Search for: All records

Transgenic expression of a double-stranded RNA in plants can induce silencing of homologous mRNAs in fungal pathogens. Although such host-induced gene silencing is well documented, the molecular mechanisms by which RNAs can move from the cytoplasm of plant cells across the plasma membrane of both the host cell and fungal cell are poorly understood. Indirect evidence suggests that this RNA transfer may occur at a very early stage of the infection process, prior to breach of the host cell wall, suggesting that silencing RNAs might be secreted onto leaf surfaces. To assess whether Arabidopsis plants possess a mechanism for secreting RNA onto leaf surfaces, we developed a protocol for isolating leaf surface RNA separately from intercellular (apoplastic) RNA. This protocol yielded abundant leaf surface RNA that displayed an RNA banding pattern distinct from apoplastic RNA, suggesting that it may be secreted directly onto the leaf surface rather than exuded through stomata or hydathodes. Notably, this RNA was not associated with either extracellular vesicles or protein complexes; however, RNA species longer than 100 nucleotides could be pelleted by ultracentrifugation. Furthermore, pelleting was inhibited by the divalent cation chelator EGTA, suggesting that these RNAs may form condensates on the leaf surface. These leaf surface RNAs are derived almost exclusively from Arabidopsis, but come from diverse genomic sources, including rRNA, tRNA, mRNA, intergenic RNA, microRNAs, and small interfering RNAs, with tRNAs especially enriched. We speculate that endogenous leaf surface RNA plays an important role in the assembly of distinct microbial communities on leaf surfaces. 

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.