Message in a Bubble: Shuttling Small RNAs and Proteins Between Cells and Interacting Organisms Using Extracellular Vesicles

Abstract

Communication between plant cells and interacting microorganisms requires the secretion and uptake of functional molecules to and from the extracellular environment and is essential for the survival of both plants and their pathogens. Extracellular vesicles (EVs) are lipid bilayer-enclosed spheres that deliver RNA, protein, and metabolite cargos from donor to recipient cells and participate in many cellular processes. Emerging evidencehas shown that both plant and microbial EVs play important roles in cross-kingdom molecular exchange between hosts and interacting microbes to modulate host immunity and pathogen virulence. Recent studies revealed that plant EVs function as a defense system by encasing and delivering small RNAs (sRNAs) into pathogens, thereby mediating cross-species and cross-kingdom RNA interference to silence virulence-related genes. This review focuses on the latest advances in our understanding of plant and microbial EVs and their roles in transporting regulatory molecules, especially sRNAs, between hosts and pathogens. EV biogenesis and secretion are also discussed, as EV function relies on these important processes.

Keywords: cell-to-cell communication; cross-kingdom RNAi; exosome; extracellular vesicles; plant immunity; small RNA.

Figure 1

EV-mediated cross-kingdom RNAi is a communication mechanism in plant–microbe interactions. (a) Plants have at least three known EV subtypes. TET-positive exosomes are released into extracellular space by MVB fusion with the PM and the subsequent release of ILVs. The biogenesis pathway of PEN1-positive EVs remains unknown. The EXPO produces EVs by fusion with the PM to release the inner vesicles into the extracellular space. MVs may also be secreted by plant cells through outward budding directly from the PM. (b) During pathogen infection, plants secrete EVs into the extracellular space. These EV-encased sRNAs can be internalized by pathogens, where they target pathogen virulence-related genes to suppress pathogen virulence. At the same time, pathogens can deliver sRNA effectors into host plant cells to suppress host immunity. EVs have been observed in the PAS where plants and AM fungi interact. We predict that pathogens may also utilize EVs to secrete and transport sRNAs into host cells. The question mark indicates a prediction that has not yet been validated experimentally. Abbreviations: AM, arbuscular mycorrhizal; EHMx, extrahaustorial matrix; ER, endoplasmic reticulum; EV, extracellular vesicle; EXPO, exocyst-positive organelle; ILV, intraluminal vesicle; LE, late endosome; MV, microvesicle; MVB, multivesicular body; PAS, periarbuscular space; PEN1, Penetration 1; PM, plasma membrane; RNAi, RNA interference; sRNA, small RNA; TET, Tetraspanin; TGN/EE, trans-Golgi network/early endosome.

Figure 2

Microbial EV formation and secretion pathways. (a) OMVs are secreted by Gram-negative bacteria from blebbing of the outer membrane. CMVs are produced by Gram-positive bacteria from blebbing of the cytoplasmic membrane. (b) Fungi can produce EVs to pass through the outer thick cell wall, although the mechanisms of fungal EV release are largely unknown. (c) Microbial EVs contain functional components including microbe-derived RNA, lipids, proteins, nucleic acids, and metabolites. The question mark represents a prediction that has not yet been validated experimentally. Abbreviations: CMV, cytoplasmic membrane vesicle; EE, early endosome; ER, endoplasmic reticulum; EV, extracellular vesicle; ILV, intraluminal vesicle; mRNA, messenger RNA; MV, microvesicle; MVB, multivesicular body; OMV, outer membrane vesicle; sRNA, small RNA.

Figure 3

RNAi and roles for EVs in crop protection. HIGS and SIGS approaches are used to deliver dsRNA/siRNA trigger molecules to crop pests and pathogens including viruses, fungi, nematodes, and insects. Following cellular internalization, RNAi trigger molecules suppress target gene expression and generate host resistance. The HIGS approach produces dsRNA in plant cells via genetic modification, with export, transport, and uptake in pest and pathogen cells likely involving EVs. For SIGS approaches where dsRNA is applied exogenously, nanocarriers such as clays, liposomes, and EVs protect the RNAi cargo and are proposed to enhance uptake. Figure adapted from images created with BioRender.com. Abbreviations: dsRNA, double-stranded RNA; EV, extracellular vesicle; HIGS, host-induced gene silencing; mRNA, messenger RNA; RISC, RNA-induced silencing complex; RNAi, RNA interference; SIGS, spray-induced gene silencing; siRNA, small interfering RNA.