skip to main content

Explore Research Products in the NSF-PAR

Title: The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum
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
Award ID(s):
1842685 1645745
NSF-PAR ID:
10367599
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Journal of Extracellular Vesicles
Volume:
11
Issue:
5
ISSN:
2001-3078
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ( , Nature Communications)
    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
  2. ; ( , Extracellular Vesicles and Circulating Nucleic Acids)
    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. 
    more » « less
  3. ; ; ( , Current Protocols)
    Abstract

    Extracellular vesicles (EVs) in plants have emerged as key players in cell‐to‐cell communication and cross‐kingdom RNAi between plants and pathogens by facilitating the exchange of RNA, proteins, and other molecules. In addition to their role in intercellular communication, plant EVs also show promise as potential therapeutics and indicators of plant health. However, plant EVs exhibit significant heterogeneity in their protein markers, size, and biogenesis pathways, strongly influencing their composition and functionality. While mammalian EVs can be generally classified as exosomes that are derived from multivesicular bodies (MVBs), microvesicles that are shed from the plasma membrane, or as apoptotic bodies that originate from cells undergoing apoptosis, plant EVs remain poorly studied in comparison. At least three subclasses of EVs have been identified inArabidopsisleaves to date, including Tetraspanin‐positive exosomes derived from MVBs, Penetration 1 (PEN1)‐positive EVs, and EVs derived from exocyst‐positive organelles (EXPO). Differences in the plant starting material and isolation techniques have resulted in different purities, quality, and compositions of the resulting EVs, complicating efforts to better understand the role of these EVs in plants. We performed a comparative analysis on commonly used plant EV isolation methods and have identified an effective protocol for extracting clean apoplastic washing fluid (AWF) and isolating high‐quality intact and pure EVs ofArabidopsis thaliana. These EVs can then be used for various applications or studied to assess their cargos and functionality in plants. Furthermore, this process can be easily adapted to other plant species of interest. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Isolation of EVs from the apoplastic fluid ofArabidopsis thaliana

    Basic Protocol 2: Density gradient fractionation of EVs

    Basic Protocol 3: Immuno‐isolation of EVs usingArabidopsistetraspanin 8 (TET8) antibody

     
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al ( , Journal of Extracellular Biology)
    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
  5. ; ; ; ; ; ; ( , The Plant Cell)
    Abstract Previously, we have shown that apoplastic wash fluid (AWF) 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 and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email