Extracellular Vesicles Isolated from the Leaf ApoplastCarry Stress-Response Proteins1[OPEN]

Brian D. Rutter and Roger W. Innes*

Department of Biology, Indiana University, Bloomington, Indiana 47405

ORCID IDs: 0000-0002-4354-9832 (B.D.R.); 0000-0001-9634-1413 (R.W.I.).

Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteinsand small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVshave not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from theapoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highlyenriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced inplants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs mayrepresent an important component of plant immune responses.

Eukaryotic cells secrete three main classes of extra-cellular vesicles (EVs), each with a distinct mechanismof biogenesis. Apoptotic bodies are the largest (1,000–5,000 nm in diameter) and most heterogenous of thethree classes. They are products of membrane blebbingreleased from cells during the late stages of pro-grammed cell death. The other two classes of EVs aremicrovesicles (100–1,000 nm in diameter) and exosomes(30–150 nm in diameter). Microvescicles are shed di-rectly from the plasma membrane (PM), while exo-somes are released from a cell after the fusion of amultivesicular body (MVB) with the PM (György et al.,2011; van der Pol et al., 2012; Akers et al., 2013)

Exosomes were originally thought to function as awaste disposal system, but more recent findings in-dicate that they mediate intercellular communicationand are capable of modulating immune responses.Exosomes secreted by immune cells present antigenson their surface and contribute to adaptive immunityby activating T cells (Raposo et al., 1996; Théry et al.,2002; Giri and Schorey, 2008). In certain cases,antigen-bearing exosomes provide an effective means

of vaccination (Wolfers et al., 2001; Aline et al., 2004;Altieri et al., 2004) and can serve as a source of diag-nostic biomarkers for various diseases (Welton et al.,2010; Foulds et al., 2012; Saman et al., 2012). Exosomesalso can down-regulate immune cells, facilitate normalgrowth and development (Hedlund et al., 2009), orpromote the spread of tumors and viruses (Skog et al.,2008; Meckes et al., 2010; Hood and Wickline, 2012).

Exosomes also mediate intercellular communicationby shuttling mRNAs and various species of smallnoncoding RNAs between cells. These molecules re-main functional after delivery and can elicit effects inthe recipient cell (Pegtel et al., 2010; Mittelbrunn et al.,2011; Ridder et al., 2014). The RNA content of exosomesvaries widely depending on the cell type and state ofpathology. For example, exosomes from malignant tu-mor cells have a microRNA content representative ofmetastatic tumors. Their RNA profiles can serve as anadditional diagnostic biomarker and help distinguishbetween malignant and benign cells (Taylor andGercel-Taylor, 2008; Pigati et al., 2010). Furthermore,the ability of exosomes to shuttle RNA from cell to cellsuggests that they could be used therapeutically toadminister nucleic acid drugs (Ohno et al., 2013).

Despite the many promising uses for exosomes,many basic biological questions regarding their bio-genesis, loading, secretion, and uptake remain unan-swered. To date, the majority of research on exosomeshas been performed using mammalian cell cultures,with just a few studies describing exosome productionin standard model organisms such as Caenorhabditiselegans (Liégeois et al., 2006) andDrosophila melanogaster(Beckett et al., 2013; Corrigan et al., 2014). Unfortu-nately, the plant kingdom has been largely absent fromthe field of exosome research, in spite of the fact thatexosome release had been observed in plant cells15 years before it was discovered in mammalian cells(Halperin and Jensen, 1967).

1 This work was supported by the National Institute of GeneralMedical Sciences of the National Institutes of Heath (grant no. R01GM063761 to R.W.I.), by the Bridge Funding program of the IndianaUniversity Office of the Vice Provost for Research, and by a MillerFellowship from the Indiana University Foundation (to B.D.R.).

* Address correspondence to rinnes@indiana.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Roger W. Innes (rinnes@indiana.edu).

R.W.I. conceived the idea of isolating exosomes from apoplasticwashes; B.D.R. developed the methods and performed all experi-ments and data analysis; B.D.R. wrote the article and prepared thefigures, with editing provided by R.W.I.

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The small amount of data on plant exosomes comesmainly from studies using transmission electron mi-croscopy (TEM). These studies show that MVBs pro-liferate in plant cells during pathogen attack and arefrequently observed in various states of fusion with thePM. The majority of these fusion events occur at or nearpapillae, extracellular defense structures that blockpathogen entry into the cell (Zeyen and Bushnell, 1979;An et al., 2006a, 2006b). GTPases that promote fusionbetween MVBs and the PM also accumulate aroundsites of infection and are required for the successfulformation of papillae (Böhlenius et al., 2010; Ebine et al.,2011; Nielsen et al., 2012). Furthermore, TEM hasshown that the papillary matrix contains small vesiclessimilar to those observed inside MVBs (Politis andGoodman, 1978). Combined, these data suggest thatexosome secretion in plants contributes to the devel-opment of early defense structures in response topathogens.Plant exosomes appear to mediate the transport of

various compounds and proteins into the extracellularspace. Important defense compounds, such as hydro-gen peroxide and callose (a b-1,3-Glc polymer), aretrafficked to the PM inside MVBs and accumulate in-side papillae (Xu and Mendgen, 1994; An et al., 2006a).The synthase responsible for producing papillary cal-lose, POWDERY MILDEW RESISTANT4, also is em-bedded within papillae, together with the syntaxinAtSYP121/PENETRATION1 (PEN1) and the ATP-binding cassette transporter PEN3 (Meyer et al., 2009;Ellinger et al., 2013; Underwood and Somerville, 2013).Significantly, mutation of PEN1 delays the formation ofpapillae (Assaad et al., 2004). In addition, exosome-mediated transport may facilitate the secretion ofmany other proteins in plants. On average, 50% of ex-tracellular proteins lack a signal peptide normally re-quired for secretion through the standard secretorypathway (Agrawal et al., 2010). A number of theseleaderless secretory proteins have antimicrobial activi-ties and are released in response to pathogens.Exosomes may also mediate the transport of small

interfering RNAs from plant cells into fungal cells.During host-induced gene silencing, small RNAsexpressed in a plant are able to reduce the expressionof target transcripts in invading fungi, specifically inthe haustoria (Nowara et al., 2010). Interestingly,TEM has revealed the presence of numerous vesiclesin the extrahaustorial matrix (Micali et al., 2011).While it is unclear if these vesicles originated in thefungus or the plant, they may mediate the transfer ofsmall RNAs between the two organisms. In supportof this hypothesis, it was reported recently that fun-gal microRNAs target genes in host plant cells, indi-cating that there exists a mechanism for the secretionof fungal microRNAs that are then taken up by thehost cell (Weiberg et al., 2013). Parasitic plants alsoform haustoria and can transport mRNAs into hosttissues. Parasitic plant mRNA can travel large dis-tances from the invading haustoria, suggesting that itmay be packaged inside exosomes (Kim et al., 2014).

Mammalian exosomes are regularly isolated fromculturedmedium and biological fluids, including blood(Caby et al., 2005), saliva (Palanisamy et al., 2010), se-men (Vojtech et al., 2014), urine (Pisitkun et al., 2004),milk (Admyre et al., 2007), cerebrospinal fluid (Streetet al., 2012), synovial fluid (Skriner et al., 2006), bron-choalveolar fluid (Prado et al., 2008), amniotic fluid(Asea et al., 2008), and fecal matter (Koga et al., 2011).To our knowledge, however, only a single laboratoryhas attempted to isolate plant exosomes. Regente et al.(2009) observed vesicles 50 to 200 nm in diameter in thefluids collected fromwater-imbibed sunflower (Helianthusannuus) seeds. To expand the current knowledge of plantexosomes,we purified vesicles from the apoplasticfluid ofArabidopsis (Arabidopsis thaliana) rosettes. These EVs areenriched in proteins related to stress and defense, includ-ing PEN1. The secretion of these EVs is enhanced duringinfection with a virulent strain of the bacterial pathogenPseudomonas syringae and in response to salicylic acid (SA).Our data suggest that exosomes contribute to innate im-munity and may mediate intercellular communication inplants as well as in animals.

RESULTS AND DISCUSSION

Vesicle-Like Structures Are Present in ArabidopsisApoplastic Fluid

EVs produced by mammalian cells are routinelyisolated from cell culture media and numerous bio-logical fluids. Isolation methods typically use a com-bination of filtration and differential centrifugationsteps. We applied these same techniques to isolate EVsfrom the apoplastic fluid of Arabidopsis rosettes.Briefly, whole rosettes were harvested and vacuuminfiltrated with a pH 6 MES buffer adapted from thatdescribed by Regente et al. (2009; see “Materials andMethods”). The infiltrated plants were carefully pack-aged inside syringes and centrifuged at a low speed.The resulting apoplastic wash was collected, filtered,and centrifuged successively at 10,000g, 40,000g, and100,000g. The 40,000g and 100,000g pellets (P40 andP100 fractions, respectively) were retained and resus-pended in buffer for further analysis.

To determine if vesicles were present in the apo-plastic fluid, we examined the P40 and P100 fractionsusing negative staining and TEM. The P40 fractioncontained numerous cup-shaped structures (approxi-mately 100 nm in diameter) reminiscent of vesicles(Fig. 1A). The cupped shape of these objects is an artifactof negative staining (Thery et al., 2006). The P100 fraction,however, was generally devoid of vesicle-like structuresand instead was dominated by small (approximately12 nm in diameter) circular objects (Fig. 1B).

We used light scattering to determine the size range ofparticles in each pellet prior to fixation. Particles in theP40 fraction ranged in size from approximately 50 to300 nm in diameter (Fig. 1C), with the most abundantspecies around 150 nm in diameter (Fig. 1D). Particles inthe P100 fractionweremuch smaller, ranging in size from

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approximately 10 to 17 nm in diameter (Fig. 1C), withthe most abundant species around 12 nm in diame-ter (Fig. 1D). These results demonstrate that vesicle-likestructures can be isolated from apoplastic fluid and that40,000g is a sufficient speed for their isolation.

PEN1 Is Associated with Apoplastic Vesicles

Based on the mammalian literature, EV preparationscontain a heterogenous mixture of vesicles originating

from different sources and possessing diverse func-tions. In animal systems, protein markers are used tohelp distinguish among various classes of EVs and ruleout contaminating cellular debris. For example, mam-malian exosomes are enriched in a particular subset ofproteins, including major histocompatibility complexesI and II, tetraspanins (CD9, CD63, CD81, and CD82),and heat shock proteins (HSC70 and HSP90; Wubboltset al., 2003). In plants, the protein content of EVs has notbeen described; however, the syntaxin PEN1 has been

Figure 1. The apoplastic wash from Arabidopsisleaves contains vesicle-like structures. A and B,Negative staining and TEM of the P40 and P100fractions derived from the apoplastic wash. C andD, Light-scattering charts showing peak intensitiesand percentages of differently sized particles inthe P40 and P100 fractions. The experiments wererepeated a minimum of three times with similarresults.

Figure 2. Arabidopsis EVs contain thesyntaxin PEN1. A, Confocal microscopyimages (inverted) of the P40 and P100fractions derived from the apoplastic washof nontransgenic (Columbia-0 [Col-0]) andGFP-PEN1 transgenic plants. Bars = 10mm.B, Quantification of fluorescent foci fromconfocal microscopy images. Letters sig-nify which values are significantly differentfrom each other based on a two-tailed un-paired Student’s t test (P , 0.05). C, Im-munoblots of the P40 and P100 fractionsconfirm that GFP-PEN1 is present only inthe P40 fraction of transgenic GFP-PEN1plants. The lysate lane indicates whole-leafprotein extracts. D, GFP-PEN1 expressedunder the control of a native promoter (NP)also accumulates in the P40 fraction. E,Detergent treatment removes GFP-PEN1from the P40 fraction. P40 fractions derivedfrom GFP-PEN1 plants were treated withbuffer alone or buffer plus TX100 followedby recentrifugation. Treatment with TX100removed GFP-PEN1 from the pellet. F,GFP-PEN1 in the P40 fraction is protectedfrom trypsin degradation in the absence ofdetergent. All experiments were repeated aminimum of three times with similar re-sults. Error bars in B indicate SD; n = 4; P,0.05 using a two-tailed unpaired Student’st test.

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shown to associate with extracellular membranousmaterial, suggesting that it may be packaged into exo-somes (Meyer et al., 2009; Nielsen et al., 2012).To determine if PEN1 is associated with the P40

fraction, we isolated vesicles from plants constitutivelyexpressing GFP-PEN1. When we examined the P40fraction using confocal fluorescence microscopy, weobserved numerous fluorescent spots on the surface ofthe slide. This fluorescent signal was not present in theP100 fraction when using GFP-PEN1 plants or in eitherpellet when using nontransgenic plants (Fig. 2, A andB). In agreement with these findings, an immunoblotfor GFP revealed the presence of GFP-PEN1 exclusivelyin the P40 fraction derived from GFP-PEN1 plants (Fig.2C). Furthermore, GFP-PEN1 was present in the P40fraction regardless of whether it was expressed underthe control of a 35S or native promoter (Fig. 2D), indi-cating that its presence in the apoplast-derived pellet isnot a result of overexpression.To test if the presence of GFP-PEN1 in the P40 frac-

tion is dependent on membranous structures, wewashed the pellet with either buffer alone or buffercontaining Triton X-100 (TX100) and recentrifuged atthe same speed. Treating the pellet with detergent be-fore the second centrifugation greatly reduced theamount of detectable GFP-PEN1 (Fig. 2E). This sug-gests that GFP-PEN1 is pelleting in association with theobserved vesicles.Papillary GFP-PEN1 fluorescence lasts for days after

its secretion, despite the unfavorably acidic conditionswithin the apoplast (Meyer et al., 2009). This observa-tion suggests that PEN1 is protected inside the lumen ofEVs. If GFP-PEN1 is associated with vesicles in the P40

fraction, then it should be largely shielded from prote-ase digestion. To test this hypothesis, we performed aprotease protection assay. When the P40 fraction wastreated with trypsin alone, GFP-PEN1 was protectedfrom digestion (Fig. 2F). Pretreating the pellet withTX100 followed by trypsin, however, completely elim-inated the GFP-PEN1 signal, indicating that PEN1 issheltered within the lumen of apoplastic vesicles.

Apoplastic Vesicles Are Enriched for PEN1

PEN1 was shown previously to mediate traffickingbetween the Golgi complex and the PM (Geelen et al.,2002). Therefore, GFP-PEN1-associated vesicles in theP40 fraction could be derived from intracellular vesiclesreleased by cellular rupture that could potentially occurduring vacuum infiltration or from reconstituted frag-ments of the PM. To test whether apoplastic vesiclesmight be artifacts produced by damaged cells, we iso-lated vesicles from plants expressing GFP-PEN1, thetrans-Golgi network/early endosome (TGN/EE)marker SYNTAXIN PROTEIN61 (SYP61-CFP; Robertet al., 2008), the late endosome marker ARA6-sYFP(Ueda et al., 2004), a marker for Golgi bodies(GmManI49-sYFP; Gu and Innes, 2012), or the PMmarker GFP-LOW TEMPERATURE INDUCED6b(LTI6b; Cutler et al., 2000). We used an immunoblot toprobe each P40 fraction for its respective fusion proteinand compared the intensity of the signals in the P40fraction with those obtained using whole-cell proteinextract. The results show that the P40 fraction isenriched for GFP-PEN1 and not the other markerstested (Fig. 3).

Figure 3. Apoplastic EVs are enriched for PEN1. A, Transgenic lines constitutively expressing the indicated endomembrane andPMmarkers were subjected to EV isolation, and the P40 fractions were analyzed by immunoblot. The lysate blot indicates whole-leaf protein extracts. Only GFP-PEN1 was readily detectable in the P40 fraction. B, Quantification of marker proteins in the P40fraction. Band intensitieswere quantified, and the ratio between the P40 and lysate bandswas calculated for each protein. PATL1,an EV-localized protein that we identified by mass spectrometry (Supplemental Table S1), was used to normalize for EV con-centrations in the P40 fractions. The specificity of the anti-PATL1 antibody is shown in Supplemental Figure S1. This experimentwas repeated a minimum of three times with similar results.

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While GFP-PEN1 localizes to the PM, the P40 fractionwas not enriched for GFP-LTI6b, suggesting that thevesicles are not reconstituted fragments of PM, nor arethey generated directly from the PM. Therefore, wesurmise that their origins are endosomal in nature. TheP40 fraction, however, was not enriched for the TGN/EE marker SYP61-CFP, the late endosome markerARA6-sYFP, or the Golgi marker GmMANI49-sYFP,suggesting that the P40 vesicles are not liberated en-dosomes or Golgi (Fig. 3). The absence of SYP61 isparticularly significant, because PEN1 and SYP61colocalize and interact on early endosomes (Drakakakiet al., 2012; Hachez et al., 2014). Combined, our resultssuggest that the vesicles in the P40 fraction are enrichedfor the exosome marker PEN1 but not for markers thatmay indicate cellular damage. Although some cellulardamage may have occurred during the isolation ofapoplastic wash, it is most likely not significant enoughto account for the presence of PEN1-rich vesicles.

Plant EVs Float in an Iodixanol Density Gradient

To further purify EVs and estimate their density, weused a discontinuous iodixanol (OptiPrep) densitygradient to isolate apoplastic vesicles from GFP-PEN1plants. After 17 h of centrifugation, we divided thegradient into 12 fractions. Each fraction was pelleted at100,000g, washed, and pelleted again.We examined thepellets derived from each fraction using an immunoblotfor GFP. The signal for GFP-PEN1 was spread out overseveral fractions (fractions 2–7) andwasmost concentrated

at densities ranging from 1.029 to 1.056 gmL21 (fractions2–4; Fig. 4A). Light scattering detected particles withinthese fractions with an average diameter of 165 nm (Fig.4B). We confirmed the presence of GFP-PEN1 fluores-cence in fractions 2 to 4 using confocalmicroscopy (Fig. 4,C and D) and observed vesicles using both negativestaining TEM and cryo-electron microscopy (Fig. 4, Eand F). The latter confirmed the presence of a lipidbilayer (Fig. 4F).

EV Recovery Is Not Affected by Treatment withBrefeldin A

The accumulation of GFP-PEN1 at powdery mildewinfection sites can be inhibited by pretreating leaveswith brefeldin A (BFA; Nielsen et al., 2012), a fungaltoxin that inhibits endomembrane trafficking by tar-geting ADP ribosylation factor-GTP exchange factorproteins (Nebenführ et al., 2002). We thus investigatedwhether pretreatment of Arabidopsis rosette leaveswith BFA would reduce the recovery of EVs in apo-plastic washes. Infiltration of 300 mM BFA (the concen-tration used by Nielsen et al. [2012]) into leaves 20 hprior to EV isolation did not reduce EV recovery basedon the detection of GFP-PEN1 (Supplemental Fig. S1). Itis difficult to interpret this result, however, because wedo not know the rate of EV turnover; thus, the EVs re-covered after 20 h of BFA treatment could have beenpresent prior to BFA treatment. In addition, PEN1 lo-calization to fungal penetration sites appears to bemediated, at least in part, by the recycling of PEN1 from

Figure 4. Density gradient purification ofEVs. A, Immunoblot detection of GFP-PEN1in fractions from the iodixanol density gra-dient. Fraction 1 consisted of 3.5mL, fraction2 was 1.7 mL, fractions 3 to 7 were 0.6 mLeach, and fractions 8 to 12 were 0.7 mLeach. B, Light-scattering charts showing peakintensities for pooled fractions 2 to 4. C,Confocal microscopy images (inverted) ofTris-HCl, pH 7.5, control (left) and pooledfractions 2 to 4 resuspended in Tris-HCl,pH 7.5 (right). Bars = 10 mm. D, Quantifi-cation of confocal images in C. Letters sig-nify values that are significantly differentfrom each other based on a two-tailed un-paired Student’s t test (P , 0.0001). E, Neg-ative staining and TEM of fractions 2 to 4.Bar = 10mm. F, Cryo-electronmicroscopy offractions 2 to 4. Note the lipid bilayer in thebottom right vesicle. Bar = 100 nm. All ex-periments were repeated at least two timeswith similar results. Error bars indicate SD;n = 3; P , ,, 0.001 using a two-tailedunpaired Student’s t test.

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the PM (Nielsen and Thordal-Christensen, 2012). BFA isknown to inhibit the endocytic pathway in plant cells(Emans et al., 2002) and, thus, might be expected toinhibit PEN1 relocalization by interfering with therecycling of PEN1 rather than by inhibiting the releaseof EVs.

Protein Content of Plant EVs

To better understand the protein content of plant EVsand gain insight into their potential roles, we analyzeddensity gradient-purified EVs from GFP-PEN1 plantsusing mass spectrometry. Our analysis identified 598proteins from two replicates. One hundred seventy ofthese proteins were common to both replicates and hadan average peptide count of at least two. Among theselected proteins, the chimeric GFP-PEN1was one of themost prominent, confirming that GFP-PEN1-associatedEVs are represented in the proteome (SupplementalTable S1).We next chose to analyze the EV proteome for Gene

Ontology (GO) terms related to biological processes.Compared with the whole Arabidopsis proteome, theEV proteomewas highly enriched for proteins involvedin biotic and abiotic stress responses. Approximately26% of the EV proteome was categorized under the GOterms responses to biotic or abiotic stimulus and re-sponse to stress. The genome-wide frequency for pro-teins with these GO terms is 11%, suggesting that EVs

are specialized for roles in defense and stress adapta-tion (Fig. 5).

Among the defense-related proteins, we recognizedseveral proteins involved in signal transmission, many ofwhich are highly induced in response to stress and/orcontribute to immunity. One of the most noteworthy ofthese proteins is RPM1-INTERACTING PROTEIN4(RIN4). RIN4 interacts with the disease resistance proteinsRESISTANCE TO PSEUDOMONAS SYRINGAE pv.MACULICOLA1andRESISTANCETOPSEUDOMONASSYRINGAE2. Modification of RIN4 by bacterial effectorsactivates resistance protein signaling and initiates an im-mune response (Mackey et al., 2002, 2003). Interestingly,wealso detected several proteins known to interact with RIN4,including ATPASE2 (AT4G30190), EARLY-RESPONSIVETO DEHYDRATION4 (AT1G30360), REMORIN(AT3G61260), and DELTA(24)-STEROL REDUCTASE(AT3G19820; Mackey et al., 2002; Liu et al., 2009). Thepresence of RIN4 and other proteins involved in immunesignaling suggests that EVs may play a role in microbe-associated molecular pattern- or effector-triggered immu-nity. It is tempting to speculate that, similar tomammalianexosomes, plant EVs modulate pathogen recognition bypromoting the extracellular trafficking of key signalingproteins. Such trafficking may rapidly remove proteinsfrom a cell to down-regulate signaling or spread signals toneighboring cells to amplify pathogen detection.

Other defense-related proteins included membersof the myrosinase-glucosinolate system, such as the

Figure 5. Plant EVs are enriched for stress-response proteins. The entire Arabidopsis proteome (top), the EV proteome fromuninfected plants (bottom left), and the EV proteome from P. syringae-infected plants (bottom right) were categorized based onGO terms through The Arabidopsis Information Resource Web site (www.arabidopsis.org). These analyses are based on the datagiven in Supplemental Table S1 and are derived from two biological replicates.

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glucosinolate transporters PEN3 (AT1G78900; Stein et al.,2006; Meyer et al., 2009; Underwood and Somerville,2013) and NRT1/PTR FAMILY2.10 (AT3G47960; Nour-Eldin et al., 2012) and themyrosinase EPITHIOSPECIFICMODIFIER1 (AT3G14210; Zhang et al., 2006). Myrosi-nases and glucosinolates are localized to separate com-partments in the cell and typically only interact afterdamage (Bones and Rossiter, 1996). Plant exosomes,however,may provide safe compartments for the storageand secretion of bioactive metabolites in intact cells(Underwood and Somerville, 2013). Once secreted, theseEVs may function as concentrated packets of antimicro-bial compounds. In this context, it is noteworthy thatPEN3 is one of the most abundant EV proteins based onpeptide counts (Supplemental Table S1) and has beenshown to accumulate around powderymildewhaustoria(Underwood and Somerville, 2013).

The EV proteome also contained proteins involvedin reactive oxygen species (ROS) signaling, such asPHOSPHOLIPASE Da (PLDa; AT4G35790) and PLDd(AT3G15730; Sang et al., 2001; Zhang et al., 2003), andoxidative stress responses, such as ANNEXIN1(AT1G35720), ASCORBATE PEROXIDASE1 (AT1G07890),andGLUTATHIONES-TRANSFERASEPHI2 (AT4G02520;Wagner et al., 2002; Davletova et al., 2005; Koussevitzkyet al., 2008; Konopka-Postupolska et al., 2009). Combined,these proteins suggest that EVsmay helpmodulate levelsof ROS or contribute to ROS signaling. Glucosinolatemetabolites induce extracellular ROS production (Hossainet al., 2013), so the pairing of proteins involved in themyrosinase-glucosinolate system and ROS signaling/protection in EVs may be functionally significant.

The EV proteome also was enriched for variousmembrane-trafficking proteins, including the syn-taxins PEN1 (SYP121), SYP122 (AT3G52400), SYP132(AT3G11820), and SYP71 (AT3G09740). PEN1, SYP122,and SYP132 belong to the same subfamily and facilitatetransport to the PM. Significantly, all three have beenshown to contribute to immunity (Nühse et al., 2003;Assaad et al., 2004; Kalde et al., 2007). Interestingly,SYP71 is localized mainly to the endoplasmic reticulumand is required for the infection of Arabidopsis by Turnipmosaic virus, where it plays a role in the fusion of virus-induced vesicles with chloroplasts (Wei et al., 2013). InLotus japonicus, LjSYP71 contributes to symbiotic nitro-gen fixation in nodules (Hakoyama et al., 2012).

Other trafficking proteins included various RABGTPases as well as PATELLIN1 (PATL1; AT1G72150)and PATL2 (AT1G22530). PATL1 and PATL2 both bindphosphoinositides and are thought to mediate vesicletransport and/or fusion (Petersen et al., 2014). PATL1has been localized to the cell plate in dividing cells butalso associates with PLASMODESMATA-LOCATEDPROTEIN1 (PDLP1) during downy mildew infectionand may colocalize with PDLP1 at the extrahaustorialmembrane (Caillaud et al., 2014). We confirmed thepresence of both PATL1 and PATL2 in the P40 fractionusing anti-PATL antibodies (Peterman et al., 2004; Denget al., 2007; Fig. 6A; Supplemental Fig. S1). We alsoshowed that PATL1 is associated with membranous

structures in the P40 fraction (Fig. 6B) and is protectedfrom proteolytic digestion, which suggests that PATL1 isprotected within the lumen of EVs (Fig. 6C). Thesefindings not only validate our mass spectrometry databut also provide an additional marker for identifyingEVs.

Finally, the EVs contained numerous proteins forthe transport of ions, water, sugar, and other sub-strates. The abundance of proton pumps in the EVproteome suggests that they actively transport ionsand may regulate their own membrane potentials.Alternatively, vacuolar ATPases could have a dualrole in vesicle trafficking and EV secretion. In C. ele-gans, the V0-ATPase complex promotes exosome se-cretion by mediating the fusion of MVBs with theapical PM (Liégeois et al., 2006). Plant EVs containnearly all of the proteins required to form a completeV1/V0-ATPase complex.

Comparisons with Other Proteomes

To better understand the potential origins and contentof EVs, we compared the list of EV proteins with otherpublished proteomes for various subcellular regions/compartments, including the PM, plasmodesmata, Golgi

Figure 6. Plant EVs are associated with PATL1 and PATL2. A, Immu-noblots of the P40 fraction and whole-leaf protein extracts using PATL1and PATL2 antibodies. B, Detergent treatment removes PATL1 from theP40 fraction. P40 fractions derived from Col-0 plants were treated withbuffer alone or buffer plus TX100. Following recentrifugation, thesample treated with TX100 containedmuch less PATL1. C, PATL1 in theP40 fraction is protected from trypsin degradation in the absence ofdetergent. All experiments were repeated a minimum of two times withsimilar results.

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complex, TGN/EE, MVBs/late endosomes, tonoplast,and clathrin-coated vesicle pits (Alexandersson et al.,2004; Fernandez-Calvino et al., 2011; Floerl et al., 2012;Heard et al., 2015). Overall, the EV proteome was mostsimilar to compartments associated with RABF2b/ARA7, a RAB GTPase distributed between the TGN/EE andMVBs. Approximately 59% of EV proteins were

reported to be present in the TGN/EE/MVB proteome(Table I). This level of similarity is consistent with ourhypothesis that EVs are derived from the intraluminalvesicles ofMVBs and, thus, are equivalent tomammalianexosomes. The EV proteome had no more than approxi-mately 54% overlapwith the remaining proteomes (TableI). Notably, we identified 23 EV proteins that were absent

Table I. Comparison of the EV proteome with other plant subcellular proteomes

Shared protein values indicate the percentages of EV proteins that were found in the indicated publishedproteomes.

Proteome Source Shared Proteins Publication Source

RABF2b/ARA7 (TGN/EE/late endosome) 58.58% Heard et al. (2015)RABD2a/ARA5 (Golgi/TGN/EE/secretory vesicles) 53.85% Heard et al. (2015)Plasmodesmata 53.25% Fernandez-Calvino et al. (2011)RABG3f (LE/MVB/tonoplast) 50.89% Heard et al. (2015)RABF1/ARA6 (LE/MVB) 49.70% Heard et al. (2015)PM 49.11% Alexandersson et al. (2004)CLC2 (clathrin-coated vesicle pits) 47.93% Heard et al. (2015)GOT1 (Golgi) 44.97% Heard et al. (2015)VAMP711 (tonoplast) 44.38% Heard et al. (2015)

Figure 7. EV secretion is enhanced during P.syringae infection. A, Total membrane content inthe P40 fraction increases over 2-fold followingspray inoculationwith a virulent P. syringae strain.Col-0 Arabidopsis plants were sprayed with eitherP. syringaeDC3000 (pVPS61:empty) at an opticaldensity at 600 nm of 0.2 or a control solutionlacking bacteria. EVs were isolated 3 d after theinitial infection from three sets of plants pertreatment. Each sample of EVs was stained with3,39-dihexyloxacarbocyanine iodide (DiOC6),washed, and repelleted. Fluorescence intensitywas quantified (error bars indicate SD; n = 3; P,,0.001 using a two-tailed unpaired Student’s t test).B to D, PATL1 and GFP-PEN1 contents in the P40fraction increase over 2-fold following P. syringaeinfection. B, Representative P40 fractions for bothtreatments, as well as samples of total cellularlysate, were immunoblotted for full-length PATL1.The levels of PATL1 are higher in the P40 fractionfrom infected plants compared with the P40fraction from mock-infected plants, as indicatedby a more intense band around 130 kD and theappearance of strong degradation products around20 and 10 kD. C and D, Quantification of PATL1and GFP-PEN1 band intensities from B. The ex-periment was repeated three times with similarresults. The results of only one experiment areshown.

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from the other endomembrane proteomes (SupplementalTable S2). These may represent useful markers for futurework on EVs.

When we compared GO terms for the EV proteomewith the other endomembrane proteomes, we foundthat the percentages for various biological processesremained similar across all proteomes. However, the EVproteome had a higher percentage of stress-responseproteins (Supplemental Table S3), suggesting that EVsmay play a role in adapting to biotic and abiotic stress.

We also used software to predict signal peptides in theEV proteome. We found that only 16% of the proteins inthe EV proteome have a predicted signal peptide, whichsupports our hypothesis that EV proteins reach theapoplast via a nonconventional secretory pathway.Combined, the above analyses suggest that plant EVsconstitute a distinct subcellular compartment.

The Plant EV Proteome Changes Little in Response toP. syringae Infection

Mammalian cells modify the composition and func-tion of exosomes during stress (Clayton et al., 2005;

Beninson et al., 2014). We questioned whether plantcells also might modify the protein content of EVsduring bacterial infection. In parallel with our proteo-mic analysis of EVs from uninfected plants, we exam-ined the protein content of EVs from plants infectedwith P. syringae pv tomato strain DC3000 (pVSP61:empty), which is virulent on Col-0 (Axtell et al., 2003).Three days after spray inoculating GFP-PEN1 plantswith P. syringae, we isolated and purified EVs for massspectrometry analysis. Our analysis identified 647 pro-teins from two replicates, 142 of whichwere common toboth replicates and had an average peptide count ofat least two. These were chosen for further analysis(Supplemental Table S1).We also analyzed our samplesfor proteins belonging to P. syringae to assess whetherwe had copurified bacterial outer membrane vesicles.However, we failed to reproducibly detect P. syringaeproteins between the two replicates.

Similar to the EV proteome from uninfected plants,EVs from infected plants were enriched for proteinsinvolved in stress and defense. Approximately 29% ofthe proteome had GO terms of responses to biotic orabiotic stimulus or response to stress, a slight increase

Figure 8. EV secretion is enhanced after SAtreatment. A, Total membrane content in the P40fraction increases over 2-fold following treatmentwith SA. Col-0 Arabidopsis plants were sprayedwith either 2 mM SA or a control solution lackingSA. EVs were isolated 12 h after spraying fromthree sets of plants per treatment. Each sample ofEVs was stained with DiOC6, washed, andrepelleted. Fluorescence intensity was quantified(error bars indicate SD; n = 3; P,, 0.001 using atwo-tailed unpaired Student’s t test). B to D, PATL1and GFP-PEN1 levels in the P40 fraction increaseover 3-fold following SA treatment. B, Represen-tative P40 fractions, as well as samples of totalcellular lysate, were immunoblotted for full-length PATL1. The levels of PATL1 are higher in theP40 fraction from SA-treated plants, as indicatedby a more intense band around 130 kD. C and D,Quantification of PATL1 and GFP-PEN1 band in-tensities from B. The experiment was repeatedthree times with similar results. The results of onlyone experiment are shown.

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from the first proteome (Fig. 5). However, when wecompared the two proteomes for protein content, wefound that they differed very little. When we tookinto consideration proteins that were identified inonly one replicate for either treatment, the differencesbetween the two proteomes essentially vanished.Only three proteins were unique to the EV proteomefrom infected plants, while eight proteins were uniqueto the EV proteome from uninfected plants. None ofthe proteins unique to either treatment have strongconnections to immunity. While the overall identity ofproteins associated with EVs did not vary significantlywith treatment, some proteins did have a much higheraverage peptide count in samples from infected plants.This suggests that, while alterations to the EV proteincontent may occur under stress, these changes areslight and likely involve differences in quantity ratherthan identity.

EV Secretion Is Enhanced during Biotic Stress

MVBs proliferate in plant cells challenged withpathogens, suggesting an increase in exosome secretion(An et al., 2006a, 2006b; Wang et al., 2014). Mammalianexosome secretion also is enhanced during stress(Clayton et al., 2005; Hedlund et al., 2011). To testwhether plant EVs are secreted in greater abundanceduring infection, we infected GFP-PEN1 Arabidopsiswith P. syringae. Three days after inoculation, we iso-lated EVs, quantified total membrane content in the P40fraction, and probed it for GFP-PEN1 and PATL1 usingan immunoblot. These analyses revealed a dynamicincrease in EV secretion during P. syringae infection.Infected plants secreted over twice as many EVs asuninfected controls (Fig. 7A). This increase in vesiclecontent was corroborated by an increase in GFP-PEN1and PATL1 signal in the P40 fraction of infected plants(Fig. 7, B–D).In order to determine if enhanced EV secretion is a

general immune response or a specific reaction toP. syringae, we treated GFP-PEN1 plants with the de-fense hormone SA and isolated EVs 12 h later. Similar toP. syringae-infected plants, the P40 fraction derivedfrom SA-treated plants contained twice as muchmembranous material as the P40 fraction from mock-treated plants (Fig. 8A). An immunoblot of the P40fractions revealed a similar increase in GFP-PEN1 andPATL1 (Fig. 8, B–D). Interestingly, during an infection,the majority of the PATL1 signal in P40 fractions waspresent as degradation products of 10 and 20 kD. Thesebands were not present in the P40 fractions from SA-treated plants. Combined, the data suggest that en-hanced EV secretion is a general immune response.

CONCLUSION

Reports of plant EVs date back to the late 1960s, buttheir function and composition remain poorly under-stood. To our knowledge, this is the first study toisolate and purify plant EVs from leaves. We also have

established plant EV markers and methods for thequantification of secreted vesicles. Our analyses haverevealed that plant EV production is enhanced in re-sponse to biotic stress and that they are enriched fordefense/stress-related proteins. Based on these findings,we expect plant EVs to play a prominent role in immu-nity. Previous studies have shown that EVs contributeto the formation of defensive papillae. They also mayfunction to deliver antimicrobial compounds into invad-ing pathogens or possibly stimulate immune responses inneighboring cells. How exosomes pass through the plantcell wall is not clear, but the fact that we can isolate themfrom apoplastic wash fluids confirms that they do.

Past research has only been able to observe localizedGFP-PEN1 secretion in the context of powdery mildewinfections, but our findings suggest that GFP-PEN1 isconstitutively secreted at some level inside EVs. Thefocal accumulation of GFP-PEN1 around fungal haus-toria is easily observable using confocal microscopy. Incontrast, EV secretion under normal circumstances islikely diffuse and, therefore, not as easy to detect.Constitutive EV secretion could provide some basallevel of apoplastic defense. However, EV secretion maybe a rapid process, and we cannot yet rule out thepossibility that buffer infiltration somehow stimulatesEV secretion.

The ability to isolate Arabidopsis exosomes representsan exciting opportunity to better understand plant im-munity and intercellular signaling. In this study, we havelaid the groundwork for asking bigger questions aboutplant EVs, such as howEVs contribute to plant immunity,which genetic pathways contribute to EV biogenesis,secretion, and uptake, and, most importantly, whetherEVs play a role in intercellular RNA transport.

MATERIALS AND METHODS

Plant Lines and Growth Conditions

The following transgenic plant lines were used in this study (all Col-0background): 35S::GFP-PEN1 (Meyer et al., 2009), native promoter::GFP-PEN1(Yang et al., 2014), SYP61-CFP (Robert et al., 2008), ARA6-sYFP andGmMANI49-sYFP (Gu and Innes, 2012), and GFP-LTI6b (Cutler et al., 2000).Arabidopsis (Arabidopsis thaliana) seeds were surface sterilized with 50% bleachand plated on 0.53 Murashige and Skoog medium containing 0.8% agar. Theseedswere vernalized for 2 d at 4°C before beingmoved to short-day conditions(9-h days, 22°C, and 150 mE m22 s21). After 1 week, the seedlings were trans-ferred to Pro-Mix PGX. Plants were grown for 5 to 7 weeks before harvest.

Vesicle Isolation

Vesicles were isolated from the apoplastic wash of Arabidopsis rosettes.Whole rosettes were harvested at the root and vacuum infiltrated with vesicleisolation buffer (VIB; 20mMMES, 2mMCaCl2, and 0.1 MNaCl, pH 6). This bufferwas based on that used by Regente et al. (2009), who isolated vesicles fromimbibed sunflower (Helianthus annuus) seeds. We modified their buffer byswitching from a Tris buffer at pH 7.5 to a MES buffer at pH 6, adding 2 mM

CaCl2, and eliminating b-mercaptoethanol. These changes were made in orderto more closely mimic native apoplastic fluids, with the expectation that wewould do less damage to cell walls and cells. However, we observed no obviousdifference in our ability to extract vesicles, and no further optimization wasattempted.

Infiltrated plants were carefully blotted to remove excess fluid, placed inside30-mL syringes, and centrifuged in 50-mL conical tubes at 700g for 20min at 2°C

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(JA-14 rotor, Avanti J-20 XP centrifuge; Beckman Coulter). The resulting apo-plastic wash was filtered through a 0.45-mm membrane and centrifuged suc-cessively at 10,000g for 30 min, 40,000g for 60 min, and 100,000g for 60 min at2°C (TLA100.3, Optima TLX ultracentrifuge; Beckman Coulter). The 40,000g and100,000g pellets (P40 and P100, respectively) were retained and resuspended inVIB for further analysis. For a majority of the experiments, only the 40,000gpellet was isolated. In these cases, the pellet was washed once with 3 mLof chilled VIB and recentrifuged at 40,000g for 60 min before final suspensionin VIB.

Discontinuous OptiPrep Gradient Vesicle Purification

To prepare discontinuous iodixanol gradients (OptiPrep; Sigma-Aldrich),solutions of 40% (v/v), 20% (v/v), 10% (v/v), and 5% (v/v) iodixanol werecreated bydiluting an aqueous 60%OptiPrep stock solution inVIB. The gradientwas formed by carefully layering 3 mL of 40% solution, 3 mL of 20% solution,3 mL of 10% solution, and 2 mL of 5% solution in a 14- 3 89-mm ultra-clearcentrifuge tube (Beckman Coulter). Approximately 0.5 mL of vesicles resus-pended in VIB was layered on top of the gradient. Centrifugation was per-formed at 100,000g for 17 h at 2°C (SW41, Optima XPN-100 ultracentrifuge;Beckman Coulter). When the vesicles were initially purified to determine theirdensity, 12 fractions were manually collected from the top of the gradient to thebottom. The first fraction consisted of 3.5 mL. The second fraction was 1.7 mL.Fractions 3 to 7 were 0.6 mL each, and fractions 8 to 12 were 0.7 mL each. Thedensity of each fraction was determined using a mock density gradient as de-scribed by Schröder et al. (1997), except that the fractionswere diluted 1:1,000 indeionized water and A244 was measured using a NanoDrop 2000 (ThermoScientific). When vesicles were purified for mass spectrometry, the first 4.5 mLat the top of the gradient was discarded. After that, 3 volumes of 0.7 mL werecollected. These fractions were each brought up to 3.5 mL with VIB andcentrifuged at 100,000g for 60 min at 2°C (TLA100.3, Optima TLX ultracentri-fuge; Beckman Coulter). The pellets were washed with 3.5 mL of VIB andrepelleted using the same centrifugation conditions. The resulting pelletswere combined by resuspending them in a total of 50 mL of VIB or 150 mL of20 mM Tris-HCl, pH 7.8, for immunoblot and mass spectrometry analyses,respectively.

Pseudomonas syringae Infections and SA Treatment

P. syringae strain DC3000 (pVSP61:empty) was grown as a lawn on King’sMedium B agar overnight at 30°C. The bacterial lawn was scraped from theplate and resuspended to an optical density at 600 nm of 0.2 using 10mMMgCl2plus 0.01% Silwet L77. Col-0 Arabidopsis plants were sprayedwith the bacterialsolution or a control solution lacking bacteria. Plastic domes were placed overthe plants overnight to maintain high humidity and removed the followingmorning. Three days after the initial infection, apoplastic wash was collectedfrom three sets of plants of both infected andmock-infected plants. The samplesfrom each set of plants were kept separate from each other during the course ofthe experiment to control for both biological and technical variations.

For SA treatment, plants were sprayed with either 20 mM MES, pH 6,+ 0.01% Silwet (control solution) or 2 mM SA, 20 mM MES, pH 6, + 0.01% Silwet.Plastic domeswere placed over the plants overnight tomaintain high humidity.Twelve hours after SA treatment, apoplastic wash was collected from three setsof plants of both SA- and mock-treated plants. The samples were kept separatefrom each other to control for both biological and technical variations.

Immunoblots

For immunoblots, 40 mL of resuspended vesicles in VIB was combined with10 mL of 53 SDS loading buffer (250 mM Tris-HCl, pH 6.8, 8% SDS, 0.1% Bro-mophenol Blue, 40% glycerol, and 400mM dithiothreitol) and heated at 95°C for5 min. Leaf lysate samples were used as positive controls. The lysate was pre-pared by freezing 250 mg of leaf tissue in liquid nitrogen and grinding with amortar and pestle. Ground leaf tissue was extracted in 500 mL of protein ex-traction buffer (150 mM NaCl, 50 mM Tris HCl, pH 7.5, 0.1% Nonidet P-40, and1% plant protease inhibitor cocktail [Sigma-Aldrich]) and centrifuged at10,000 rpm for 5 min at 4°C to pellet debris. Forty microliters of leaf lysate wascombined with 10 mL of 53 SDS loading buffer, and the mixture was heated at95°C for 5 min. All samples were loaded on 4% to 20% Precise Protein Gels(Thermo Scientific) and separated at 100 V for 1 h in BupH Tris-HEPES-SDSrunning buffer (Thermo Scientific). The proteins were transferred to a nitro-cellulose membrane (GE Water & Process Technologies). Ponceau staining was

used to confirm the equal loading of samples and successful transfer. Mem-branes were washed with 13 Tris-buffered saline (50 mM Tris-Cl and 150 mM

NaCl, pH 7.5) containing 0.1% Tween 20 (TBST) and blocked with 5% DifcoSkimMilk (BD) overnight at 4°C. Membranes were incubated with monoclonalmouse anti-GFP or anti-mCherry antibody (Abcam) at a 1:3,000 dilution orpolyclonal anti-PATL1 (Peterman et al., 2004; Deng et al., 2007) at a 1:5,000dilution for 1 h, washed with TBST, and incubated with horseradishperoxidase-labeled goat anti-mouse or anti-rabbit antibody (Abcam) at a 1:5,000dilution for 1 h. After a final wash in TBST, protein bands were imaged usingImmuno-Star Reagents (Bio-Rad) or Supersignal West Femto Maximum Sen-sitivity Substrates (Thermo Scientific) and x-ray film. Band intensities werequantified using the FlourChem E system with AlphaView SA software (ver-sion 3.4.0; ProteinSimple).

Fluorometric Quantification of EVs

For fluorometric assays, P40 fractions were resuspended in 100 mL of 10 mM

DiOC6 (ICN Biomedicals) diluted with MES buffer (20 mM MES, pH 6) plus 1%plant protease inhibitor cocktail (Sigma-Aldrich) and 2 mM 2,29-dipyridyl di-sulfide. The resuspended P40 fractions were incubated at 37°C for 10 min,washed with 3 mL of MES buffer, repelleted (40,000g, 60 min, at 2°C; TLA100.3,Optima TLXUltracentrifuge; Beckman Coulter), and resuspended in freshMESbuffer. DiOC6 fluorescence was recorded using an Appliskan plate reader(Thermo Electron) and 96-well, half-area plates (lot no. 16915037; Costar).Fluorescence intensity was measured at 485 nm excitation and 535 nm emission(using filters C00005X/E00035M) for 500 ms.

Dynamic Light Scattering

Light-scattering analyseswereperformedusingaZetasizerNano-S (MalvernInstruments) and a 50-mL quartz cuvette. Samples were equilibrated at 20°C for2 min followed by two readings consisting of 10 measurements each.

Confocal Microscopy and TEM

To detect vesicle-associated GFP fluorescence in ultracentrifuge fractions,resuspended pellets were examined using a Leica SP5 AOBS inverted confocalmicroscope (LeicaMicrosystems) equippedwith 320 numerical aperture 0.7 and363 numerical aperture 1.3 water objectives. GFP fluorescence was detectedusing an argon laser (488 nm excitation) and a 495- to 550-nm emission range.

For TEM, 6 mL of vesicles resuspended in VIB was applied to 3.05-mmcopper Formvar-carbon-coated electron microscopy grids (Electron Micros-copy Sciences). Prior to applying the samples, the grids were glow dischargedfor 45 s. Samples were wicked off using filter paper, and the grids were washedin 60 mL of 2% uranyl acetate. The grids were allowed to air dry and imaged at80 kV using a JEM-1010 transmission electron microscope (JEOL USA).

For cryo-electron microscopy, 6 mL of vesicles was applied to copperQuantifoil grids (R1.2/1.3; Electron Microscopy Sciences). Prior to freezing, thegrids were glow discharged for 45 s on each side. Grids were frozen using aVitrobot (22°C, 100% humidity, blot time of 4 s, drain time of 2 s, and 22-mmoffset to the grid’s position; FEI). The frozen samples were analyzed using aJEM 3200FS transmission electron microscope (JEOL USA).

Protease Protection Assay

To test if GFP-PEN1 is protected within the lumen of plant vesicles, P40fractions derived from35S::GFP-PEN1plantswere resuspended in 30mLof Tris-HCl, pH 7.8. Resuspended vesicles received one of three different treatments:(1) no treatment; (2) 1% TX100; or (3) 1% TX100 followed by 1 mg mL21 trypsin(Promega). For treatment with TX100, 6 mL of 6% TX100 was added. Thesamples were kept on ice for 30 min with occasional mixing. For trypsin di-gestion, 4 mL of 10 mg mL21 trypsin in 1 mM HCl was added to TX100-treatedsamples. The samples were incubated at 37°C for 1 h before being processed foran immunoblot.

BFA Treatment

For BFA treatment, a 50 mM stock solution of BFA (ApexBio) was preparedusing methanol. The stock solution was diluted in deionized water to create aworking solution of 300 mM BFA. Arabidopsis plants were hand infiltrated withthe working BFA solution or a mock solution containing an equivalent amount

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of methanol. The plants were covered with a dome after infiltration to maintainhumidity. Twenty hours after infiltration, apoplastic wash was collected fromthree biological replicates for both treatments. EVs were isolated from equalamounts of apoplastic wash for each sample. The P40 fractions were probed forGFP-PEN1 using an immunoblot.

Mass Spectrometry

EVs were isolated from native promoter::GFP-PEN1 transgenic Arabidopsisthat contained a T-DNA insertion in the native PEN1 gene (Collins et al., 2003).Samples were denatured in a solution of 8 M urea, then incubated for 45 min at57°C with 2.1 mM dithiothreitol to reduce Cys residue side chains. These sidechains were then alkylated with 4.2 mM iodoacetamide for 1 h in the dark at21°C. The urea was diluted to 1 M, then 1 mg of trypsin was added and thesamples were digested for 12 h at 37°C. The resulting peptides were desaltedusing a ZipTip (Millipore). The samples were dried down and injected into anEksigent HPLC device coupled to an LTQ Velos mass spectrometer (ThermoFisher Scientific) operating in top-eight data-dependent tandem mass spectrom-etry selection. The peptides were separated using a 75-mm, 15-cm column packedin house with C18 resin (Michrom Bioresources) at a flow rate of 300 nLmin21. A2-h gradient was run from buffer A (2% acetonitrile and 0.1% formic acid) to60% buffer B (100% acetonitrile and 0.1% formic acid). The resulting data weresearched in Protein Prospector against the Arabidopsis proteome in UniProt(downloaded January 21, 2015). Carbamidomethylation of Cys residueswas setas a fixed modification. Protein N-terminal acetylation, oxidation of Met, andpyro-Gln formation were set as variable modifications. A total of two variablemodifications were allowed. The mass tolerance for parent and precursor ionswas set to 0.6 D.

Proteins thatwere present in both replicates for a given treatment and had anaverage peptide count of no less than two were selected for further analysis. Allproteomes were categorized based on GO annotation using The ArabidopsisInformation Resource bulk data retrieval and analysis tools (https://www.arabidopsis.org/tools/bulk/go/index.jsp). Comparisons between the list ofEV-associated proteins and other published proteomes were accomplishedusing the Bioinformatics & Evolutionary Genomics Venn Diagram software(bioinformatics.psb.ugent.be). Signal peptides were predicted using SignalP4.1Server (http://www.cbs.dtu.dk/services/SignalP/; Petersen et al., 2011).

Accession Numbers

Arabidopsis Genome Initiative accession numbers for all proteins identifiedin our EVs are provided in Supplemental Table S1.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The anti-PATL1 antibody is specific for PATLprotein.

Supplemental Figure S2. Pretreatment with BFA does not affect EV recovery.

Supplemental Table S1. Plant EV proteome.

Supplemental Table S2. EV proteins not found in published endomem-brane proteomes.

Supplemental Table S3. Comparison of GO terms among Arabidopsissubcellular proteomes.

ACKNOWLEDGMENTS

We thank Dr. Claire Walczak for the use of equipment and Dr. StephanieEms-McClung for technical advice; Dr. Jonathan Trinidad and the Laboratoryfor Mass Spectrometry at Indiana University for the proteomic analysis andhelp describing the methods involved; Indiana University’s Electron Micros-copy Center, especially Drs. Barry Stein and DavidMorgan, for help with TEM,as well as the Indiana University LightMicroscopy Imaging Center for access tothe Leica SP5 confocal microscope; Drs. WilliamUnderwood and XangdouWeifor providing 35S::GFP-PEN1 and NP::GFP-PEN1 seeds, respectively; and Dr.Kaye Peterman for providing antisera to PATL1 and PATL2.

Received August 10, 2016; accepted November 3, 2016; published November 8,2016.

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Apoplastic Vesicles Carry Stress-Response Proteins