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. 
                        more » 
                        « less   
                    
                            
                            Growing pains: addressing the pitfalls of plant extracellular vesicle research
                        
                    
    
            Summary Extracellular vesicles (EVs) are small, membrane‐enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long‐sought‐after mechanism for host‐induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguishbona fideEV cargo from merely co‐purifying contaminants. 
        more » 
        « less   
        
    
    
                            - PAR ID:
- 10455365
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- New Phytologist
- Volume:
- 228
- Issue:
- 5
- ISSN:
- 0028-646X
- Page Range / eLocation ID:
- p. 1505-1510
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
- 
            
- 
            Abstract Extracellular vesicles (EVs) secreted by human‐induced pluripotent stem cells (hiPSCs) have great potential as cell‐free therapies in various diseases, including prevention of blood–brain barrier senescence and stroke. However, there are still challenges in pre‐clinical and clinical use of hiPSC‐EVs due to the need for large‐scale production of a large quantity. Vertical‐Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC‐EVs using a scalable aggregate or microcarrier‐based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3‐D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA‐seq, respectively. Thein vitrofunctional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3‐D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA‐seq. The microcarrier cultures had at least 17–23 fold higher EV secretion, and EV collection in mTeSR had 2.7–3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA‐seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt‐related pathways). hiPSC‐EVs demonstrated the ability of stimulating proliferation and M2 polarization of microgliain vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti‐aging study.more » « less
- 
            Abstract Extracellular vesicle (EV)‐carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA‐binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non‐vesicular form in serum and can be an EV contaminant. In addition, RNA‐binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV‐containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra‐ and extra‐vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.more » « less
- 
            Abstract 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 phytopathogenColletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, includingArabidopsis 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 strains 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 onArabidopsisleaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.more » « less
- 
            Abstract Small RNAs (sRNAs) of the fungal pathogenBotrytis cinereacan enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate thatB. cinereautilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). TheB. cinereatetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerousArabidopsisclathrin-coated vesicles (CCVs) aroundB. cinereainfection sites and the colocalization ofB. cinereaEV marker BcPLS1 andArabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and theB. cinerea-secreted sRNAs are detected in purified CCVs after infection.Arabidopsisknockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance toB. cinereainfection. Furthermore, Bc-sRNA loading intoArabidopsisAGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.more » « less
 An official website of the United States government
An official website of the United States government 
				
			 
					 
					
