Extracellular vesicles: cross-organismal RNA trafficking in plants, microbes, and mammalian cells

Abstract

Extracellular vesicles (EVs) are membrane-enclosed nanometer-scale particles that transport biological materials such as RNAs, proteins, and metabolites. EVs have been discovered in nearly all kingdoms of life as a form of cellular communication across different cells and between interacting organisms. EV research has primarily focused on EV-mediated intra-organismal transport in mammals, which has led to the characterization of a plethora of EV contents from diverse cell types with distinct and impactful physiological effects. In contrast, research into EV-mediated transport in plants has focused on inter-organismal interactions between plants and interacting microbes. However, the overall molecular content and functions of plant and microbial EVs remain largely unknown. Recent studies into the plant-pathogen interface have demonstrated that plants produce and secrete EVs that transport small RNAs into pathogen cells to silence virulence-related genes. Plant-interacting microbes such as bacteria and fungi also secrete EVs which transport proteins, metabolites, and potentially RNAs into plant cells to enhance their virulence. This review will focus on recent advances in EV-mediated communications in plant-pathogen interactions compared to the current state of knowledge of mammalian EV capabilities and highlight the role of EVs in cross-kingdom RNA interference.

Keywords: Extracellular vesicles; cross-kingdom RNA interference; plant-microbial interaction; small RNAs; spray-induced gene silencing.

Figure 1

Cross-kingdom RNAi in plant–microbial interactions. Some fungal pathogens, such as B. cinerea and V. dahlia, deliver sRNAs into the plant cells to silence host genes that are involved in plant immunity^[88,91]. Cross-kingdom RNAi is bidirectional, and plants secrete TET8/9-positive EVs to transport host functional sRNAs into pathogens to silence fungal genes involved in virulence^[29]. TET8/9-positive EVs contain a variety of RBPs, including AGO1, RHs, and ANNs, which contribute to the selection or stabilization of sRNAs in EVs^[56]. Cross-kingdom RNAi also exists in bacteria-plant interaction. Rhizobia tRNA-derived short fragments act as functional sRNAs moving into plant cells to silence target genes related to nodulation^[113]. sRNAs from fungal pathogen and bacterium rhizobia were all found to be loaded into plant host AGO1 to silence host target genes. Fungi and bacteria are predicted to secrete and transport sRNAs into host cells by EVs. The question mark indicates a prediction that has not yet been validated experimentally. EVs: extracellular vesicles; MVB: multivesicular body; ILV: intraluminal vesicle; OM: Outer membrane; PM: plasma membrane; sRNA: small RNA; TE: Transposable element; tRFs: tRNA-derived fragments.

Figure 2

Heterogeneous populations of EVs isolated from plants. There are at least four known EV populations that have been isolated from plants: TET-positive EVs, PEN1-positive EVs, autophagy-related EVs, and pollenosomes. They have different sizes, densities, cargoes, and intracellular origins. Pathogen infection induces secretion of both TET-positive EVs and PEN1-positive EVs^[56]. In the process of EV isolation, final centrifugation of 40,000 × g (P40) pellets larger and heavier vesicles such as PEN1-positive EVs and large EVs, non-vesicular free RNA, and RNA-protein complexes^[30,79]. Small EVs, such as TET-positive EVs, are mainly present in the supernatant after 40,000 × g centrifugation and require a higher speed of ultracentrifugation at 100,000 × g for collection (P100)^[30,56]. Autophagy-related EVs marked with ATG8a were collected using 100,000 × g from plants during the autophagy process within cells^[64]. Pollenosomes secreted during pollen germination and pollen tube growth were collected at 100,000 × g from in-vitro pollen gemination media^[59]. EXPO-derived EVs originate from the plant-specific organelle EXPO, are marked by the protein Exo70E2, and have not yet been isolated from plants^[71]. This figure was created with https://www.biorender.com/.

Figure 3

Potential Application for Plant EVs. Plant-derived EVs and artificial vesicles have been developed for agricultural crop protection and advances in human medicine. Artificial vesicles have been used to load and stabilize pathogen and pest-targeted sRNAs on plants^[163], as well as being used in drug delivery mechanisms for human medicine^[77,78]. Plant-derived EVs have been explored for their native anticancer, anti-inflammatory, and other medicinal uses in humans, as well as their potential to uptake and deliver drugs through biological barriers within the body. This figure was created with https://www.biorender.com/.