The Potyviral P3 Protein Targets Eukaryotic Elongation ...The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN]

The Potyviral P3 Protein Targets Eukaryotic ElongationFactor 1A to Promote the Unfolded Protein Response andViral Pathogenesis1[OPEN]

Hexiang Luan, M.B. Shine, Xiaoyan Cui, Xin Chen, Na Ma, Pradeep Kachroo, Haijan Zhi*, andAardra Kachroo*

National Center for Soybean Improvement, National Key Laboratory for Crop Genetics and GermplasmEnhancement, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China (H.L., N.M., H.Z.);Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546 (H.L., M.B.S., P.K., A.K.);and Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China (X.Cu., X.Ch.)

ORCID ID: 0000-0002-8401-5634 (H.L.).

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement,and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component ofthe translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viralpathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibitsaccumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducingchemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding theeEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance,suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets hostelongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stressresponse during pathogen infection.

Soybean mosaic virus (SMV) is a member of thelargest and most successful genus of plant pathogenicviruses, Potyvirus (Adams et al., 2005). SMV is seed-borne and aphid-transmitted, and causes mosaic andsevere necrosis in soybean. It affects both seed yield andquality and has caused up to ;35% reduction in yieldBased on the differential phenotypic reactions of spe-cific soybean cultivars, seven distinct SMV strains have

been described in North America (G1–G7, Cho andGoodman, 1979), and twenty-one strains in China(SC1–SC21; Wang et al., 2003; Li et al., 2010a). Resis-tance to the North American SMV strains is derivedfrom the Rsv (Resistance to SMV) loci, which comprisedominant resistance (R) genes (Buss et al., 1999; Hayeset al., 2000; Gunduz et al., 2002; Liao et al., 2002; Goreet al., 2002; Zheng et al., 2005). Of these, resistance de-rived from Rsv1 and Rsv3 is effective against strainsG1–G6 and G5–G7, respectively (Gunduz et al., 2002).Rsv4was initially thought to provide resistance againstall North American strains of SMV but was later shownto exhibit a late susceptible phenotype to strains G1 andG2 (Chen et al., 1993; Ma et al., 1995; Ma et al., 2002;Gunduz et al., 2004). Although none of the Rsv geneshave been cloned, some downstream signaling com-ponents that regulate Rsv-derived resistance have beenidentified (Fu et al., 2009; Zhang et al., 2012;Wang et al.,2014). The microRNA and small interfering RNApathways are also known to regulate defense againstSMV (Chen et al., 2015). Resistance to the strains fromChina is derived from the Rsc loci, and these have beenmapped to chromosomes 2, 13, and 14 in the cultivarsKefeng 1, Qihuang, and Dabaima, respectively (Wanget al., 2011; Ma et al., 2011; Zheng et al., 2014).

The SMV genome is a single-stranded positive-senseRNA, which like other potyviruses, is processed as asingle large polypeptide that is later cleaved by viral

1 This work was supported by the Kentucky Soybean PrmotionBoard, the National Natural Science Foundation of China(31371646, 31571690), the National Soybean Industrial TechnologySystem of China (CARS-004), Jiangsu Collaborative Innovation Cen-ter for Modern Crop Production (JCIC-MCP) and the Fund of Trans-genic Breeding for Soybean Resistance to Soybean mosaic virus(2016ZX08004-004).

* Address correspondence to [email protected] or [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Aardra Kachroo ([email protected])

H.L. and M.B.S. performed most of the experiments; X.Cu, X.Ch.,and N.M. provided technical assistance; P.K. conceived some exper-iments and complemented the writing; H.Z. and A.K. conceived theproject and supervised the experiments; and A.K. wrote the manu-script with contributions from H.L. and M.B.S.

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encoded proteases to yield mature proteins. SMV en-coded mature proteins include P1 (proteinase), helpercomponent-proteinase (HC-Pro), P3, 6K1, cylindricalinclusion (CI), 6K2, viral protein genome-linked (VPg),nuclear inclusion a proteinase (NIa-Pro), nuclear in-clusion b (NIb, RNA-dependent RNApolymerase), andcoat protein (CP). Transcriptional slippage at a singlenucleotide insertion site within the P3 cistron resultsin an additional polypeptide, P3N-PIPO. This poly-peptide is only produced by a very small fraction ofthe viral RNA, which contains the single nucleotideinsertion (Olspert et al., 2015). P3N-PIPO contributesto cell-to-cell movement of the virus in susceptiblehosts (Chung et al., 2008; Wen and Hajimorad, 2010).HC-Pro, along with P3 and Cl, regulates SMV virulence(Eggenberger et al., 2008; Hajimorad et al., 2008; Seoet al., 2009; Zhang et al., 2009; Hajimorad et al., 2011;Seo et al., 2011; Chowda-Reddy et al., 2011; Wen et al.,2011). The precise biochemical function of SMV P3, andindeed the potyviral P3 protein in general, is largelyuncharacterized due to the lack of structural or func-tional motifs in its sequence. Roles in viral replication,movement, virulence activity, and in one case, avir-ulence, have been suggested (Rodriguez-Cerezo et al.,1993; Sáenz et al., 2000; Desbiez et al., 2003; Jenner et al.,2003; Choi et al., 2005; Hajimorad et al., 2005, 2006;Hjulsager et al., 2006; Eggenberger et al., 2008;). Somepotyviral P3 proteins were detected in the nucleoli ofearly-infected cells, whereas others were detected ex-clusively in the cylindrical inclusions of infected cells(Rodriguez-Cerezo et al., 1993; Langenberg and Zhang,1997). Some potyviral P3 proteins interact with the NIa,HC-Pro, P1, and NIb of the respective virus, but theseinteractions have not yet provided major insights intothe function of P3. In case of SMV, P3 from the G7Hstrain did not interact with any other viral protein,whereas the Pinellia ternata isolate showed weak inter-action with NIa and NIb (Guo et al., 2001; Kang et al.,2004; Lin et al., 2009).

Here, we attempted to determine the role of the SMVP3 in soybean-SMV pathogenesis. We find that theER-localized SMV P3 interacts with eukaryotic elon-gation factor 1 alpha (eEF1A), which is essential forviral virulence in the susceptible host. eEF1A is a cy-toplasmic protein that delivers aminoacylated tRNAsto ribosomes during polypeptide elongation whenbound to GTP. After tRNA transfer, eEF1A is recycledto its active GTP-bound form by the guanine nucleotideexchange factor, eEF1B. eEF1A interacts with the RNAdependent RNApolymerases (RdRp) of tobaccomosaicvirus and turnip mosaic virus and is known to be as-sociated with the replication complexes of potyviruses,tobamoviruses, and tombusviruses (Yamaji et al., 2006;Nishikiori et al., 2006; Thivierge et al., 2008; Li et al.,2010b).We also show that SMV replication is associatedwith the unfolded protein response (UPR) and thateEF1A contributes to this. UPR is associated with ERstress and involves transduction of the ER stress signalvia the ER membrane receptor protein kinase-like ERkinase (PERK), the inositol-requiring transmembrane

kinase and endonuclease 1a (IRE1), and the activa-tion of transcription factor 6 (ATF6) in mammals(Walter and Ron, 2011). The plant UPR comprises twobranches, one mediated by IRE1 and the other byATF6-like receptors, which involves the transcriptionfactors bZIP17 and 28 (Howell, 2013). Viral infection inplants is known to induce the IRE1 arm of UPR, whichinvolves IRE1-mediated splicing of bZIP60 mRNA,resulting in the synthesis of a functional bZIP proteinthat can activate ER stress-inducible promoters in thenucleus (Ye et al., 2011, 2013; Verchot 2014). Examplesinclude induction of bZIP60 and UPR chaperone geneexpression by the ER associated movement protein ofpotato virus X (Ye and Verchot, 2011) and the turnipmosaic virus 6K2 protein-induced splicing of bZIP60mRNA (Zhang et al., 2015). We show that plantslacking eEF1A or the associated eEF1B show reducedUPR in response to SMV infection and better resistviral accumulation.

RESULTS

SMV Infection Is Associated with the Induction of UPR-Associated Gene Expression in Soybean

In an effort to better understand the process of SMVinfection in soybean, we carried out ultrastructuralanalysis of SMV-infected soybean leaves from resistantand susceptible cultivars at 10 and 20 d postinfection(dpi) with SMV. At these time points, the resistantcultivar did not exhibit visual symptoms of infection,whereas the susceptible cultivar showed clear mosaicsymptoms (data not shown). In contrast to the resistantcultivar, the SMV-inoculated susceptible plants showedpinwheel-type inclusions in the cytoplasm, and therewas a progressive increase in these inclusion structuresat later time points of infection (Fig. 1A). No othermajor cellular changes were noticeable between themicroscopic ultra sections prepared fromSMV-inoculatedresistant and susceptible cultivars. In wheat, thepinwheel structures associated with Wheat spindlestreak virus infection are considered to be derivedfrom the endoplasmic reticulum (ER; Langenberg andSchroeder, 1973).

To test if there was any association between SMVand the ER membrane, we fractionated total extractsfrom SMV-infected soybean leaves on a Suc gradientand examined the presence of SMV coat protein (CP)in various subcellular fractions. Western blot analysisshowed that SMV CP co-purified with fractions con-taining the ER-localized binding protein (BiP, chape-rone protein that restores proper protein folding duringER stress, Fig. 1B). We next tested if ER membrane re-organization due to pinwheel structure formation inSMV infected plants was associated with UPR, in-volving the accumulation of unfolded proteins at theER membrane. For this, we examined the mRNA levelsof genes associatedwith UPR, including those encodingBiP; IRE1, the ER resident protein sensor; bZIP, the

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downstream transcription factors that activate genesrequired for coping with UPR and the associated ERstress; and NRP, the cell death domain-containingAsn-rich protein that is associated with the ER stress-induced cell death response. qRT-PCR analysisshowed that the soybean orthologs (Silva et al., 2015)of these genes BiP-D, NRP-A, bZIP68 (ortholog ofAtbZIP60, downstream regulator of the IRE branch ofUPR), and four isoforms of IRE1 were significantlyinduced in SMV (G5)-infected plants (cv Essex, Fig.1C, left and middle panels). Interestingly, SMV-infected plants also showed induction of bZIP37 andbZIP38 (ortholog of AtbZIP17/28, Fig. 1C, rightpanel), which function in the ATF6-like receptorbranch of UPR. Induction of UPR was further con-firmed in western blot analyses of BiP protein, whichwas significantly higher in the SMV infected plants(Fig. 1D).We next tested whether ER stress altered response to

SMV. Soybean (Glycine max cv Essex) plants weretreated with the ER stress-inducing agents, DTT andtunicamycin. DTT disrupts disulphide bonding, while

tunicamycin inhibits N-linked glycosylation of secretedglycoproteins, both of which affect protein foldingresulting in ER stress (Howell, 2013). Control plantswere treated with water or DMSO, respectively. Inter-estingly, SMV was detected earlier and accumulated atmuch higher levels in plants treated with DTT or tuni-camycin as compared to control plants treated withwater or DMSO, respectively (Fig. 1, E and F). In con-trast, DTT or tunicamycin did not alter the in plantalevels of two other viruses that are not associated withthe ERmembrane (Fig. 1, G andH). Turnip crinkle virus(TCV) replicates on the outer mitochondrial membrane(Russo and Martelli, 1982; Blake et al., 2007), whereascucumber mosaic virus (CMV) is thought to replicate inthe cytoplasm (Zitter and Murphy, 2009). Accumula-tion of neither TCV nor CMVwas significantly differentin DTT- or tunicamycin-treated Arabidopsis (Arabi-dopsis thaliana) plants as compared to the respectivecontrol plants (Fig. 1, G and H). Together, these resultssuggested that SMV infection induces ER stress insoybean, which in turn promotes virus accumulation inthe plant.

Figure 1. ER stress inducers promote SMV accu-mulation in soybean. A, Transmission electronmicrograph of mesophyll cells from infectedleaves of resistant (R, 4.03 magnification) andsusceptible (S, 5.03 magnification) plants at10 dpi. M, Mitochondria; Ch, chloroplast; OG,osmophilic globule; VP, virus particles; PW, pinwheel structures. Scale bars = 1 mm. B, Westernblot analysis of protein extracts from SMV-infected(4 dpi) plants separated on a Suc gradient. Lanenumbers indicate pools of consecutive fractions.SMV CP and BiP were visualized using protein-specific antibodies. C, Relative mRNA levels ofthe indicated ER stress marker genes in SMVinfected soybean plants as determined by qRT-PCR. D, Western blot analysis showing levels ofthe ER localizing BiP-D protein at indicated dpiwith SMV. E, ELISA of SMV levels in soybeanplants (cv Essex) treated with water, DTT, DMSO,or tunicamycin (Tun) at indicated dpi with SMV.Error bars indicate SD (n = 3). Asterisks denotesignificant difference from the correspondingcontrol plants (water for DMSO and DTT for Tun),t test, P , 0.0001. F–H, Western blot analysisshowing coat protein (CP) levels of SMV (F), TCV(G), and CMV (H) in plants (soybean for SMV,Arabidopsis for TCV and CMV) were treated within water, DTT, DMSO, or tunicamycin (Tun) andsampled at indicated days postinfection with therespective virus. Experiments were done using thecultivar Essex and G5 strain of SMV. Results arerepresentative of 2–3 independent experiments.

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EF1A Regulates UPR and SMV Resistance



SMV P3 Interacts with GmEF1A

Based on the above results, we considered the pos-sibility that SMV virulence proteins might target plantproteins to induce ER stress. Therefore, we conducted ayeast two-hybrid (Y2H) screen to identify soybeanproteins that interacted with SMV P3 protein, becauseP3 is associated with virulence and localizes to theER (see below). Of the several P3-interacting proteinsidentified, we proceeded to characterize the P3 inter-actorGmEF1A (Glyma17g186600) because itwas identi-fied multiple times in the Y2H screen. The predictedGmEF1A protein sequence contained conserved se-quences of the eukaryotic eEF1A protein family, in-cluding the GTP/Mg+2 binding site (Andersen et al.,2001), the EF1B alpha binding site, the switch I and IIregions that undergo conformational changes uponGTP binding (Sprang, 1997), and the G box motifs

(GDP/GTP-binding motif elements) of the Ras super-family GTPases (Colicelli, 2004; Wennerberg et al.,2005; Supplemental Fig. S1A).

The interaction of full length GmEF1A with SMVP3 was confirmed using bimolecular florescence com-plementation (BiFC) and coimmunoprecipitation (IP)assays. For BiFC assays, proteins were tagged with re-ciprocal N- or C-terminal halves of enhanced yellowfluorescent protein (nEYFP and cEYFP) and transientlyco-expressed in Nicotiana benthamiana. Coexpression ofGmEF1A with SMV P3 cloned from G5 stain resultedin reconstitution of EYFP, indicating positive interac-tion (Fig. 2A). Likewise, GmEF1A also interacted withP3 from SMV strains G7 and SC15 (SupplementalFig. S1B). In contrast, P3 did not interact with GST(glutathione-S-transferase), and GmEF1A did not in-teract with SMVHC-Pro (Fig. 2A), although all proteins

Figure 2. eEF1A interacts with the P3 protein of SMV. A, Bimolecular fluorescence complementation (BiFC) assay showing inplanta interactions. 403 magnification of micrographs at 48 h postinfiltration from plants co-expressing nYFP-GmEF1A withcYFP-P3, cYFP-HC-Pro, or cYFP-GSTare shown. TransgenicN. benthamiana expressing CFP-H2B (nuclear localized histone 2B)were used for the BiFC assays. Images are representative of three separate infiltrations from two independent experiments for eachinteraction using both combinations of n/cYFP fused proteins. Scale bars = 10 mm. B, Immunoprecipitation assay using proteinscoexpressed in N. benthamiana. MYC-tagged SMV P3 was co-expressed with FLAG-tagged GmEF1A. Proteins were immuno-precipitated (IP-FLAG) from total extracts using a-FLAG antibodies and visualized using tag-specific antibodies. Results arerepresentative of two independent repeats. C, C-terminal YFP/CFP-tagged proteins were transiently expressed individually(GmEF1A-CFP, P3-YFP) or co-expressed (P3-YFP, GmEF1A-CFP) in N. benthamiana via Agro-infiltration. Scale bars = 10 mm.GmEF1A-CFP was expressed in transgenic plants expressing RFP-H2B (nuclear localized). P3-YFP was expressed in transgenicplants expressing RFP localized to the ER via the N-terminal signal sequence of Arabidopsis basic chitinase and a carboxy-ter-minal HDEL ER-retention signal. Images in two different focal planes are shown for P3-YFP to enable ideal visualization of the ERaswell as the nucleus (indicated bywhite arrowhead in the lowermost left panel). Bottom italicized letters indicate the fluorescentchannel used for imaging. D, MYC-tagged SMV P3 or GmEF1A were expressed individually in N. benthamiana. Total (T) leafextracts and membrane (M) or soluble (S) fractions were analyzed using MYC-specific antibodies. TIP (tonoplast intrinsic protein,membrane specific) and Hsc70 (cytosolic heat shock protein 70) were used to test purity of the fractions. Blots are representativefor all experimental repeats.


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were adequately expressed inN. benthamiana (SupplementalFig. S1C). For IP assays, proteins were transiently co-expressed in N. benthamiana as MYC- or FLAG-taggedderivatives. IP assays confirmed the interaction betweenP3 and GmEF1A (Fig. 2B). The GmEF1A-P3 interactionis unlikely to involve P3N-PIPO because this interac-tion was detected using heterologously expressed P3,whereas P3N-PIPO is specifically made from viral RNAvariants generated by the potyviral replicase (Olspertet al., 2015, Rodamilans et al., 2015).GmEF1A is predicted to be a cytoplasmic protein

(PlantLoc probability: 1.0, http://cal.tongji.edu.cn/PlantLoc/index.jsp). Indeed, transiently expressedGmEF1A did localize primarily to the cell periphery(Fig. 2C, middle panel), and was detected in the solubleprotein fraction, suggesting cytoplasmic localization(Fig. 2D). On the other hand, P3 co-localized primarilywith the ERmarker in planta (Fig. 2C, lower panel) andwas detected in the membrane fractions of leaf extracts(Fig. 2D). However, the localization of GmEF1A andP3 proteins was inconsistent with the BiFC result thatshowed interaction in the cell periphery as well as inthe nucleus (Fig. 2A). To address this, we assayedlocalization in leaves co-expressing both GmEF1A andP3 proteins. Interestingly, consistent with BiFC data,co-expression of GmEF1A and P3 resulted in increasednuclear localization of both GmEF1A and P3 (Fig. 2C,top panel). Together, these results suggested that P3and GmEF1A promote each other’s relocalization to thenucleus, which explains their interaction in the nuclearcompartment.

GmEF1A Proteins Negatively Regulate SoybeanResistance to SMV

The Glycine max genome contains four otherGmEF1A-like sequences. These includeGlyma05g089000,Glyma10g212900, Glyma16g068000, and Glyma19g052400,the predicted protein sequences of which shared;86% identity with GmEF1A (Supplemental Fig.S1A). Quantitative (q) RT-PCR analysis showed thattranscript levels forGmEF1A (Glyma17g186600) was themost abundant in every tissue analyzed (cotyledon,leaf, stem, flower, and roots), and these were expressedto highest levels in leaf tissues (Supplemental Fig.S2A). Furthermore, SMV infection resulted in sig-nificant (P , 0.0001) induction of Glyma17g186600expression (Supplemental Fig. S2B). AlthoughSMV infection also induced Glyma05g089000 ex-pression, its levels were much lower than those ofGlyma17g186600.We next tested if GmEF1A contributed to soybean

resistance against SMV. For this, we knocked down theGmEF1A genes in soybean using the bean pod mottlevirus (BPMV)-based VIGS (virus-induced gene silenc-ing) vector (Zhang and Ghabrial, 2006; Kachroo andGhabrial, 2012). We generated a vector derived fromGlyma17g186600 (Supplemental Fig. S3A), which wasexpected to knock down the expression of all isoforms

simultaneously because the various isoforms share.90% nucleotide identity. Plants (cv Essex) infectedwith the vector were tested for GmEF1A transcriptlevels using qRT-PCR. Notably, mRNA levels of all butone (Glyma16g068000) GmEF1A isoforms were signif-icantly reduced in the GmEF1A-knockdown plants(SEF1A) as compared to that in plants infected with aBPMV control vector (contains a nonspecific sequence,V; Supplemental Fig. S3B). Consistent with the reducedtranscript levels, GmEF1A protein levels were alsosignificantly reduced in the SEF1A plants as comparedto V plants (Fig. 3A).

Knockdown of GmEF1A expression did not alterthe morphology of soybean plants (Fig. 3B, top panel).The V and SEF1A plants (cvs. Essex and Essex-Rsv1) wereinfectedwith the G5 isolate (virulent on Essex, avirulenton Essex-Rsv1) of SMV. The SEF1A plants developedsignificantly reduced SMV-related symptoms as com-pared to V plants (Fig. 3B, lower panel). This correlatedwith the protein gel blot and ELISA analysis of totalprotein extracts, which showed that SMV accumulationin the inoculated as well as systemic leaves of SEF1Aplants was much delayed compared to V plants ofEssex cultivar (Fig. 3, C and D). Likewise, the levelsof SMV positive-sense RNA were also significantlylower in the SEF1A plants than in V plants at 4 and 7 dpi(Fig. 3E), even though both plants contained compa-rable levels of SMV RNA at the time of infection(Supplemental Fig. S4A). Furthermore, SMV infectionresulted in much higher induction of PR1 expression inthe SEF1A plants than in V plants (Fig. 3F). Similar toSMV G5, the G7 strain of SMV, which is virulent onboth Essex and Essex-Rsv1, was detected at much latertime points in these backgrounds (Supplemental Fig.S4B). In contrast to the Essex plants, SMV G5 was notdetected in either V or SEF1A Essex-Rsv1 plants (datanot shown), indicating that GmEF1A did not contributeto Rsv1-derived resistance to SMV. Notably, the SEF1Aplants showed reduced accumulation of another ER-associated virus, TRSV (tobacco ring spot virus; Hanand Sanfaçon, 2003; Fig. 3G), but accumulated similarlevels of BPMV (Fig. 3C) and Bean yellow mosaic virus(BYMV) as did control plants (Supplemental Fig. S5A).Like TCV and CMV, the ER stress-inducing agenttunicamycin did not affect BYMV accumulation(Supplemental Fig. S5B). Together, these results sug-gest that GmEF1A specifically affects SMV accumula-tion in susceptible plants.

Knockdown of GmEF1A Expression Renders PlantsInsensitive to SMV- or Chemical-Induced UPR

Our results that ER stress-inducing chemicals in-hibited SMV replication, and that SEF1A plants showedenhanced resistance to SMV, prompted us to test thepossible link between eEF1A and UPR. We determinedthe response of the SEF1A plants to chemically inducedER stress. The V and SEF1A plants (cv Essex) were treatedwith the ER stress-inducing agent DTT, and control



















plants were treated with water. Plants were then as-sessed for expression of the BiP-D transcript usingqRT-PCR analysis. BiP-D mRNA levels increased by;1.6-fold in DTT-treated V plants as compared to thewater-treated counterparts. In contrast, BiP-D mRNAlevels showed minimal increase in response to DTTtreatment in the SEF1A plants (Fig. 4A). This suggestedthat the SEF1A plants might be impaired in chemical-induced ER stress response. We next tested the tran-script levels of other genes associated with UPR,including BiP-D, IRE1a-d, and bZIP37, 38, and 68, andNRP-A. SMV infection significantly (P , 0.0001) in-duced the mRNA levels of all of these markers in Vplants (Fig. 4, B and C). In contrast, SMV-infected SEF1Aplants showed induction of only bZIP68 and bZIP38mRNA at levels comparable to V plants. The SMV-infected SEF1A plants also showed induction of IRE1band IRE1c mRNA, though at significantly lower levelsthan V plants; IRE1b/c mRNA levels in SMV-infectedSEF1A plants were comparable to their respective levelsin uninfected V plants (Fig. 4C). This suggested thatSMV-induced UPR was attenuated in the SEF1A plants.Consistent with their gene expression data, and unlikeV plants, the SEF1A plants did not accumulate the BiPprotein in response to SMV infection (Fig. 4D). Wetested whether the ER stress inducers DTT or tunica-mycin altered response to SMV in the SEF1A plants be-cause these ER stress-inducing agents promoted SMVaccumulation in wild-type plants. Both DTT and tuni-camycin treatments resulted in increased SMV accu-mulation in V plants similar to wild-type plants (Fig. 1,E and F, and Fig. 4). In contrast, these chemicals did notsignificantly increase SMV accumulation in the SEF1Aplants (Fig. 4E). Together, these results indicate thatknockdown of GmEF1A expression alters the plantsability to induce ER stress, which in turn correlates withtheir enhanced resistance to SMV.

GmEF1A Is Required for Pathogen- and Chemical-InducedCell Death

SMV G7 induces a typical systemic cell death phe-notype termed lethal systemic hypersensitive response(LSHR) on plants containing the Rsv1 locus (Hajimoradand Hill, 2001, Hajimorad et al., 2003). We noticed thatSMV G7 did not induce visible or microscopic LSHR onSEF1A plants (Fig. 5, A and B). This in turn correlatedwith reduced induction of the HR marker gene Hsr203jin SMV G7-infected SEF1A plants (Fig. 5C). Together

Figure 3. Silencing GmEF1A alters soybean response to SMV. A,Western blot analysis showing GmEF1A protein levels in plants infectedwith the control BPMV vector (V) and the GmEF1A knockdown (SEF1a)plants. GmEF1A was detected using plant eEF1A specific antisera.Ponceau staining was used as control for protein loading. B, Visualsymptoms of V and SEF1A plants (cv Essex) before (upper panel) and after(lower panel) infection with SMV-G5. C, Western blot detecting SMVcoat protein (CP) in inoculated (I) and systemic leaves (S) of V and SEF1Aplants at indicated days postinfection (dpi) with SMV. Proteins weredetected using antibodies specific to SMVor BPMVCP. Total extracts of Iand S leaveswere used. Ponceau stainingwas used as control for proteinloading. D, ELISA of SMV CP levels in V and SEF1A plants (cv Essex) atindicated dpi (4, 6 dpi from I, and 10 dpi from S) with SMV (G5 or G7).Error bars indicate SD (n = 3). Asterisks denote significant difference

from V, t test, P , 0.0001. E and F, Quantitative RT-PCR analysisshowing relative levels of SMV positive sense RNA (E) or PR1mRNA (F)in V and SEF1A plants at indicated dpi with SMV. Error bars indicate SD(n = 3). Asterisks denote significant differences from V for each timepoint, t test, P , 0.0001. G, ELISA of Tobacco ring spot virus (TRSV)levels in Vand SEF1A plants at indicated dpi. Results are representative of2–3 independent repeats.


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these results suggested a correlation between celldeath and ER stress and that EF1A was required forSMV-induced cell death. The SEF1A plants also exhibitedimpaired cell death when treated with paraquat (1,1’-dimethyl-4,4’- bipyridinium dichloride), which inducescell death by promoting the formation of reactiveoxygen species (ROS; Farrington et al., 1973; Hiyamaet al., 1993); paraquat induced cell death on V, butnot on SEF1A plants (Fig. 5D). Expression analysis ofgenes encoding ROS-quenching enzymes showed ele-vated basal levels of ascorbate peroxidase (APX) andglutathione-S-transferase (GST) in SEF1A plants (Fig. 5E),which correlated with their increased tolerance toparaquat.We next evaluated if silencing GmEF1A alters HR-

associated cell death that occurs during the pathogenresistance response because ROS contributes to the cell-death response. We chose to assay HR response againstthe bacterial pathogen Pseudomonas syringae pv. glycinea(Psg) expressing avrB because Rsv1 conferred extremeresistance that is not associated with visible or micro-scopic cell death against SMV. To this end, we gener-ated SEF1A plants in the Rpg1-b background (cv Merit)and infected themwith Psg avrB. Rpg1-b is the R proteinthat specifies resistance to Psg avrB in soybean (Ashfield

et al., 2004). As expected, Psg avrB induced HR-likemicroscopic cell death on V plants, but no cell deathwas observed in the Psg avrB-infected SEF1A plants (Fig.5F). Consistent with their microscopic phenotype, SEF1Aplants showed reduced ion leakage (Fig. 5G) and in-creased susceptibility to Psg avrB (Fig. 5H). This in turncorrelated with induction of the Glyma17g23900 iso-form of GmEF1A in response to infection by Psg avrB(Fig. 5I). Notably, Psg avrB infection induced transcriptlevels of the ER stress marker BiP-D in V plants butnot in SEF1A plants (Fig. 5J), suggesting that Psg avrB-induced cell death is also associated with ER stress.

eEF1B Participates in ER Stress and SMV Resistance

Because eEF1A is a subunit of the eukaryotic trans-lation elongation complex, it is possible that the role ofeEF1A in ER stress and SMV resistance is linked to itsrole in translation elongation. eEF1A recruits codon-specific aminoacyl-tRNAs to the ribosome duringtranslation elongation, and this involves conversion ofGTP to GDP. The eEF1B subunit, a guanine nucleotideexchange factor, then restores the GTP moiety to reac-tivate eEF1A. Thus, lack of eEF1B should potentially

Figure 4. Knockdown of GmEF1A expressioninhibits soybean response to ER stress inducers.A–C, Quantitative RT-PCR analysis showing (A)fold increase in the mRNA levels of BiP-D in DTT-treated plants (cv Essex) knocked down forGmEF1A mRNA expression (SEF1A) or infectedwith the control BPMV vector (V), or (B and C)relative expression of mRNA levels of indicatedgenes in V and SEF1A plants at 0 and 4 dpi withSMV G5. Error bars indicate SD (n = 3). Asterisksdenote significant difference from V, t test, P ,0.0001. D and E, Western blot analysis withponceau staining used as control for proteinlevels. D, BiP levels in V and SEF1A plants at indi-cated dpi with SMV G5. E, SMV coat protein (CP)in water- or tunicamycin (Tun)-treated V and SEF1Aplants at indicated dpi with SMV G5. Results arerepresentative of 2–3 independent repeats.





render eEF1A inactive, and these plants should there-fore produce phenotypes similar to those displayed byGmEF1A knockdown plants. To test this, we firstidentified the GmEF1B homolog (Glyma13g073200)that contained the eEF1B guanine nucleotide exchangedomain (cd00292, Andersen et al., 2000) and a GSTC-terminal domain (pfam00043, Dixon et al., 2002).The soybean genome contained three other sequencesincluding Glyma02g276600, Glyma04g195100, andGlyma14g039100 that showed ;86% amino acid iden-tity to GmEF1B (Supplemental Fig. S6A). Although therelative transcript abundance of the GmEF1B isoformsdiffered in different tissues (Supplemental Fig. S6C, leftpanel), transcripts of all four isoforms were inducedin response to SMV infection (Supplemental Fig. S6C,middle panel).

We knocked down the expression of GmEF1B insoybean using the BPMV-based vector by selecting aregion that was common between all GmEF1B iso-forms (Supplemental Fig. S6B). Plants (cv Essex)infected with the BPMV-GmEF1B vector were testedfor knockdown of the respective genes using qRT-PCR. As expected, transcript levels of all GmEF1Bisoforms were reduced in the GmEF1B-knockdownplants (SEF1B; Supplemental Fig. S6C, right panel) butthis did not alter the morphology of the plant (datanot shown). The V, and SEF1B plants (cv Essex) wereinfected with the virulent G5 isolate of SMV and virusaccumulation was monitored in the inoculated andsystemic tissues over time. Western blot and ELISAanalysis of total protein extracts showed that SMVaccumulation in the inoculated as well as systemicleaves of SEF1B plants was much delayed compared toV plants (Fig. 6, A and B). Likewise, the SEF1B plantsalso showed enhanced resistance against the virulentG7 strain of SMV, but not against BYMV (Fig. 6, Aand C). As seen for SEF1A plants, the SEF1B plants ac-cumulated higher mRNA levels of defense markergene PR1 in response to SMV infection than the Vplants (Fig. 6D).

Next, we tested the response of the SEF1B plants tochemically induced ER stress. The V, and SEF1B (cvEssex) plants were treated with DTT or tunicamycinand assessed for transcript levels of the marker genesBiP-D and NRP-A using qRT-PCR analysis. As seen forSEF1A plants, both BiP-D and NRP-A were induced tohigh levels in V plants treated with DTT or tunicamy-cin, but not in SEF1B plants (Fig. 6E). Similarly, SMV

Figure 5. Knockdown of GmEF1A expression inhibits the cell deathresponse in soybean. A and D, Visual phenotypes on leaves of Essex-Rsv1 GmEF1A knockdown plants (SEF1A) or those infected with thecontrol BPMV vector (V) in response to infection by SMV-G7 (A) or spottreatment with paraquat (D). B and F, Trypan blue staining showingmicroscopic cell death in V and SEF1A leaves (cv Essex-Rsv1) infectedwith SMV-G7 (B) or Rpg1-b plants (cv Merit) infected with Psg avrB (F).C and E, Relative mRNA levels of the hypersensitive response markergene Hsr203j (C), or the antioxidant genes ascorbate peroxidase (APX)and glutathione-S-transferase (GST) (E) as determined by quantitativeRT-PCR. Error bars indicate SD (n = 3). Asterisks denote significantdifference from M or V plants of the corresponding genotype as deter-mined by t test. C, Expression in V (white bars) and SEF1A (black bars)plants (cv Essex-Rsv1) treated with buffer (M) or infected with SMV (G7)and sampled at 7 dpi, P , 0.0002. E, Expression in M (white bars) orparaquat black bars)-treated plants, P , 0.0001. G, Electrolyte leakagein V and SEF1A plants at 24 h post infiltration of MgCl2 or infection withPsg avrB. Error bars indicate SD (n = 5). Very small error bars are notvisible. H, Bacterial counts in V and SEF1A plants (Rpg1-b, cv Merit)

infiltrated with Psg Vir, or avrB (105 cfu/ml). LOG10 values of colonyforming units (cfu) per unit leaf disc from infected leaves at 0 (whitebars) or 4 (gray/black bars) days postinoculation (dpi) are presented.Error bars indicate SD (n = 5). Asterisks denote significant difference fromV plants infected with the corresponding strain (Student’s t test P ,0.001). I and J, Relative mRNA levels of GmEF1A isoforms (I) or the ERstress marker gene BiP-D (J), in Rpg1-b (cv Merit) plants at indicated dpiwith Psg avrB, and as determined by quantitative RT-PCR. Asterisksdenote significant difference from 0 dpi (Student’s t test, P , 0.0001).Results are representative of 2–3 independent experiments.


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infection did not induce expression of any of the UPR-associated genes in SEF1B plants (Supplemental Fig. S7).Furthermore, the ER stress inducers DTT or tunicamy-cin resulted in increased accumulation of SMV in Vplants, but not in SEF1B plants (Fig. 6, F and E, datashown for tunicamycin). This suggested that, likeSEF1A, the SEF1B plants were impaired in the chemical-and SMV-induced UPR. Together, these results in-dicate that GmEF1A and GmEF1B contribute to UPR,and thereby SMV resistance. Notably, unlike eEF1A, P3did not interact with eEF1B in BiFC or Co-IP assays(Supplemental Fig. S8), suggesting that GmEF1B likelyfunctions via GmEF1A in mediating UPR.

DISCUSSION

Eukaryotic elongation factor 1A (eEF1A) is an es-sential protein that is not only involved in proteintranslation but also performs a variety of cellular func-tions including nucleocytoplasmic trafficking, proteindegradation, cytoskeletal regulation, and activationof phosphatidylinositol 4-kinase. Here, we show a newrole for eEF1A as an important regulator of UPR. UPR

involves the accumulation of unfolded proteins at theER and often occurs during the ER stress response,an important coping mechanism for plants under-going biotic or abiotic stresses (Liu and Howell, 2010).Depending on the severity and length of stress condi-tions, UPR can either induce autophagy as a mecha-nism of cell survival or result in cell death (Williamset al., 2014). We show that the potyvirus SMV inducesUPR in soybean, and this in turn promotes virus accu-mulation in the plant. We further show that the eEF1Asubunit of the translation elongation machinery is es-sential for this response and thereby SMV proliferationin soybean.

Considering its abundance (1%–3% of total protein)and multifunctional properties, eEF1A is an idealpathogen target (reviewed by Li et al. [2013]). For in-stance, binding of eEF1A to aminoacylated viral RNAhas been suggested to enhance viral protein translationwhile repressing viral minus-strand RNA synthesis(Matsuda et al., 2004). eEF1A has also been suggested tostabilize viral replicase complexes and facilitate viralreplicase complex or particle assembly (Ott et al., 2000;Li et al., 2010b; Warren et al., 2012). This correlates with

Figure 6. Knockdown of GmEF1B expression al-ters soybean response to SMV. A, C, and G,Western blot showing SMV or BYMV coat protein(CP) in GmEF1B knockdown plants (SEF1B), orthose infected with the control BPMV vector (V) atindicated days postinfection (dpi) with virus.Plants were infected with (A) SMV G5 or G7, (C)BYMV, and (F) SMV G5. Proteins were detectedusing antibodies specific to SMV or BYMV CP.Total extracts from inoculated (0, 4 dpi), or sys-temic (7, 10, 14 dpi) leaves were used. Ponceaustaining was used as control for protein levels. B,ELISA of SMV levels in soybean plants (cv Essex) atindicated dpi (4, 6 dpi from inoculated, and 10 dpifrom systemic) with SMV (G5). Error bars indicateSD (n = 3). Asterisks denote significant differencefromV, t test, P, 0.0001. D, Relative levels of PR1mRNA in SMV-infected plants at 0 (white bars)and 7 dpi (black bars) as determined by quanti-tative RT-PCR analysis. Error bars indicate SD (n =3). Asterisks denote significant difference from V, ttest, P , 0.0001. E, Fold increase in BiP-D andNRP-AmRNA in V and SEF1B plants in response totreatment with the ER stress inducer DTT. Errorbars indicate SD (n = 3). Asterisks denote signifi-cant difference from V, t test, P , 0.0005. F,Western blot analysis of BiP levels in V and SEF1Bplants at 4 dpi with SMV G5. Ponceau stainingused as control for protein levels. Results arerepresentative of 2 to 3 independent experiments.







the fact that eEF1A binds RdRps of some viruses(Yamaji et al., 2006; Li et al., 2013). Indeed, in addition toSMV P3, GmEF1A also interacts with the SMV RdRP,NIb (Supplemental Fig. S9). This is similar to thebinding of eEF1A with multiple HIV-1 (human immu-nodeficiency virus) proteins (Cimarelli and Luban,1999; Allouch and Cereseto, 2011; Abbas et al., 2014Warren et al., 2012). In particular, the altered localiza-tion of GmEF1A in the presence of SMV P3 and theeffect of GmEF1A on pathogen/paraquat-induced celldeath is strikingly similar to the effect of the HIV-1 Nefprotein on eEF1A and apoptosis. Nef associates witheEF1A and promotes its nuclear-cytoplasmic transportin monocyte-derived macrophages, which in turn in-hibits ER stress-induced apoptosis (Abbas et al., 2014).Thus, P3 might affect GmEF1A function by promotingincreased nuclear localization of the protein in infectedcells. This, together with the comparable virulence-related roles of SMV P3 and HIV-1 Nef (enhancesHIV-1 replication and survival in infected cells and fa-cilitates in vivo spread of the virus) suggest possibleparallel mechanistic functions for these proteins. Forinstance, Nef is known to bind tRNAs and promoteannealing between primer tRNA and HIV-1 RNA(Ecchari et al., 1997). This in turn could be linked to theability of eEF1A to bind aminoacylated tRNAs. Testingthe ability of P3 to bind tRNAs and GmEF1A to bindSMV RNA might help determine the role of the P3-GmEF1A interaction in SMV pathogenesis. Clearly,the interaction between P3 and eEF1A does not ap-pear to be important for R protein-derived signalingbecause P3 from several SMV strains is able to interactwith eEF1A, and silencing GmEF1A did not inhibitresistance derived from the Rsv1 locus.

It is also possible that the interaction with P3affects posttranslational modifications and therebythe function of GmEF1A. For example, the additionof a phosphatidylethanolamine (PE) moiety can affectthe activity of eEF1A (Hiatt et al., 1982; Whiteheartet al., 1989; Rosenberry et al., 1989; Venema et al., 1991;Ransom et al., 1998; Zobel-Thropp et al., 2000). Indeed,all the five GmEF1A isoforms contain two PE bindingsites that are highly conserved in plant eEF1A proteinsbut have not been reported in the yeast EF1A. Inter-estingly, in yeast, GmEF1A localizes to the nucleus inthe absence of SMV P3 (Supplemental Fig. S10). Thisraises the possibility that PE binding could be impor-tant for the extra-nuclear localization of eEF1A, whichmay be impaired in the presence of P3 thereby facili-tating the retention of eEF1A within the nucleus inplanta. The biological significance of the nuclear local-ization of eEF1A or P3 proteins remains unknown atpresent.

SMV-induced UPR correlates with the copurificationof SMV CP with the ER membrane fraction and isconsistent with a previous report suggesting the ER asthe initial site of replication of the potyvirus Turnipmosaic virus (TuMV;Wei et al., 2010). Notably, only theIRE branch of UPR, but not the bZIP17/28 branch, wasshown to regulate TuMV pathogenesis (Zhang et al.,

2015). In contrast, SMV infection resulted in the in-duction of IRE1 and its downstream regulator bZIP68,as well as that of bZIP37/38, orthologs of AtbZIP17/28that function in the other branch of UPR (Silva et al.,2015). Functional analysis of bZIP37 and bZIP38 inSMV-infected plants would help resolve their true in-volvement in SMV pathogenesis. The mechanism un-derlying ER stress activation by plant viruses or themechanism of viral perception by ER stress sensorsremain unknown. Our results suggest that binding ofplant eEF1A with the viral P3 protein could at least bepart of such a stress response leading to ER stress.Consistent with this notion, knockdown of GmEF1Aexpression inhibited the plants ability to induce UPR inresponse to chemical ER stress inducers or pathogeninfection. Thus, UPR promotes SMV accumulation inwild-type plants and knock down ofGmEF1A results inenhanced resistance. The role of GmEF1A in ER stressand SMV resistance is likely linked to its function intranslation elongation because silencing genes encod-ing the GmEF1B subunit of the translation elonga-tion complex also enhanced resistance to SMV andinhibited ER stress. Testing the ER stress response inplants lacking other members of the translation elon-gation complex, and analyzing response to SMV inplants expressing the GTPase-defective derivative ofGmEF1A, could further elucidate the precise relation-ship between translation elongation and the ER stressresponse. Notably, the GmEF1A/B knockdown plantsdid not exhibit enhanced resistance to another potyvi-rus, BYMV. The replication site for BYMV is not known,but it is possible that the involvement of eEF1A/B in ERstress affects plant response to only those viruses thatrecruit the ER membrane for proliferation.

Reduced BiP-D transcript in the GmEF1A andGmEF1B knockdown plants suggests that eEF1A andeEF1B regulate the mRNA levels of BiP, which in turn isthought to negatively regulate UPR (Leborgne-Castelet al., 1999; Alvim et al., 2001; Carvalho et al., 2014).Notably, overexpression of BiP in soybean and tobaccoamplified the HR response (Carvalho et al., 2014) andconversely BiP-silenced tobacco plants showed atten-uated HR. Furthermore, 35S::BiP plants showed higherSA levels and increased expression of SA-responsivemarker gene PR-1 (Carvalho et al., 2014). Together,these results suggest that BiP levels are critical forUPR as well as plant defense. Consistent with resultsreported for BiP-silenced tobacco plants (Carvalhoet al., 2014), GmEF1A knockdown plants showed re-duced expression of BiP and thereby attenuated HRthus placing eEF1A upstream of BiP. Interestingly, likeSMV, Psg infection also induced ER stress in soybean.Reduced expression of BiP in GmEF1A knockdownplants likely enhanced susceptibility to Psg because BiPnegatively regulates ER stress. It is possible that Psg-induced ER stress is dependent on pathogen-encodedeffectors that are directed to the ER (Wang and Fobert,2013). Indeed, at least one P. syringae effector (HopD1)is known to localize to the ER (Block et al., 2014). In fact,the conserved bacterial elongation factor Tu is itself a


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well-characterized PAMP in Arabidopsis and otherplants of the Brassicaceae (Zipfel et al., 2004). Together,these further highlight the importance of elongationfactors in regulating host-pathogen interactions.

MATERIALS AND METHODS

Plant Growth Conditions

Soybean [Glycine max (L.) Merr.] cultivarsMerit (Rpg1-b), Essex (rsv1), Essex-Rsv1 (Rsv1), Nannong 1138-2 (rsc3), and Qihuang 1 (Rsc3) were grown in thegreenhouse with day and night temperatures of 25°C and 20°C, respectively.Arabidopsis plants (ecotype Col-0) were grown in MTPS 144 Conviron(Winnipeg, MB, Canada) walk-in-chambers at 22°C, 65% relative humidity,and 14 h photoperiod.

Construction of Viral Vectors, in Vitro Transcription, andPlant Inoculation

For silencing experiments, construction of silencing vectors, in vitrotranscription, rub-inoculation of recombinant BPMV vectors on soybeanleaves, and confirmation of silencing was carried out as described previously(Kachroo et al., 2008; Kachroo and Ghabrial, 2012). A 180 bp fragment (L63-G122) of Glyma17g186600 was used to generate vectors targeting GmEF1A. A225 bp fragment (L134-P208) of Glyma13g073200 was used to generate vectorstargeting GmEF1B.

Sequence Accessions and Phylogenetic Analysis

Database accessions for sequences used here are PR1 (AI930866), b-tubulin(M21297), BiP-D (Glyma5g219600),NRP-A (Glyma20g066100), IRE1a (Glyma01g157800),IRE1b (Glyma16g111800), IRE1c (Glyma11g08720), IRE1d (Glyma04g195100),bZIP68 (Glyma02g161100), bZIP37 (Glyma19g126800), bZIP38 (Glyma03g123200),Hsr203j (Glyma06g310100), APX (L10292), GST (AF243364), the GmEF1Afamily (Glyma17g186600, Glyma05g089000, Glyma10g212900, Glyma16g068000,Glyma19g052400), and the GmEF1B family (Glyma13g073200, Glyma02g276600,Glyma04g195100, and Glyma14g039100). Sequence alignment and phylogeneticanalysis were carried out using the Megalign program in the DNASTAR package(Swofford, 2000).

Pathogen Infection Assays and Chemical Treatments

For SMV or BYMV inoculations, infected plant tissue was homogenized in0.01 M phosphate buffer mixed with a small amount of carborundum and usedfor rub-inoculation of leaves. For ELISA assays of SMV levels, 0.3 g leaf tissuefrom inoculated plants was collected. Total extracts were prepared by grindingin PBST buffer containing 0.14 M NaCl, 3 mM KCl, 4.5 mM Na2HPO4, 2 mM

KH2PO4, 1% Tween 20, and centrifuged at 12,000 g. Extract corresponding to200 ng of proteinwas used for ELISA assayswith purified IgG against SMV coatprotein. Bacterial strains and plant inoculations were carried as describedpreviously (Selote et al., 2013). For ER stress treatments, plants (V2 stage) werevacuum-infiltrated with 4 mM DTT or 10 mM Tunicamycin. Control plants wereinfiltrated with water or 0.1 mMDMSO, respectively. Leaf tissue was sampled at6 h, 12 h, and 24 h post infiltration for gene expression analysis. SMV was in-oculated 24 h post infiltration for pathogenicity assays.

Cell Death Assays

For ion leakage, soybean leaves (V2 stage)were infiltratedwith 13105CFU/mLof Psg avrB (cvMerit), rub-inoculated with SMVG7 (cv Essex-Rsv1), or spot-treatedwith 50 mM paraquat (6, 20 ml droplets each/leaf). 6 leaf discs per treatment(d = 0.7 cm) were collected at 24 h postinfection of Psg, 7 d postinfection withSMV, or 24 h posttreatment with paraquat, washed in distilledwater for 30min,and transferred to tubes containing 10 ml of distilled water. Conductivity ofthe solution was measured every 2 h for 24 h with an NIST traceable digitalConductivity Meter (Fisher Scientific, Waltham, MA). SD was calculated fromthree replicate measurements per treatment per experiment. Results are rep-resentative of three independent experiments.

RNA Extraction and Quantitative RT-PCR Analysis

RNA from leaf tissues of soybean plants at V2/V3 growth stage was extractedusing the TRIzol reagent (Invitrogen, Carlsbad, CA), per manufacturer’s instruc-tions. Reverse transcription (RT) and first-strand cDNA synthesis were carried outusing Superscript II (Invitrogen, Carlsbad, CA). Two to three independent RNApreparations were analyzed by quantitative RT-PCR to evaluate relative differ-ences in transcript levels. Primers were designed to amplify gene-specific PCRproducts of ,200 bp in size. Actin was employed as an internal control to nor-malize the cDNA. qRT-PCR was carried out in 96 well plate using SYBR GreenMix, with cycling conditions described previously (Wang et al., 2014). Gene ex-pression was quantified using the relative quantification (DDCt) method as de-scribed before (Livak and Schmittgen, 2001). Each sample or treatment was testedin at least three biological repeats and the same experiment was performed twice.

Protein Extraction and Coimmunoprecipitation Assays

Proteins were expressed as N-terminal epitope-tagged fusions in N. ben-thamiana using the pSITE vectors (Martin et al., 2009). Total extracts of plantproteins were prepared by grinding 1g leaf tissue in buffer containing 50 mM

Tris-HCl (pH 7.5), 10% glycerol, 150mMNaCl, 10mMMgCl2, 5mMEDTA, 5mM

DTT, and 13 protease inhibitor cocktail (Sigma- Aldrich, St. Louis, MO). Totalprotein extract was centrifuged at 10,000 g followed by a second centrifugationat 125,000 g. For immunoblot analysis, proteins (10–50 mg) were fractionated on7–10% SDS-PAGE gels and subjected to immunoblot analysis using a-MYC,a-FLAG (Sigma-Aldrich, St. Louis, MO) or antibodies specific to coat proteinsof respective viruses. Immunoblots were developed using ECL detection kit(Roche) or alkaline-phosphatase-based color detection. For Ponceau-S staining,PVDF membranes were incubated in Ponceau-S solution (40% methanol [v/v],15% acetic acid [v/v], 0.25% Ponceau-S.) The membranes were destained usingdeionized water. IP assays were performed by incubating total protein extractswith M2 FLAG-affinity beads (unless noted otherwise), followed by extensivewashing with extraction buffer lacking PVPP. Proteins were separated on10% SDS-PAGE at 100V for 1–2 h and detected by western blot analysis usingprotein/tag-specific antibodies. Expected Mrs of proteins: GmEF1A ; 50 kD,FLAG-GmEF1A ; 50 kD, MYC-GmEF1A ; 64 kD, MYC-P3 ; 44 kD, FLAG-P3 ;39 kD,MYC-NIb; 40 kD, SMVCP; 29 kD, BPMV-CP; 40 kD, CMV-CP; 24 kD,TCV-CP; 38 kD, BYMV-CP; 30 kD, BiP; 74 kD.

Membrane Protein Isolation

Total microsomal membranes were obtained by homogenization of N.benthamiana leaves transiently expressing 33FLAG-GmEF1A/B or SMV P3 (viaAgrobacterium-mediated expression). Leaf tissues were homogenized in buffercontaining 250 mMTris-HCl (pH 8.5), 290 mM Suc, 25mM EDTA, 10mMDTT, 13Protease inhibitor cocktail (Roche), 1 mM PMSF and 2% PVPP. Crude lysate wasfiltered through four layers of cheese-cloth and centrifuged at 10,000 g for 10 minat 4°C. The clear supernatant constitutes the total protein (T) fraction. Total proteinfractions were centrifuged at 100,000 g for 1 h at 4°C to obtain supernatantcomprising the soluble (S) fraction and pellet comprising the microsomal (M)fraction. The microsomal membrane pellet was washed with homogenizationbuffer (lacking PVPP) to remove soluble protein contaminations. The total (T),soluble (S), andmembrane (M) protein fractions were analyzed by standard SDS-PAGE (10% gel) followed by immunoblotting with anti-FLAG M2 antibody(GmEF1A, or P3), anti-TIP (tonoplast intrinsic protein,membrane protein control)and anti-Hsc70 (soluble protein control, Agrisera). The expected Mrs of controlproteins were: TIP ; 23 kD, and Hsc70 ; 70 kD.

ER membrane fractionation was carried out as described before (Edwardsand Lloyd, 1977). Briefly, total proteins were extracted in homogenizationbuffer (0.5 M Suc, 10 mM KCI, 1 mM EDTA, 1 mM MgCl2, 2 mM DTT, 0.1 mM

phenylmethyl-sulfonyl fluoride, 150 mM Tricine/KOH pH 7.5). 5 mL of theprotein extract was layered onto a preformed Suc gradient (8 mL step of 20% on4 mL each of 30%, 40%, 50%, and 60% weight/volume of Suc. The tubes werecentrifuged at 25,000 rpm, for 2 h at 2°C. Fractions were collected from eachlayer and used for western blot analysis using antibodies specific to SMV-CP orBiP (luminal binding protein, Hsp70 family ER chaperone).

Yeast Two-Hybrid Assay

A soybean cDNA library (;0.68 3 107 clones) from SMV-SC15 infected soy-bean (cv Nannong1138-2) was cloned into the modified vector pPR3-N usingGateway technology. The SMV SC15 P3 protein cloned in pBT3 was used as bait





to screen the library (33 clones) by cotransformation in yeast (NMY51). Yeasttransformants expressing P3-interacting proteins were selected on syntheticdropout medium lacking tryptophane, Leu, His, and adenine. Yeast strainsexpressing P3 interactors were further assessed for b-galactosidase activity. Pos-itive interactors including GmEF1A were identified based on sequence identity.

Nuclear Localization Assay

Full-length P3, eEF1A, and eEF1B were cloned into the pNIA-Ca vector(Zaltsman et al., 2007), which produced in-frame fusion of the proteins to the Cterminus of the mLexA-Gal4AD chimera. mLexA-Gal4AD induces reportergene expression only if the test protein is able to enter the nucleus. The con-structs were transformed into yeast strain L40 and selected for growth onsynthetic dropout medium lacking Trp, Leu, His, and adenine at 29°C for 2–4 d.

Bimolecular Fluorescence Complementation Assays

BiFC assays were carried out as described before (Selote and Kachroo, 2010).Briefly, the various proteins were fused to the N/C-terminal halves of EYFPusing the pSITE-n/cEYFP vectors (Martin et al., 2009) and introduced inAgrobacterium tumefaciens strain LBA4404. Agrobacterium strains expressingproteins fused to reciprocal halves of EYFP were co-infiltrated into CFP-H2B-tagged N. benthamiana plants (transgenic plants expressing nuclear localizedCFP). 48 h later, water-mounted sections of leaf tissue were examined byconfocal microscopy using a water-immersion PLAPO60XWLSM (NA 1.0)objective. Positive interactions were detected as yellow florescence upon re-constitution of the complete EYFP. CFP and YFP overlay images (403 magni-fication) are shown. All interactionswere confirmed using both combinations ofreciprocal nEYFP/cEYFP fusion proteins in two separate experiments (threereplicates per experiment). Protein localization assays were done using agro-infiltration of C-terminal YFP/CFP tagged proteins in transgenicN. benthamianaplants expressing RFP-H2B or RFP tagged with the amino-terminal signal se-quence of Arabidopsis thaliana basic chitinase and a carboxy-terminal HDELER-retention signal (Goodin et al., 2007).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Amino acid sequence alignment of GmEF1Aisoforms.

Supplemental Figure S2. qRT-PCR analysis of eEF1A mRNA levels insoybean.

Supplemental Figure S3. Knockdown of GmEF1A in soybean.

Supplemental Figure S4. Effect of GmEF1A-knockdown on soybean re-sponse to SMV.

Supplemental Figure S5. Effect of GmEF1A-knockdown on soybean re-sponse to BYMV.

Supplemental Figure S6. Amino acid sequence alignment of GmEF1Bisoforms.

Supplemental Figure S7. Effect of GmEF1B-knockdown on UPR-relatedgene expression.

Supplemental Figure S8. eEF1B does not interact with SMV P3.

Supplemental Figure S9. GmEF1A interacts with SMV Nib.

Supplemental Figure S10. Nuclear import assay of GmEF1A and SMV P3.

ACKNOWLEDGMENTS

We thank Said Ghabrial and Reza Hajimorad (helpful discussions), PaulVincelli (critical review of the manuscript), Michael Goodin (help with confocalimaging and nuclear import assay), Todd Pfeiffer (amplifying soybean seedstock), AimingWang (SMVNIb clone), and Amy Crume (management of plantgrowth facilities) for their generous help. The information reported in thisarticle (No. 16-12-048) is part of a project of the Kentucky Agricultural Exper-iment Station and is published with the approval of the Director.

Received March 30, 2016; accepted June 14, 2016; published June 29, 2016.

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