Functions of Extracellular Vesicles in Immunityand Virulence1[OPEN]
Katarzyna Rybak and Silke Robatzek2,3
LMU Biocentre, Ludwig-Maximilian-University of Munich, Grosshaderner Strasse 4, 82152 Martinsried,Germany
ORCID ID: 0000-0002-9788-322X (S.R.).
Extracellular vesicles (EVs) are lipid bilayer-enclosed,cytosol-containing spheres that are released by all eu-karyotes and prokaryotic cells into the extracellularenvironment. Primarily, EVs act in cell-to-cell commu-nication, delivering cargo from donor to recipient cellsand modulating their physiological condition. SinceEVs transport a plethora of protein, nucleic acid, andlipid cargoes, they play roles in multiple signalingpathways, including those determining the interactionoutcome between plants and microbes. Increasing evi-dence indicates that microbial EVs play a prominentrole in modulating plant immunity and that plant-derived EVs control microbial infection at variouslevels. In this review, the importance of both microbialand plant-derived EVs is discussed in terms of patho-genesis and the establishment of immunity, with aspecial focus on modulation of the immune system andplant defense.
Cell-to-cell communication is ubiquitous in all bio-logical systems. As a means to manage species inter-actions, secretion, and delivery of molecular signals inthe extracellular environment is essential for speciessurvival. A major way to achieve cell-to-cell commu-nication is through EVs, which are cytosol-containingmembrane spheres that provide selection, storage, andprotection against degradation of enclosed cargoes in ahighly dynamic and environmental cue-responsivemanner. EVs also offer the opportunity for directedcargo delivery to dedicated recipient cells. EVs havebeen well characterized in human cells and human-infecting bacteria. Both modes of release and uptakehave been frequently studied, and the molecular com-ponents of these pathways are defined. This contrastsmarkedly with the current understanding of EVs inplants and plant-infecting microbes, including bacteria,fungi, and oomycetes, where our knowledge remainsrudimentary. This is partly due to major technicalchallenges, such as the proper detection of EVs, as well
as the belief that EVs cannot be released and taken upby plant cells because of their cell walls.
Half a century ago, EVs were originally described asexcreted particles from Vibrio cholerae cultures andmatrix vesicles present in the epiphyseal plate of mice(Chatterjee and Das, 1967; Anderson, 1969). Interestincreased in the 1980s when EVs were found acrossboth pathogenic and nonpathogenic Gram-negativebacterial species and in biological fluids (i.e. bloodfrom multicellular organisms; Trams et al., 1981;Johnstone et al., 1987; Kuehn and Kesty, 2005). More-over, cancer cells were found to discharge largeamounts of EVs to promote tumor growth (Dvoraket al., 1981; Ruivo et al., 2017). Since EVs are a heter-ogenous class of nano- to microscale vesicles (20–1,000nm) of diverse origins and are present outside the cells,they were named according to their size (i.e. nano-vesicles, nanoparticles, microvesicles, microparticles)and biogenesis (i.e. membrane vesicles and outermembrane vesicles, or exosomes). For example, mem-brane vesicles and outer membrane vesicles are formedby budding and shedding of the (outer) plasma mem-brane (PM) in eukaryotic cells and Gram-negativebacteria, respectively (Raposo and Stoorvogel, 2013;Jan, 2017). MVs can also be produced by endolysin-triggered cell lysis as observed in Gram-positive
AADVANCES
• An emerging theme in plant-pathogen
interactions is the inter-species communication
by extracellular vesicles (EVs).
• Both plants and their infecting pathogens release
EVs.
• Pathogen EVs exhibit dual activities: provoking
prototypic pattern-triggered immune responses
and supporting infection.
• Plants release and load EVs in response to
infection.
• Plant EVs function as a defense system: delivering
sRNAs into pathogens and thereby mediating
cross-kingdom RNA interference.
1S.R. is supported by the German Research Foundation (DFG)with a Heisenberg fellowship.
2Author for contact: [email protected] author.K.R. and S.R. wrote the paper.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.18.01557
1236 Plant Physiology�, April 2019, Vol. 179, pp. 1236–1247, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon January 9, 2020 - Published by Downloaded from
bacteria (Toyofuku et al., 2018). Exosomes, however,originate from multivesicular bodies (MVBs) throughinward budding of the endosomal membrane (Raposoand Stoorvogel, 2013). MVBs are single-membranecompartments with intraluminal vesicles. They are or-ganelles of the endocytic pathway in eukaryotes, typi-cally mediating the transport from the trans-Golginetwork (TGN) to vacuoles (Cui et al., 2018). Yet, MVBtrafficking can be redirected to the PM in order to re-lease their intraluminal vesicles to the extracellularspace, referred to as exosomes (Raposo and Stoorvogel,2013). Other mechanisms of unconventional secretion,such as lysosomal exocytosis, secretory autophagy, andexocyst-dependent secretion, have also been discussedto contribute to the extracellular release of EVs, eitherdirectly or indirectly through interaction with MVBs(Wang et al., 2010; Hessvik and Llorente, 2018; Rutterand Innes, 2018). Consequently, EVs contain moleculesof their donor cells and are specifically enriched inproteins associated with their biogenesis, often used asEV biomarkers (Hessvik and Llorente, 2018). As insuf-ficient biomarkers are available for convincingly prob-ing their origin, their heterogeneity challenges thediscrimination in particular subpopulations, and wewill therefore collectively refer to these vesicles as EVs.Initially, EVs were proposed to maintain cellular
homeostasis by eliminating waste products (Hessvikand Llorente, 2018). However, studies of EVs of di-verse origins support a common function in cell-to-cellcommunication, which implies that EVs secreted bydonor cells interact with recipient cells to induce acellular response. The interaction of EVs with recipientcells is mediated by surface components. For exam-ple, tetraspanins, transmembrane proteins regulatingmembrane fusion and cell adhesion processes, for ex-ample, are commonly expressed on EVs in eukaryotesand involved in exosomal uptake in target cells (Caiet al., 2018b; Sims et al., 2018; Vora et al., 2018). Afterattachment to recipient cells, EVs can fuse with the PMor become internalized by clathrin-dependent and-independent endocytosis (Abels and Breakefield,2016; O’Donoghue and Krachler, 2016). Ligands onthe EVs and receptors on recipient cell surfacesthereby confer specificity to the attachment and themode of EV uptake (Colombo et al., 2014). Fusion withthe PM represents a direct pathway by which EVs candischarge their contents into the cytosol of target cells.Release of contents from internalized EVs requiresescape from the endosomal compartment, which couldinvolve contact events between the endoplasmic retic-ulum and exosome-containing endosomes, and retro-grade trafficking (Heusermann et al., 2016; Bielaszewskaet al., 2017).In the context of host-microbe interactions, EVs are
secreted by both organisms and enable bidirectionalcommunication across kingdoms. By delivering theircontents, microbial EVs can modulate the host immunesystem and EVs from host cells can participate in anti-microbial immunity. Although their role in cell-to-cellcommunication has been well-established in diverse
human-pathogen interactions, attention has turned toEVs regulating the interaction between plants and mi-crobes. In this review, we focus on EVs in the context ofplant-microbe interactions and will discuss both thepotential of microbial EVs to modulate the plant’s im-mune system and the function of plant-derived EVs inantimicrobial immunity.
TECHNIQUES FOR EV ISOLATION
Given that EVs are heterogeneous in size and mo-lecular composition and thus in density and charge, it isuseful to discuss the different isolation methods, whichmay influence the nature of EVs. The standard tech-nique and most widely used is differential ultracentri-fugation with consecutive steps of low centrifugalforces (g) to discard cellular debris and high-speedforces (i.e. 40,000g and 100,000g) to collect EVs basedon density (Li et al., 2017; Rutter and Innes, 2017).Differential ultracentrifugation is used as a standalonetechnique but more often combined with density gra-dient centrifugation (i.e. Suc and Optiprep) to reducecontaminants and thus purify refined EVs (Sunkaraet al., 2016; Li et al., 2017; Rutter et al., 2017). This iso-lation technique was used to purify EVs from diversecultured plant bacteria and fungi (Table 1), plant cells,and fromplant extracellular fluids collected from leavesand seedlings (Table 2). Applying differential ultra-centrifugation with or without density gradient cen-trifugation depends on the grade of desired purity andthe purpose of EV isolation, i.e. proteomic and tran-scriptomic profiling. The choice of starting material andquantity should be carefully considered when purify-ing EVs. For example, medium should be collectedfrom bacterial cultures in the exponential versus thestationary growth phase to reduce the amount of debrisfrom dying cells, and extracellular plant (apoplastic)fluids should be used instead of plant fluids obtainedbymechanical disruption of tissues as thesewill containa mixture of extracellular and intracellular vesicles(Pérez-Bermúdez et al., 2017). EVs can also be isolatedbased on size bymembrane filtration, i.e. size-exclusionchromatography, which was used to isolate EVs fromplant-infecting Xylella fastidiosa subsp. pauca 9a5c(Santiago et al., 2016). Alternatively, EVs can be isolatedusing immunoaffinity capture and advanced imagingflow cytometry (Li et al., 2017; Mastoridis et al., 2018).However, the latter two approaches have not been de-scribed for the isolation of EVs from plants and plant-interacting microbes (Tables 1 and 2), particularlybecause they depend on suitable EV biomarkers.
TECHNIQUES FOR EV VISUALIZATION
Proper detection of EVs represents a major challenge.Initial discoveries of EVs in the interaction betweenplants and microbes were from transmission electronmicroscopy (TEM). EVs were observed to concentrate
at bacterial and fungal infection sites, for example uponattempted hyphal penetration of Blumeria graminis f. sp.hordei into barley epidermal cells, and successful inva-sion of Golovinomyces orontii and Botrytis cinerea hyphaeinto Arabidopsis (Arabidopsis thaliana) leaf cells (Anet al., 2006b; Micali et al., 2011; Solé et al., 2015; Caiet al., 2018b). TEM analysis also revealed plant MVBsfused with the PM (An et al., 2006b; Cai et al., 2018b),which suggested that EVs are released by MVB-mediated secretion. Consistent with this pathway ofunconventional secretion, the EV proteome is enrichedin proteins lacking a predicted signal peptide (Rutterand Innes, 2017).In addition, most information on EV morphology
and size is derived from TEM measurements, whichreveal EVs as spheres ranging in size from 10 to 400 nmin plant-interacting bacteria and from 10 to 300 nm inplant fluids (Tables 1 and 2). Immunogold electronmicroscopy and cryo-electron microscopy provide ad-ditional information on EV contents and morphology(Wang et al., 2010; Prado et al., 2014; Solé et al., 2015;Sunkara et al., 2016). Whereas electron microscopy-based methods provide information on the purity ofisolated EVs and whether the vesicles are intact, theyrequire sample fixation and dehydration. This affectsthe true morphology and size of EVs, meaning that EVscould appear much smaller than that under hydratedconditions. Dynamic light scattering and nanoparticletracking analysis measure EVs directly in solution andare therefore more likely to determine the true size ofEVs and the size distribution of a population of vesicles(Sunkara et al., 2016). For example, the average diam-eter of EVs from Arabidopsis apoplastic fluids isaround 12 nm (P100 fraction) and 100 nm (P40 fraction),as measured by TEM (Rutter and Innes, 2017). Yet,dynamic light scattering analysis revealed EV diame-ters ranging from 10 to 17 nm (P100 fraction) and 50 to300 nm (P40 fraction), with the most abundant vesiclesaround 150 nm (Rutter and Innes, 2017). Moreover,nanoparticle tracking analysis is suited to quantify EVs(Ionescu et al., 2014). Atomic force microscopy alsodetects EVs in solution and was used to characterizeX. fastidiosa ssp. pauca 9a5c EVs (Santiago et al., 2016).Standard protein assays can determine total EV
protein contents, such as that from bacterial EVs(Chowdhury and Jagannadham, 2013; Mendes et al.,2016), but may not reflect true protein amounts due tothe vesicle’s nature. Most commonly, EVs are charac-terized by immunoblotting and immunogold electronmicroscopy based on biomarkers initially identifiedin proteomic profiling being enriched in EVs (Sidhuet al., 2008; Matsumoto et al., 2012; Chowdhury andJagannadham, 2013; Regente et al., 2017; Rutter andInnes, 2017; Cai et al., 2018b). However, this is limitedas very few biomarkers have been described for EVs ofplants and plant-interacting microbes (Tables 1 and 2).For plant-derived EVs, biomarkers include tretaspa-nin8 (TET8) and molecular components related totheir biogenesis, i.e. the penetration1 (PEN1) syntaxinand the exocyst subunit Exo70E2 in plant-derived EVsT
(Table 2). Components related to the outer membrane(i.e. XadA1) and secreted proteins (i.e. LesA and Ax21)are used as biomarkers for EVs from plant-interactingbacteria (Table 1). Notably, although most plant-derived EVs have a diameter between 10 and 300 nm(Table 2), which is considerably small for detection bystandard light-based microscopy, green fluorescenceprotein (GFP) tagging of biomarkers enabled the directvisualization of EVs using confocal laser scanning mi-croscopy upon EV isolation and in planta (Rutter andInnes, 2017; Cai et al., 2018b). To which extent this re-flects the observation of single vesicles versus aggre-gates and/or the intrinsic brightness of geneticallyencoded and highly expressed GFP-fused biomarkersis unknown. In addition, dyes labeling EV lipids, suchas FM4-64 and DiO, were used to visualize plant-derived EVs (Regente et al., 2017; Rutter and Innes,2017; Cai et al., 2018b). As EV composition reflectsthat of their producing donor cells, these vesicles canincorporate nucleic acids, i.e. in the form of small in-terfering (s)RNAs, exemplified by micro (mi)RNA822that was revealed by RT-PCR in plant-derivedEVs positive for TET8 (Cai et al., 2018b). Given thatthere is no single biomarker that can uniquely identifyEVs, multiple techniques are best to isolate andcharacterize EVs.
PRESENCE OF EVs INPLANT-INTERACTING MICROBES
Some of the best-characterized EVs from microbesare those produced by Gram-negative bacterial patho-gens that interact with humans. The vesicles assist inintermicrobial and host-microbe interactions, i.e. bio-film formation, surface attachment, and immunomo-dulation of host cells. The interaction outcome isdetermined through the delivery of molecules at higherconcentration and over longer distances while beingprotected in a membrane sphere (MacDonald andKuehn, 2012). By contrast, surprisingly little is knownabout EVs from plant-interacting microbes, althoughthe presence of EVs has been observed in culturedphytopathogens and in plant samples infected withmicrobes (Table 1). Since the 1980s, EVs were observedby electron microscopy in cultures of Erwinia amylovoraand E. carotovora, necrotrophic pathogens of the Enter-obacteriaceae that cause fire blight in fruits of Rosaceaeand bacterial soft rot in a wide range of vegetables,respectively (Laurent et al., 1987; Yaganza et al., 2004).Several hemibiotrophic pathogens of the Xanthomo-nadaceae release EVs in culture and during plant in-fection as determined by biochemical purification andelectron microscopy (Table 1): (1) culturedXanthomonascampestris pv campestris (strains 33913 and B100), thecausal agent of black rot disease in crucifers, releaseslipopolysaccharide (LPS)-positive EVs from the outermembrane (Sidhu et al., 2008; Bahar et al., 2016);(2) EVs from X. campestris pv vesicatoria strain 85-10,which causes bacterial leaf spot on tomatoes (Solanum
lycopersicum) and pepper (Capsicum spp.), have beenobserved in bacterial cultures and during the infectiousprocess in pepper leaves (Solé et al., 2015); (3) EVs havebeen purified from cultured X. oryzae pv oryzae strainPXO99, the causal agent of bacterial blight in rice (Oryzasativa; Bahar et al., 2016) and (4) cultured X. citri ssp.citri strain 306, which causes citrus canker (Ionescuet al., 2014); (5) EVs were observed in cultures and ex-tracellular fluids from plants infected with Xylella fas-tidiosa ssp. fastidiosa Temecula 1, responsible for Pierce’sdisease in grapevine (Vitis vinifera; Matsumoto et al.,2012; Ionescu et al., 2014; Nascimento et al., 2016);and (6) in cultured X. fastidiosa ssp. pauca (strains 9a5cand J1a12), causing citrus variegated chlorosis (Mendeset al., 2016; Santiago et al., 2016). Pathovars of thehemibiotroph Pseudomonas syringae, which infect Ara-bidopsis and tomato causing bacterial speck disease,were also reported to release EVs (Chowdhury andJagannadham, 2013; Bahar et al., 2016; E. Stigliano,K. Rybak, M. Janda, S. Robatzek unpublished data), aswell as Acidovorax citrulli M6, a biotrophic pathogenthat causes seedling blight and bacterial fruit blotchin cucurbits (Bahar et al., 2016). The fact that plant-interacting Gram-negative bacteria produce EVs, whichwere also observed in planta during infection (Soléet al., 2015), suggests that these EVs are involved incross kingdom communication between bacteria andplant cells (Fig. 1).
Whether other plant-interacting bacteria such aspathogenic Agrobacteria, Ralstonia, and beneficial Rhi-zobia produce EVs is unclear. Similarly, EVs have notbeen observed from the many plant-interacting fungiand oomycetes, including pathogens accounting forserious economic losses inmajor crops. However, it wasshown that both Phytophthora infestans andMagnaportheoryzae discharge virulence-associated proteins thatfunction inside plant cells, so-called cytoplasmic effec-tors, via unconventional protein secretion (Wang et al.,2010; Giraldo et al., 2013; Liu et al., 2014). Given thatEVs participate in unconventional protein secretionand that yeast and other fungi associated with humandiseases have been shown to release EVs (Miura andUeda, 2018), it is plausible to speculate that plant-interacting oomycetes and fungi produce EVs duringthe infectious process. Indeed, we recently detected EVsproduced from germinating spores of cultured M. ory-zae (P. Carranca Nunes Rosa, E. Stigliano, N. Talbot,S. Robatzek, unpublished data). Supporting evidence ofEVs released from plant-infecting fungi has beenobtained by TEM analysis. In a susceptible interaction,numerous EVs are present in the extrahaustorial ma-trix, the space at the interface of the plant cell and theinvading fungal haustorium, which remain of un-known origin (Micali et al., 2011; Fig. 1). Since abundantMVBs were observed in haustoria, it suggests thatG. orontii could release EVs into the extrahaustorialmatrix (Micali et al., 2011). Another insight into thepotential presence of fungal EVs comes from the find-ing that necrotrophic B. cinerea produces sRNAs thatsilence plant genes for promoting pathogen infection
(Weiberg et al., 2013; Wang et al., 2016, 2017), a processthat could involve EVs for delivery of the sRNAs.
IMMUNE MODULATION BY MICROBIAL EVS
It has been recognized that microbes produce EVs toaid infection success when colonizing a host. X. fastid-iosa ssp. fastidiosa Temecula 1 releases EVs to mediatebacterial detachment from plant cells, thus enablingmovement along and between xylem vessels (Ionescuet al., 2014). EV production in X. fastidiosa ssp. fastidiosaTemecula 1 is suppressed by the diffusible signal factor,an unsaturated fatty acid that accumulates as bacterialnumbers increase. Consequently, DrpfF mutants lack-ing diffusible signal factors exhibit hypervesiculationand are more virulent compared with wild-type strains(Newman et al., 2004). In this case, EVs represent amechanism of regulating bacterial-to-plant cell attach-ment to promote explorative colonization.Another benefit of EV production to pathogens is the
secretion of virulence factors that provide protectionagainst the degradative environment of extracellularplant fluids. Preliminary proteomic analysis identifiedthat EVs of cultured X. campestris pv campestris strainB100 are enriched in virulence determinants such ascellulase and xylosidase (two cell-wall-degrading en-zymes), components of the type-III-system and the se-creted proteins (Sidhu et al., 2008). X. campestris pvvesicatoria strain 85-10 produces EVs that contain
degradative enzymes, i.e. a putative protease andxylanase, and type-II-secreted virulence-associatedproteins (Solé et al., 2015). Similarly, the type-II-secreted lipase/esterase LesA is present in EVs of cul-tured X. fastidiosa ssp. fastidiosa Temecula 1 (Nascimentoet al., 2016). Consistent with a function as a virulencefactor, DlesAmutants display reduced infection rates ingrapevine. Preliminary proteomic analysis of EVs fromcultured P. syringae pv tomato T1 identified type-III-secreted effectors, i.e. AvrA1 and HopI1, known tosuppress plant immune responses (Chowdhury andJagannadham, 2013). Interestingly, we observed thatEVs purified from P. syringae pv tomato DC3000 cul-tures have immune-suppressing activities such thatthe vesicles impair prototypic pattern-triggered re-sponses and full antibacterial immunity (E. Stigliano,K. Rybak, M. Janda, and S. Robatzek, unpublisheddata; Fig. 1). Although evidence for EVs in the secretionof virulence factors is growing and the presence of EVshas been observed during the infectious process inleaves (Solé et al., 2015), it is unknown whether mi-crobial EVs discharge their cargo to the plant’s extra-cellular space and/or have the ability to interact withplant cells to deliver cargo into the cytosol (Fig. 1). Itwas also noted that the production and composition ofEVs differed depending on culture conditions (Sidhuet al., 2008; Ionescu et al., 2014). This is in agreementwith EV production representing an envelope stressresponse of Gram-negative bacteria, i.e. as experiencedduring host infection, so that the cargo of EVs is
Figure 1. Schematic representation ofEVs functioning in plant-microbe in-teractions. In brief, EVs released frommicrobes can exert immunogenic andimmune-suppressive activities, poten-tially both inside plant cells and in theextracellular space, i.e. apoplast andEHMx, and also could play roles inmicrobe-microbe interactions. Plant-produced EVs function in antimicro-bial immunity, i.e. delivering sRNAsand papillae formation. Details areexplained in the text. Dashed lines andquestion marks refer to possible butnot yet demonstrated functions. Bluecoloring indicates roles of EVs thatpromote plant immunity; red color-ing indicates roles of EVs in the poten-tial promotion of plant susceptibility.PRR, Pattern recognition receptor;PTI, pattern-triggered immunity; EHM,extrahaustorial membrane; EHMx,extrahaustorial matrix.
regulated (MacDonald and Kuehn, 2012). Therefore, itis possible that the EV cargo differs significantly whenexamined from extracellular fluids of infected plantsversus that of cultured bacteria.
Abundant components of EVs are LPS and elonga-tion factor Tu (EF-Tu), as shown for EVs ofX. campestrispv campestris, P. syringae pv tomato T1, and X. oryzae pvoryzae, respectively (Sidhu et al., 2008; Chowdhury andJagannadham, 2013; Bahar et al., 2016). Both LPS andEF-Tu represent microbe-associated molecular patterns(MAMPs), which activate pattern-triggered immunity(PTI) upon recognition by cognate plant-encoded im-mune receptors, i.e. the EF-Tu receptor (EFR; Boutrotand Zipfel, 2017). In agreement with the presence ofMAMPs at EVs, the vesicles provoked prototypic PTIresponses in plants. EVs from X. campestris pv cam-pestris strain 33913 stimulated the production of reac-tive oxygen species (ROS), ion release, and expressionof defense genes in Arabidopsis (Bahar et al., 2016).Representing a prototypic PTI response, the ROS burstinduced by EVs was dependent on EFR and revealedthat this immune response is triggered by EF-Tu pre-sent at EVs (Bahar et al., 2016). Since EF-Tu wasdetected in both cell-free supernatant and EV prepara-tions, it indicates that this MAMP is likely present at theoutside of EVs (Bahar et al., 2016).
Unlike the ROS burst, EV-induced defense gene ex-pression was neither mediated by EFR nor by the im-mune receptors that recognize the bacterial MAMPsflagellin and peptidoglycan, as well as aMAMP specificto Xanthomonas (Bahar et al., 2016; Boutrot and Zipfel,2017). However, EV-induced defense gene expressionwas partially dependent on BRI1-associated kinase1(BAK1) and suppressor of BAK1-interacting receptor1(SOBIR1) kinase (Bahar et al., 2016). Both BAK1 andSOBIR1 are essential coreceptors common to severalPTI signaling pathways including EFR-mediated im-munity (Boutrot and Zipfel, 2017). This suggests thatEVs from X. campestris pv campestris contain severalMAMPs, including yet unidentified patterns, whichcould be recognized by BAK1- and SOBIR1-dependentimmune receptors. It is also possible that EV-induceddefense gene expression involves recognition of LPSpresent at the vesicles, since BAK1 and SOBIR1 havenot been implicated in perception of LPS (Sidhu et al.,2008; Boutrot and Zipfel, 2017). Interestingly, Protein-ase K treatments, which would remove proteins fromthe outside of EVs, i.e. EF-Tu, improved the immuno-genic activity of the EVs (Bahar et al., 2016). This sug-gests that nonproteinaceous MAMPs (i.e. LPS) couldbecome more exposed from EVs, resulting in a strongerdefense gene induction.
Induction of defense genes was observed for EVsfrom three other Gram-negative bacteria, X. oryzae pvoryzae, P. syringae pv tomato DC3000, and A. citrulliM6 (Bahar et al., 2016; E. Stigliano, K. Rybak, M. Jandaand S. Robatzek, unpublished data). The ability ofEVs to induce plant immune responses demonstratestheir immunogenic potential. In addition, the activa-tion of the immune response involves EFR, BAK1, and
SOBIR1, which are cell-surface-localized receptors(Boutrot and Zipfel, 2017), thus suggesting that EVsinteract with plant cells (Fig. 1). It is worth noting thatthe level of defense gene expression was less inducedupon stimulation with EVs from P. syringae pv tomatoDC3000 compared to that with EVs from X. campestrispv campestris, X. oryzae pv oryzae, and A. citrulli, al-though P. syringae pv tomato DC3000 produced largeamounts of EVs (Bahar et al., 2016). Since EVs containimmunogenic determinants and virulence-associatedproteins (Chowdhury and Jagannadham, 2013), EVsgenerate an immunomodulatory response in plant cellsthat does not directly mimic the effect of purifiedMAMPs or transgenic expression of an effector.
Bacterial EVs also perform functions in intermicro-bial interactions (Fig. 1). EVs of X. fastidiosa ssp. paucacontain XfYgiT, a component of the toxin-antitoxinsystem known to regulate biofilm formation and beinvolved in the survival of X. fastidiosa ssp. fastidiosaTemecula 1 (Merfa et al., 2016; Santiago et al., 2016).Moreover, when microbial communities colonize ahost, such as in the context of the plant microbiota(Durán et al., 2018), EVs could represent a mechanismby which the donor microbe influences the competitormicrobe’s ability to adapt to the host environment(Barrett et al., 2011; Hammerschmidt et al., 2014), i.e.through promoting cell lysis (MacDonald and Kuehn,2012). This way, intermicrobial competition in theroot microbiota could involve EVs, observed as directantifungal activities and redundant determinant ofbacterial root commensals that protects plants againstthe detrimental root-derived filamentous eukaryotes(Durán et al., 2018). However, whether bacterial plantcommensals produce EVs and whether the vesicles in-teract with other microbes of the microbiota and thehost plant is not yet known.
PLANT DEFENSE BY EVs
Plant-derived EVs play also important roles in cell-to-cell communication with microbes. Several studieshave shown that immune stress stimulates EV secretionfrom plant cells. Both infection with P. syringae pv to-mato DC3000 bacteria and activation of immune sig-naling in response to the defense hormone salicylic acidincreases EV abundance in Arabidopsis extracellularfluids (Rutter and Innes, 2017). An obvious function ofEVs at the plant-pathogen interface is to serve as me-chanical protection against invading pathogens (Fig. 1).Indeed, plant resistance to pathogen entry is linked tothe polarized immune response (Kwon et al., 2008a).This pathway functions by concentrating EVs in theextracellular space beneath attempted sites of pathogenpenetration, resulting in the formation of so-called pa-pillae (Kwon et al., 2008a). When the pathway is al-tered by mutation in genes affecting vesicle fusion andsecretion, pathogen entry is increased (Collins et al.,2003; Kwon et al., 2008b; Nielsen et al., 2012). Consis-tent with serving as mechanical protection, papillae
contain callose, which is a b-1,3-glucan cell wall poly-mer (Xu and Mendgen, 1994; Meyer et al., 2009) thatcontributes to resisting pathogen entry (Assaad et al.,2004; Nielsen et al., 2012). Given that callose-containingMVBs have been observed in infected cells (Xu andMendgen, 1994), EVs could be involved in the deliv-ery of papillary callose. In agreement, the callose syn-thase GSL5/PMR4 is found at papillae together withthe PEN1 syntaxin, a biomarker of EVs (Meyer et al.,2009; Rutter and Innes, 2017). This polarized immuneresponse pathway is conserved in divergent plants—barley (Hordeum vulgare), bean (Phaseolus vulgaris), andArabidopsis—and functions in a similar fashion againstdiverse fungal pathogens, i.e. Blumeria graminis f. sp.hordei, Erysiphe graminis, and Uromyces vignae (Zeyenand Bushnell, 1979; Xu and Mendgen, 1994; An et al.,2006a, 2006b).Specific components of Arabidopsis EV cargo have
recently been identified and shown to be involved inantimicrobial defense, including the glucosinolatetransporters PEN3 and NTR1 as well as the myrosinaseEPITHIOSPECIFIER MODIFIER1 (Rutter and Innes,2017). Indole glucosinolate metabolism has been asso-ciated with innate immunity in response to diversefungal pathogens as well as insects (Zhang et al., 2006;Bednarek and Osbourn, 2009; Hiruma et al., 2010;Sanchez-Vallet et al., 2010; Campe et al., 2016). PEN3 isalso required for immunity against P. syringae pv tomatoDC3000 bacteria (Xin et al., 2013). Given that themyrosinase-glucosinolate defense system is normallycompartmentalized so that hydrolysis only occurs uponpathogen attack (Shirakawa and Hara-Nishimura,2018), we can speculate that different types of EVs arereleased. In addition, proteins involved in immunesignaling, i.e. BIR2, GRP7, RIN4, SOBIR1, and the po-larized immune response pathway, i.e. PEN1, SYP122,SYP132, are cargoes of Arabidopsis EVs (Rutter andInnes, 2017). Their association with EVs could indicatethat the vesicles play roles related to regulation of cel-lular homeostasis during immune signaling and regu-lation of EV release. Interestingly, EV abundance, butnot protein composition, changed significantly whenvesicles were purified from P. syringae pv tomatoDC3000-infected leaves (Rutter and Innes, 2017), sug-gesting that a default secretory pathway is adopted forimmune responses (Kwon et al., 2008b).Recently, sRNAs present in EVs, including vesicles
positive for TET8, were characterized, showing thatEVs of Arabidopsis were able to mediate a response infungal cells (Fig. 1) in an sRNA-dependent manner(Baldrich et al., 2018; Cai et al., 2018b). TAS1c-siR483,TAS2-siR453, IGN-siR1, and miRNA166 were enrichedin EVs purified from extracellular fluids of Arabidopsisinfected with B. cinerea compared with that in EVs fromuninfected plants (Cai et al., 2018b). Pathogen infectiontherefore modifies the quality of EVs released by Ara-bidopsis in addition to their quantity (Rutter and Innes,2017). The same sRNAs accumulated in fungal cells andwere shown to silence B. cinerea target genes duringinfection (Cai et al., 2018b). Accumulation of sRNAs in
fungal cells was lost in tet8 tet9 double mutants, sug-gesting that sRNA transfer to fungal cells involvedthese tretraspanins (Cai et al., 2018b).Moreover, sRNAspresent in EVs were protected against degradativeconditions, indicating their intraluminal localization.Consistent with a role in delivering ArabidopsissRNAs, EVs, including vesicles positive for TET8 andTET9, concentrated at fungal infection sites and accu-mulated inside Botrytis cells (Fig. 1). In this context,evidence lines up that sRNAs shown to function inhost-induced gene silencing, which is the transgenicexpression of double stranded RNA in plants to silencegenes in pests and pathogens (Knip et al., 2014), areloaded into EVs and thereby transferred to the infectingorganism (Cai et al., 2018a). Notably, abundant sRNAsderived from the same mRNA precursors, such asTAS1c-siR483 and TAS2-siR453, were not enriched inEVs (Cai et al., 2018b), suggesting that sRNA loading ofEVs involves a selective process. A recent study sup-ports the selective sRNA loading of EVs, albeit not forTAS1- and TAS2-derived sRNAs but for a specificgroup of miRNAs (Baldrich et al., 2018). This study alsodiscussed a role of EVs as waste disposal for tinysRNAs (Baldrich et al., 2018). Since at least two types ofEVs, PEN1- and TET8-positive, have been identifiedfrom plant extracellular fluids (Rutter and Innes, 2017;Cai et al., 2018b), it will be interesting to see whetherPEN1- and TET8-positive EVs represent overlapping ordistinct pools of EVs, i.e. related to their purificationusing 40,000g versus 100,000g (Rutter and Innes, 2017;Cai et al., 2018b), and whether this changes uponpathogen infection.
CONCLUSION
Although discoveredmany years ago, EV biology is arelatively recent research field in plant-microbe inter-actions. The findings obtained so far have establishedthat secretion of EVs is an important biological processshaping the interaction outcome between plants andmicrobes. First, EVs derived from microbes can (1) in-duce plant immune responses, such as those related toPTI; (2) inhibit plant immune strategies by delivery of
effectors; (3) favor explorative colonization of patho-gens; and (4) potentially inhibit competitor microbes.Second, EVs from plants play roles in disease resistancethrough (1) physically preventing pathogen penetra-tion, (2) inhibiting pathogen proliferation by transmit-ting toxic molecules, and (3) potentially regulatingimmune signaling in the form of removing molecularregulators from the cell surface. It will be challenging toelucidate the specific contribution of EVs to immunityand infection, sincemutations that affect the productionof EVs in both plants and microbes will likely be lethal.However, a more comprehensive understanding of thebiology of EVs—their molecular compositions, effectson recipient cells, and uptake mechanisms—is on thehorizon once EVs can be purified from cultured mi-crobes and extracellular fluids of infected plants. Futureresearch has to clarify standards for EV purification andvisualization, including defined biomarkers. Severalfunctional mechanisms of plant EVs remain to becharacterized (see Outstanding Questions).
ACKNOWLEDGMENTS
The authors would like to thank members of the Robatzek laboratory forfruitful discussions amd the group members Egidio Stigliano, Pedro CarrancaNunes Rosa, Martin Janda, and Prof. Nicholas Talbot (The Sainsbury Labora-tory) for agreeing to communicate unpublished data.
Received December 18, 2018; accepted January 23, 2019; published January 31,2019.
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