Biology subjects

Rutter, B. D.

Publications and source records attributed to Rutter, B. D..

5 recordsLinked to original sources

bioRxiv2024

Arabidopsis produces distinct subpopulations of extracellular vesicles that respond differentially to biotic stress

Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogenous in contents and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this knowledge gap, we used high-resolution density gradient ultracentrifugation to separate distinct subpopulations of Arabidopsis EVs. We analyzed the protein content, morphology, and purity of these subpopulations, confirming the presence of three distinct EV subpopulations. The EV marker protein TETRASPANIN 8 (TET8) was detected only in medium-density EVs and was not associated with cell wall nanofilaments, which was unique among EV proteins. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted on mostly separate EV populations using total internal fluorescence microscopy. We found that EV marker proteins are differentially secreted in response to phytohormones, changes in growth temperature, and infection with fungal pathogens Colletotrichum and Golovinomyces cichoracearum. EV subpopulations marked by TET8, PEN1, and RPM1-INTERACTING PROTEIN 4 (RIN4) were highly increased as soon as one day after fungal infection, while other EV populations remained unaffected. Together these data indicate that Arabidopsis EVs are highly heterogenous and suggest that specific EV subpopulations may contribute to plant immunity.

plant biology↗

bioRxiv2023

Characterization of extracellular vesicles isolated from Sorghum bicolor reveals a conservation between monocot and eudicot extracellular vesicle proteomes

Plant extracellular vesicles (EVs) are membrane bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. In this paper, we described the isolation and characterization of extracellular vesicles from Sorghum bicolor. Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, Calcein-acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also being found in the EV proteome of Arabidopsis. These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

plant biology↗

bioRxiv2022

Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

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 phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis 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 lines 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 on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

cell biology↗

bioRxiv2021

Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

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 EVs and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they comprise both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional 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 co-immunoprecipitated 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 these extravesicular RNAs mediate host-induced gene silencing, rather than RNA inside EVs. One-sentence summaryThe apoplast of Arabidopsis leaves contains diverse small and long-noncoding RNAs, including circular RNAs, that are bound to protein complexes and are located outside extracellular vesicles.

plant biology↗

bioRxiv2018

Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10 to 17 Nucleotide \"Tiny\" RNAs

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 lipid 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 plant 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 new RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

plant biology↗