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

   https://doi.org/10.20517/evcna.2023.10  

   Cai, Qiang ; Halilovic, Lida ; Shi, Ting ; Chen, Angela ; He, Baoye ; Wu, Huaitong ; Jin, Hailing ( January 2023 , Extracellular Vesicles and Circulating Nucleic Acids)

   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. 

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2. Isolation of Extracellular Vesicles from Arabidopsis

   https://doi.org/10.1002/cpz1.352  

   Chen, Angela ; He, Baoye ; Jin, Hailing ( January 2022 , Current Protocols)

   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

    

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3. Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

   https://doi.org/10.1038/s41598-020-75108-3  

   Rodriguez, Blanca V. ; Kuehn, Meta J. ( October 2020 , Scientific Reports)

   Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such asStaphylococcus aureusand their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells.S. aureusproduce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show thatS. aureussecretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

    

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4. Extracellular RNA: mechanisms of secretion and potential functions

   https://doi.org/10.1093/jxb/erac512  

   Borniego, M Lucía ; Innes, Roger W ( January 2023 , Journal of Experimental Botany)

   Manavella, Pablo (Ed.)

   Abstract Extracellular RNA (exRNA) has long been considered as cellular waste that plants can degrade and utilize to recycle nutrients. However, recent findings highlight the need to reconsider the biological significance of RNAs found outside of plant cells. A handful of studies suggest that the exRNA repertoire, which turns out to be an extremely heterogenous group of non-coding RNAs, comprises species as small as a dozen nucleotides to hundreds of nucleotides long. They are found mostly in free form or associated with RNA-binding proteins, while very few are found inside extracellular vesicles (EVs). Despite their low abundance, small RNAs associated with EVs have been a focus of exRNA research due to their putative role in mediating trans-kingdom RNAi. Therefore, non-vesicular exRNAs have remained completely under the radar until very recently. Here we summarize our current knowledge of the RNA species that constitute the extracellular RNAome and discuss mechanisms that could explain the diversity of exRNAs, focusing not only on the potential mechanisms involved in RNA secretion but also on post-release processing of exRNAs. We will also share our thoughts on the putative roles of vesicular and extravesicular exRNAs in plant–pathogen interactions, intercellular communication, and other physiological processes in plants. 

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5. Shared TIR enzymatic functions regulate cell death and immunity across the tree of life

   https://doi.org/10.1126/science.abo0001  

   Essuman, Kow ; Milbrandt, Jeffrey ; Dangl, Jeffery L. ; Nishimura, Marc T. ( July 2022 , Science)

   BACKGROUND Diverse organisms, from archaea and bacteria to plants and humans, use receptor systems to recognize both pathogens and dangerous self-derived or environmentally derived stimuli. These intricate, well-coordinated immune systems, composed of innate and adaptive components, ensure host survival. In the late 20th century, researchers identified the Toll/interleukin-1/resistance gene (TIR) domain as an evolutionarily conserved component of animal and plant innate immune systems. Today, TIR-domain proteins are known to be broadly distributed across the tree of life. The TIR domain was first recognized as an adaptor for the assembly of macromolecular signaling complexes in mammalian innate immune pathways. Work on axon degeneration in animals—as well as on plant, archaeal, and bacterial immune systems—has uncovered additional enzymatic activities for TIR domains. ADVANCES Mammalian axons initiate a self-destruct program upon injury and during disease that is mediated by the sterile alpha and TIR motif containing 1 (SARM1) protein. The SARM1 TIR domain enzymatically consumes the essential metabolic cofactor nicotinamide adenine dinucleotide (NAD + ) to promote axonal death. Identification of the SARM1 NAD + -consuming enzyme (NADase) revealed that TIR domains can function as enzymes. Given the evolutionary conservation of TIR domains, studies investigated whether the SARM1 TIR NADase was also conserved. Indeed, bacteria, archaea, and plant TIR domains possess NADase activity. In prokaryotes, TIR NADase activity is found in an ancient antiphage immune system. In plants, identification of TIR NADase activity and linkage of TIR enzymatic products to downstream signaling components addressed the question of how nucleotide-binding, leucine-rich repeat (NLR) receptors trigger hypersensitive cell death during an immune response. Studies in plants show that their TIR domains can cleave nucleic acids and possess 2′,3′ cyclic adenosine monophosphate (2′,3′-cAMP) and 2′,3′ cyclic guanosine monophosphate (2′,3′-cGMP) synthetase activity that aids cell death programs in plant innate immunity. Thus, TIR domains constitute an ancient family of enzymes that are activated in immune and cell death pathways. OUTLOOK The discovery of TIR-domain enzyme activities carries implications for innate immunity and neurodegeneration. The identification of the SARM1 NADase defined a drug target for a wide number of neurodegenerative diseases that is being exploited in both preclinical and clinical studies. Hyperactive mutations in the SARM1 NADase have been discovered in amyotrophic lateral sclerosis (ALS) patients. Future work will seek to clarify the contribution of the SARM1 axon degeneration pathway to ALS pathogenesis. NAD + biology influences cellular processes from metabolism to DNA repair to aging. How TIR enzymes influence the NAD + metabolome and its associated pathways in bacteria, archaea, plants, and animals will be an exciting area for upcoming investigation. The discovery of the diversity of TIR enzymatic products is revealing signaling pathways across kingdoms. Discovery of TIR enzymatic function in plants and animals may yet inspire studies of enzymatic functions for Toll-like receptors in animals. We anticipate that cross-kingdom studies of TIR-domain function will guide interventions that will span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases. Conserved TIR-domain enzymatic activity. TIR-domain proteins from prokaryotes and eukaryotes cleave NAD + into nicotinamide (Nam), ADP-ribose (ADPR), cyclic ADP-ribose (cADPR), isomers of cyclic ADP-ribose (2′ or 3′cADPR), and related molecules [e.g., phosphoribosyl adenosine monophosphate (pRib-AMP)]. Plant TIR domains also possess a nuclease activity, can degrade DNA and RNA, and can function as a 2′,3′-cAMP or 2′,3′-cGMP synthetase. TIR enzymatic activity drives cell death and immune pathways across kingdoms. TIR activity can kill cells directly through NAD + depletion or indirectly using enzymatic products as signal molecules. The representative TIR domain structure shown here is Protein Data Bank ID 6O0Q. EDS1, enhanced disease susceptibility 1; ThsA, Thoeris A. 

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