiana all indicated a general role in EV secretion for EXO70 proteins (a subunit of the exocyst complex) and the immune-related protein RPM1-INTERACTING PROTEIN4 (RIN4). To confirm these hypotheses, we assessed the impact of mutations in various EXO70 family genes and in RIN4 on EV release, as well as mutations in additional genes known to regulate endomembrane trafficking and secretion. Mutation of EXO70E1, EXO70E2 or STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1; a GTP-exchange factor for RabE GTPases) reduced secretion of EVs marked by TETRASPANIN8 (TET8), PENETRATION1 (PEN1), and PATELLIN1 (PATL1), indicating that these proteins are generally required for EV secretion. In contrast, mutation of RIN4 reduced levels of TET8+ and PEN1+ EVs, but not PATL + EVs. Mutation of the small GTPase gene RABA2a specifically affected PEN1+ EV secretion, while mutations in AUTOPHAGY PROTEIN5 (ATG5) and VAMP-ASSOCIATED PROTEIN27 (VAP27) specifically affected TET8+ EV secretion. Lastly, we found that exo70 family mutants are more susceptible to infection with the fungal pathogen Colletotrichum higginsianum, underlining the importance of secretion for plant immunity. Together, our results unravel some of the complex mechanisms that give rise to EV subpopulations in plants. 

ABSTRACT Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogeneous in content and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this, we used high‐resolution density gradient ultracentrifugation and total internal fluorescence microscopy (TIRF‐M) to purify and distinguish distinct subpopulations of Arabidopsis EVs. The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium‐density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in mostly separate EV populations using TIRF‐M, while PEN1 was co‐secreted with PENETRATION 3 (PEN3) much more often. Secretion of EV subpopulations marked by TET8, PEN1 and RPM1‐INTERACTING PROTEIN 4 (RIN4) into the apoplast and onto the leaf surface was induced by phytohormones, changes in temperature and infection with fungal pathogens. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of fungal infection by altering fungal germ tube development and fungal morphology. Significantly, extracellular RNAs, including miRNAs and siRNAs, did not co‐fractionate with TET8‐labeled EVs, and instead, co‐fractionated with extravesicular ARGONAUTE proteins in high‐density fractions. Together, these data indicate that Arabidopsis EVs are highly heterogeneous and contribute to immunity but are unlikely to mediate cross‐kingdom RNA interference. 

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license . 

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host–pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum–Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral ( x-y) pixel resolution of 10 nm and an axial ( z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum–Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . 

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.