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

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.

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Small RNA trafficking at the forefront of plant–pathogen interactions

F1000Research ◽

10.12688/f1000research.15761.1 ◽

2018 ◽

Vol 7 ◽

pp. 1633 ◽

Cited By ~ 3

Author(s):

Yan Zhao ◽

Xiangxiu Liang ◽

Jian-Min Zhou

Keyword(s):

Extracellular Vesicles ◽

Small Rna ◽

Plant Pathogen ◽

Small Rnas ◽

Plant Cells ◽

Host Immunity ◽

Lytic Enzymes ◽

Pathogen Virulence ◽

Pathogenic Microbes ◽

Plant Pathogen Interactions

Plants and pathogenic microbes are engaged in constant attacks and counterattacks at the interface of the interacting organisms. Much of the molecular warfare involves cross-kingdom trafficking of proteins, nucleic acids, lipids, and metabolites that act as toxins, inhibitors, lytic enzymes, and signaling molecules. How various molecules are transported across the boundaries of plants and pathogens has remained largely unknown until now. Extracellular vesicles have emerged as likely carriers of molecular ammunition for both plants and pathogens. Recent advances are beginning to show how extracellular vesicles serve as powerful vehicles that transfer small RNAs from plants to fungal cells to diminish pathogen virulence and from fungi to plant cells to dampen host immunity.

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Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes

Science ◽

10.1126/science.aar4142 ◽

2018 ◽

Vol 360 (6393) ◽

pp. 1126-1129 ◽

Cited By ~ 222

Author(s):

Qiang Cai ◽

Lulu Qiao ◽

Ming Wang ◽

Baoye He ◽

Feng-Mao Lin ◽

...

Keyword(s):

Rna Interference ◽

Immune Responses ◽

Extracellular Vesicles ◽

Fungal Pathogen ◽

Small Rnas ◽

Virulence Genes ◽

Host Immunity ◽

Arms Race ◽

Host Cells ◽

Fungal Genes

Some pathogens and pests deliver small RNAs (sRNAs) into host cells to suppress host immunity. Conversely, hosts also transfer sRNAs into pathogens and pests to inhibit their virulence. Although sRNA trafficking has been observed in a wide variety of interactions, how sRNAs are transferred, especially from hosts to pathogens and pests, is still unknown. Here, we show that hostArabidopsiscells secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogenBotrytis cinerea. These sRNA-containing vesicles accumulate at the infection sites and are taken up by the fungal cells. Transferred host sRNAs induce silencing of fungal genes critical for pathogenicity. Thus,Arabidopsishas adapted exosome-mediated cross-kingdom RNA interference as part of its immune responses during the evolutionary arms race with the pathogen.

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Extracellular vesicles in plant host-microbe interaction

Trillium Exctracellular Vesicles ◽

10.47184/tev.2019.01.07 ◽

2019 ◽

Vol 1 (1) ◽

pp. 46-50

Author(s):

Constance Tisserant ◽

Arne Weiberg

Keyword(s):

Extracellular Vesicles ◽

Small Rnas ◽

Potential Role ◽

Bilayer Membrane ◽

Host Immunity ◽

Special Focus ◽

Mediated Communication ◽

Plant Host ◽

Biomedical Field ◽

Host Parasite

Extracellular vesicles (EVs) are secreted lipid bilayer membrane particles that are increasingly drawing attention due to their potential role in intercellular communication. EVs have been mainly reported in mammalian systems, but are also found in non-mammalian classes, such as Archeae, bacteria, fungi, oomycetes, protozoa, invertebrates and plants. Over the last decade, EV research on mammalian systems has been massively advanced driven by the interests and applications of the biomedical field, while research on non-mammalian EVs that aims to understand the biological origins and functions of EVs remains rather descriptive and premature. Nevertheless, recent pioneering works resulted in novel concepts that place EVs carrying regulatory small RNAs as central players in inter-species and cross-kingdom communication with emphasis on host-pathogen, host-parasite and host-microbiome interactions. EVs transport small RNAs from microbe/pathogen/parasite to animal or plant hosts, and vice versa, which results in the manipulation of host immunity or microbial virulence, respectively. This article highlights some of the latest discoveries regarding EV-mediated communication across species and kingdoms with a special focus on plants and their interacting microbes.

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Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways

Science ◽

10.1126/science.1239705 ◽

2013 ◽

Vol 342 (6154) ◽

pp. 118-123 ◽

Cited By ~ 510

Author(s):

Arne Weiberg ◽

Ming Wang ◽

Feng-Mao Lin ◽

Hongwei Zhao ◽

Zhihong Zhang ◽

...

Keyword(s):

Rna Interference ◽

Fungal Pathogen ◽

Small Rnas ◽

Plant Immunity ◽

Gray Mold ◽

Host Immunity ◽

Rnai Machinery ◽

Naturally Occurring ◽

Immunity Genes ◽

Argonaute 1

Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers “virulent” sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.

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Biomimetic Extracellular Vesicles Embedded with Black Phosphorus for Molecular Recognition-Guided Biomineralization

10.26434/chemrxiv.6721553.v1 ◽

2018 ◽

Author(s):

Yingqian Wang ◽

Xiaoxia Hu ◽

Lingling Zhang ◽

Chunli Zhu ◽

Jie Wang ◽

...

Keyword(s):

Molecular Recognition ◽

Extracellular Vesicles ◽

Inorganic Phosphate ◽

Physiological Activity ◽

Medical Engineering ◽

Matrix Vesicles ◽

Black Phosphorus ◽

Photothermal Effect ◽

Biological Processes ◽

Extracellular Environment

Extracellular vesicles (EVs) are involved in the regulation of cell physiological activity and the reconstruction of extracellular environment. Matrix vesicles (MVs) are a type of EVs, and they participate in the regulation of cell mineralization. Herein, bioinspired MVs embedded with black phosphorus are functionalized with cell-specific aptamer (denoted as Apt-bioinspired MVs) for stimulating biomineralization. The aptamer can direct bioinspired MVs to targeted cells, and the increasing concentration of inorganic phosphate originated from the black phosphorus can facilitate cell biomineralization. The photothermal effect of the Apt-bioinspired MVs also positively affects mineralization. In addition, the Apt-bioinspired MVs display outstanding bone regeneration performance. Considering the excellent behavior of the Apt-bioinspired MVs for promoting biomineralization, our strategy provides a way of designing bionic tools for studying the mechanisms of biological processes and advancing the development of medical engineering.<br>

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Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22052737 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2737

Author(s):

Daisy Sproviero ◽

Stella Gagliardi ◽

Susanna Zucca ◽

Maddalena Arigoni ◽

Marta Giannini ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Extracellular Vesicles ◽

Small Rnas ◽

Biomarker Discovery ◽

Toll Like Receptor ◽

Neurotrophin Signaling ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Lateral Sclerosis ◽

Glycosphingolipid Biosynthesis

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.

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Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells

Membranes ◽

10.3390/membranes11030199 ◽

2021 ◽

Vol 11 (3) ◽

pp. 199

Author(s):

Silvia Marconi ◽

Sara Santamaria ◽

Martina Bartolucci ◽

Sara Stigliani ◽

Cinzia Aiello ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Extracellular Vesicles ◽

Breast Cancer Cells ◽

Culture Supernatant ◽

High Resolution Mass Spectrometry ◽

Recombinant Antibody ◽

Target Cells ◽

Cellular Processes ◽

Erbb2 Signaling

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

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Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽

10.3390/cells10051187 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1187

Author(s):

Michael Wassenegger ◽

Athanasios Dalakouras

Keyword(s):

Dna Methylation ◽

Rna Interference ◽

Gene Silencing ◽

Plant Cells ◽

Transcriptional Gene Silencing ◽

Rnai Machinery ◽

Double Stranded Rna ◽

Post Transcriptional Gene Silencing ◽

Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

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The Yin and Yang of tumour-derived extracellular vesicles in tumour immunity

The Journal of Biochemistry ◽

10.1093/jb/mvaa132 ◽

2020 ◽

Author(s):

Takayoshi Yamauchi ◽

Toshiro Moroishi

Keyword(s):

Extracellular Vesicles ◽

Tumour Progression ◽

Tumour Microenvironment ◽

Host Immunity ◽

Biological Processes ◽

Small Particles ◽

Tumour Immunity ◽

Heterogeneous Nature ◽

Yin And Yang ◽

Cellular Components

Abstract Extracellular vesicles (EVs) are small particles that are naturally released from various types of cells. EVs contain a wide variety of cellular components, such as proteins, nucleic acids, lipids and metabolites, which facilitate intercellular communication in diverse biological processes. In the tumour microenvironment, EVs have been shown to play important roles in tumour progression, including immune system–tumour interactions. Although previous studies have convincingly demonstrated the immunosuppressive functions of tumour-derived EVs, some studies have suggested that tumour-derived EVs can also stimulate host immunity, especially in therapeutic conditions. Recent studies have revealed the heterogeneous nature of EVs with different structural and biological characteristics that may account for the divergent functions of EVs in tumour immunity. In this review article, we provide a brief summary of our current understanding of tumour-derived EVs in immune activation and inhibition. We also highlight the emerging utility of EVs in the diagnosis and treatment of cancers and discuss the potential clinical applications of tumour-derived EVs.

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Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation

Cells ◽

10.3390/cells10092308 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2308

Author(s):

Yanshe Xie ◽

Guangbin Liu ◽

Xupeng Zang ◽

Qun Hu ◽

Chen Zhou ◽

...

Keyword(s):

Extracellular Vesicles ◽

Small Rnas ◽

Target Genes ◽

Embryo Implantation ◽

Mature Embryo ◽

Luciferase Reporter ◽

Small Rna Sequencing ◽

Transmission Electron ◽

Differential Expression Pattern ◽

Endometrial Epithelium

Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs’ EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs’ EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA–mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs’ EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.

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