Cylindrical Inclusion Protein of Turnip Mosaic VirusServes as a Docking Point for the IntercellularMovement of Viral Replication Vesicles1

Nooshin Movahed,a Camilo Patarroyo,a Jiaqi Sun,a Hojatollah Vali ,b,c Jean-François Laliberté ,d

and Huanquan Zhenga,2

aDepartment of Biology, McGill University, Montreal, Quebec H3B 1A1, CanadabFacility for Electron Microscopy Research, McGill University, Montreal, Quebec H3A 0C7, CanadacDepartment of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, CanadadINRS-Institut Armand-Frappier, Laval, Quebec H7V 1B7, Canada

ORCID IDs: 0000-0001-8852-245X (N.M.); 0000-0003-4359-9896 (J.S.); 0000-0002-6934-224X (J.-F.L.); 0000-0003-2986-725X (H.Z.).

Plant viruses move from the initially infected cell to adjacent cells through plasmodesmata (PDs). To do so, viruses encodededicated protein(s) that facilitate this process. How viral proteins act together to support the intercellular movement of virusesis poorly defined. Here, by using an infection-free intercellular vesicle movement assay, we investigate the action of CI(cylindrical inclusion) and P3N-PIPO (amino-terminal half of P3 fused to Pretty Interesting Potyviridae open reading frame), thetwo PD-localized potyviral proteins encoded by Turnip mosaic virus (TuMV), in the intercellular movement of the viralreplication vesicles. We provide evidence that CI and P3N-PIPO are sufficient to support the PD targeting and intercellularmovement of TuMV replication vesicles induced by 6K2, a viral protein responsible for the generation of replication vesicles. 6K2interacts with CI but not P3N-PIPO. When this interaction is impaired, the intercellular movement of TuMV replication vesiclesis inhibited. Furthermore, in transmission electron microscopy, vesicular structures are observed in connection with thecylindrical inclusion bodies at structurally modified PDs in cells coexpressing 6K2, CI, and P3N-PIPO. CI is directed to PDsthrough its interaction with P3N-PIPO. We hypothesize that CI serves as a docking point for PD targeting and the intercellularmovement of TuMV replication vesicles. This work contributes to a better understanding of the roles of different viral proteins incoordinating the intercellular movement of viral replication vesicles.

Turnip mosaic virus (TuMV) is a member of the Poty-viridae family (Nicolas and Laliberté, 1992). It infects abroad spectrum of plants of the Brassica genus, includingthe economically important oilseed rape (Brassica napusssp. oleifera). The genome of TuMV ismonopartite positivesingle-stranded RNA (Nicolas and Laliberté, 1992), whichencodes for at least 11 different mature proteins (Nicolasand Laliberté, 1992; Chung et al., 2008). It has been shownthat, upon infection, TuMV induces the rearrangement ofthe endomembrane system and the formation of smallvesicles through the action of a transmembrane viral

protein called 6K2 (Nicolas and Laliberté, 1992; Beaucheminand Laliberté, 2007; Cotton et al., 2009; Grangeon et al.,2012). 6K2-induced vesicles contain viral RNA andseveral replication-related viral and host proteins(Beauchemin et al., 2007; Dufresne et al., 2008). More-over, the 6K2 vesicles also contain RNA polymerase anddouble-stranded viral RNAs, suggesting that the 6K2-tagged vesicular structures are viral RNA replicationsites (Wan et al., 2015).

The 6K2 replication vesicles also serve as a vehiclefor the cell-to-cell spread of TuMV (Grangeon et al.,2013). The 6K2-induced replication vesicles are formedfrom the endoplasmic reticulum (ER) in a COPII-COPI-dependent manner (Cotton et al., 2009) and then bud offfrom the ER at the ER exit sites (Cotton et al., 2009).Once budding off, these replication vesicles trafficintracellularly along microfilaments (Cotton et al.,2009) and eventually reach plasmodesmata (PD),from where they ultimately cross into the uninfectedneighboring cell (Grangeon et al., 2013). Interestingly,the 6K2-induced replication vesicles can be formedthrough the ectopic expression of 6K2, but these ves-icles are incapable of moving intercellularly in theabsence of TuMV infection (Grangeon et al., 2013),indicating that additional viral proteins are requiredfor its intercellular movement.

1 This research was supported by grants from the Natural Sciencesand Engineering Research Council of Canada (NSERC) and fromFonds Québec de la Recherche sur la Nature et les Technologies(FRQNT) to J.-F.L. and H.Z.

2 Address correspondence to hugo.zheng@mcgill.ca.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:Huanquan Zheng (hugo.zheng@mcgill.ca).

N.M., H.V., J.-F.L., and H.Z. conceived and designed the experi-ments; N.M., C.P., and J.S. performed plasmid construction; N.M.and C.P. performed confocal microscopy; N.M. performed TEM,Y2H, BIFC, and FRET experiments; N.M., H.V., J.-F.L., and H.Z.analyzed the data; N.M., C.P., and H.Z. wrote the article.

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It is commonly accepted that plant viruses spreadintercellularly through the PD. However, viral particlesare too large to pass through PD. Most plant viruses en-code for at least one protein that increases the size ex-clusion limit (SEL) of PD to allow the passage of viralparticles from cell to cell (Brandenburg andZhuang, 2007;

Benitez-Alfonso et al., 2010; Niehl and Heinlein, 2011;Ritzenthaler, 2011; Ueki and Citovsky, 2011). In the caseof TuMV, there are at least three viral proteins involved inits cell-to-cell movement: P3N-PIPO (P3-N-terminal-Pretty Interesting Potyviral Open Reading Frame), CI(Cylindrical Inclusion) helicase, and CP (Coat Protein).

Figure 1. CI and P3N-PIPO are necessary andsufficient to support the intercellular movementof 6K2 vesicles. A to D, Expression of the dualconstruct pCambia/6K2-mCherry/GFP-HDELalone (A), with P3N-PIPO-YFP (B), with CI-YFP(C), or with both CI-YFP and P3N-PIPO-YFP (D).Thewhite arrow in D points to a cell showing redfluorescence only. Bars = 10 mm. E, Quantifica-tion of 6K2 intercellular movement by countingthe percentage of neighboring cells showing only6K2-mCherry signal and measuring the meanintensity of 6K2-mCherry signals in red-onlyareas of the neighboring cells (in arbitrary units)with Imaris image-analysis software (n = numberof neighboring cells examined).

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P3N-PIPO localizes to PD and modifies the SEL of PD tofacilitate its own and potyvirus intercellular movement(Vijayapalani et al., 2012). It has been described that,different from the PD targeting of 6K2-induced replica-tion vesicles, the PD targeting of P3N-PIPO requires afunctional COPII-COPI transport system but not actinmicrofilaments or myosin motors (Wei et al., 2010b).Additionally, a plasma membrane-localized cation bind-ingprotein has been shown to be involved in the targeting

of P3N-PIPO to the PD (Vijayapalani et al., 2012). The CIhelicasewas shown to localize to PD, possibly through itsphysical interaction with P3N-PIPO (Wei et al., 2010b).Mutations in the CI helicase outside of the conservedhelicase domains from the related potyvirus Tobacco etchvirus severely delay or completely disrupt its intercellularmovement (Carrington et al., 1998). The CP also localizesto the PD together with CI and P3N-PIPO during TuMVinfection (Wei et al., 2010b).

Figure 2. CI and P3N-PIPO support the PDtargeting of 6K2 vesicles. Colocalization of 6K2-mCherry is shown with PDLP1-GFP-labeled PDduring ectopic expression of 6K2-mCherry alone(A) or when coexpressed with CI-CFP (B), P3N-PIPO-CFP (C), or both CI-CFPand P3N-PIPO-CFP(D). Insets (I–IV) for A show four selected regionsin A; insets for B to D are color-separated imagesfor selected regions in B to D, respectively. Barson the left = 4 mm; bars on the right (high-magnification images) = 2 mm.

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Despite the study of the involvement of these pro-teins in the cell-to-cell movement of TuMV, how theseproteins act together to support the intercellular spreadof TuMV is not clear. In this article, we developed aninfection-free intercellular 6K2 vesicle movement assaywith the aim of studying the action of CI and P3N-PIPOin the intercellular movement of 6K2-induced repli-cation vesicles. We found that P3N-PIPO and CI arenecessary and sufficient to facilitate the intercellularmovement of the 6K2-induced replication vesicles.P3N-PIPO and CI also are sufficient to target the 6K2vesicles to PD when expressed together, althoughP3N-PIPO and 6K2 take different routes to reach PD.6K2 can interact physically with CI but not P3N-PIPO;furthermore, a mutated CI that is unable to interactwith 6K2 but still interacts with P3N-PIPO failed tosupport the intercellular movement of 6K2. The evi-dence found suggests that CI and P3N-PIPO of TuMVcompose a minimal complex required for the inter-cellular movement of 6K2 viral replication vesiclesand that CI serves as a docking point between theP3N-PIPO-modified PD and the 6K2-coated replica-tion vesicles.

RESULTS

CI and P3N-PIPO Are Necessary and Sufficient to Facilitatethe Intercellular Movement of 6K2 Vesicles

Since both P3N-PIPO andCI proteins have been shownto play important roles in the intercellular spread ofTuMV (Wei et al., 2010b; Vijayapalani et al., 2012), wewondered if CI and P3N-PIPO, when expressed alone,could facilitate the intercellular movement of 6K2vesicles in the absence of TuMV infection. To test this,the dual cassette construct pCambia/6K2-mCherry/GFP-HDEL (Grangeon et al., 2013) was expressedalone, together with CI fused to YFP (CI-YFP), P3N-PIPO fused to YFP (P3N-PIPO-YFP), or both. The in-tercellular movement of 6K2-mCherry is observed incells emitting red fluorescence only, since the ER markerGFP-HDEL cannotmove from cell to cell (Grangeon et al.,2013). When the dual cassette construct was expressedalone (Fig. 1A) or coexpressed with either P3N-PIPO-YFP

(Fig. 1B) or CI-YFP (Fig. 1C), we never observed cellsthat expressed 6K2-mCherry red fluorescence only,indicating that there was no cell-to-cell movement of6K2-mCherry (Fig. 1, A–C and E). Interestingly whenthe dual cassette construct was coexpressed withboth CI-YFP and P3N-PIPO-YFP, 6K2-mCherry onlyfluorescence was detected (Fig. 1, D, arrow, and E),indicating that there was intercellular movement of6K2-mCherry. Of 136 neighboring cells we examined,we found that 29 cells (;22%) showed 6K2-mCherrysignal only (Fig. 1E). The mean intensity of the 6K2-mCherry signal in the red-only neighboring cells wasmeasured to be about 27 in arbitrary units (Fig. 1E), usingthe Imaris image analysis. No neighboring cell showing6K2-mCherry signals was observed in leaves expressingthe dual construct pCambia/6K2-mCherry/GFP-HDELalone (Fig. 1, A and E) or with either P3N-PIPO-YFP(Fig. 1, B and E) or CI-YFP (Fig. 1, C and E). These re-sults indicated that the presence of both CI and P3N-PIPO facilitates the intercellular movement of 6K2.

CI and P3N-PIPO Support the PD Targeting of 6K2

One explanation for both proteins being required forthe intercellular movement of 6K2 vesicles is that theyfacilitate the targeting of 6K2 to PDs. To verify this, thelocalization of 6K2-mCherry to PDLP1-GFP, a PDmarkerprotein, was examined when coexpressed with CI-CFP,P3N-PIPO-CFP, or both. Colocalization was hardly seenbetween 6K2-mCherry and PDLP1-GFP when CI-CFPand P3N-PIPO-CFP were not present (Fig. 2A) or werepresent individually (Fig. 2, B and C). However, colocal-ization of 6K2-mCherry with PDLP1-GFPwas seenwhenbothCI-CFP andP3N-PIPO-CFPwere expressed together(Fig. 2D). To confirm these results, the Pearson’s correla-tion coefficient between 6K2-mCherry and PDLP1-GFPwas measured using the ImageJ-based open-sourceprocessing package Fiji, as described by Breeze et al.(2016). Pearson correlation coefficients between sets ofdata are a measurement of how well they are linearlyrelated. The Pearson’s correlation coefficients between6K2-mCherry and PDLP1-GFP in cells with the ex-pression of 6K2-mCherry only (Fig. 3A, Ectopic), incells with 6K2-mCherry coexpressedwith both CI-CFP

Figure 3. CI and P3N-PIPO are sufficient tosupport the PD targeting of 6K2 vesicles. A,Quantification of the colocalization between6K2-mCherry and PDLP1-GFP when expressedalone (Ectopic), when coexpressed with bothCI-CFP and P3N-PIPO-CFP (+ CI & PIPO), orduring infection (Infection). The y axis showsthe Pearson’s correlation coefficient (PCC;Student’s t test, n = 20, P. 0.05). Columnswitha single asterisk show that the t test for thoserows is statistically significant. B, Colocalizationof 6K2-mCherry with PDLP1-GFP-labeled PDduring TuMV infection. Bar on the left = 15 mm;bars on the right (high-magnification images) =5 mm.

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and P3N-PIPO-CFP (Fig. 3A, CI & PIPO), and in cellsinfected by TuMV (Fig. 3A, Infection) are presented inFigure 3A. The results indicate that the colocalizationbetween 6K2-mCherry and PDLP1-GFP was signifi-cantly higher when coexpressed with CI and P3N-PIPO compared with the expression of 6K2-mCherryalone (Fig. 3A). We also examined the localization of6K2 with PDLP1 during TuMV infection (Fig. 3B). Thecolocalization of 6K2-mCherry with PDLP1-GFP (Fig.3B, I and II) was higher during TuMV infection thanwhat was observed in cells with the ectopic expressionof 6K2-mCherry alone (Fig. 3A). Interestingly, the lo-calization of 6K2-mCherry to PD was not significantlydifferent between the coexpression with both CI-CFPand P3N-PIPO-CFP and during TuMV infection (Fig.3A). These results suggest that these two proteins aresufficient to direct the 6K2 vesicles to PD.

Targeting of P3N-PIPO to PD, But Not the IntracellularMotility of 6K2, Requires FunctionalPost-Golgi Trafficking

The PD targeting of P3N-PIPO requires a functionalCOPII-COPI transport system but not actin microfila-ments or myosin motors (Wei et al., 2010b). However,the intracellular trafficking of TuMV replication vesi-cles requires actin microfilaments and myosin motors(Cotton et al., 2009; Wei et al., 2010a). Therefore, wehypothesize that the PD targeting of P3N-PIPO and theintracellular motility of 6K2 take different intracellularpathways. To test this, a dominant negative mutant ofthe small GTPase RabE1d(NI) was used. RabE1d is in-volved in the post-Golgi trafficking toward the plasmamembrane (Zheng et al., 2005; Speth et al., 2009). ThemutatedRabE1d(NI) inhibits the intercellularmovementof TuMV but not its replication (Agbeci et al., 2013). Wefirst tested whether the localization of the fluorescentlylabeled proteins P3N-PIPO-CFP and CI-CFP can be al-tered or notwhen coexpressedwith RabE1d(NI). Each ofthese fluorescent fusion proteins was coexpressed witheither RabE1d(WT) or RabE1d(NI), and its localizationwas examined at 3 d postinfiltration (dpi) as described(Zheng et al., 2005). The protein P3N-PIPO-CFP pro-duced punctate structures when coexpressed with thewild-type version of RabE1d (Fig. 4A), as describedpreviously (Wei et al., 2010b), but it showed a con-tinuous distribution resembling the plasmamembranewhen coexpressed with RabE1d(NI) (Fig. 4B). Thissuggested that the correct PD targeting of P3N-PIPOrequires a functional post-Golgi trafficking pathway.Interestingly, the localization of CI-CFP showed nonoticeable differences when coexpressed either withthe wild type or the dominant negative version ofRabE1d (Fig. 4, C and D).

We next examined the formation or intracellular trans-port of 6K2 vesicles in the presence of RabE1d(NI). 6K2-mCherry was only colocalized with GFP-HDEL in theglobular structure but not in ER tubuleswhen coexpressedwith either RabE1d (Fig. 5A) or RabE1d-(NI) (Fig. 5B), asdescribed previously (Grangeon et al., 2012). Similarly,

6K2-mCherry did not fully colocalize with ST-GFPwhen coexpressed with either RabE1d (Fig. 5C) or itsdominant negative version (Fig. 5D). These resultsindicated that the formation or intracellular transportof 6K2 vesicles is not affected by the presence ofRabE1d(NI). To further confirm this, the abundance,apparent size, and speed of movement of 6K2 vesicleswere quantified when coexpressed with either RabE1dor the dominant negative RabE1d(NI). There were nosignificant differences in the abundance (Fig. 5E), ap-parent size (Fig. 5F), and speed of movement (Fig. 5G)of the 6K2 vesicles when coexpressed with RabE1d(NI)compared with RabE1d. Taken together, these resultsindicate that, unlike the transport of P3N-PIPO, theformation and intracellular motility of 6K2 vesicles donot require a functional post-Golgi trafficking.

Inhibition of RabE1d-Mediated Post-Golgi TransportAffects PD Targeting of 6K2 Vesicles

Although the expression of the dominant negativeRabE1d(NI) does not affect the intracellular motilityof 6K2 vesicles, it disrupts the correct PD localizationof P3N-PIPO. Therefore, we wondered if the coex-pression of RabE1d(NI) would affect the targeting of6K2-mCherry to PD. Since coexpressing five differentconstructs [P3N-PIPO, CI, 6K2, PDLP1, and eitherRabE1d or RabE1d(NI)] in the same cell is difficult,the effect of the coexpression of RabE1d(NI) in the PDtargeting of 6K2 vesicles was examined during TuMV

Figure 4. Transport of P3N-PIPO takes a post-Golgi transport-dependentsecretory pathway. A and B, Localization of P3N-PIPO-CFP whencoexpressed with either wild-type (WT) RabE1d (A) or the dominantnegative RabE1d(NI) (B). C and D, Localization of CI-CFP when coex-pressed with either wild-type RabE1d (C) or the dominant negativeRabE1d(NI) (D). Bars = 5 mm.

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infection. For this purpose, the colocalization of 6K2-mCherry and PDLP1-GFP was observed and quantifiedwhen coexpressedwith either RabE1d (Fig. 6, A and C) orRabE1d(NI) (Fig. 6, B and C) during TuMV infection. Wefound that there was a statistically significant decrease ofthe colocalization between 6K2-mCherry and PDLP1-GFPwhen the dominant negative RabE1d(NI) was expressed

(Fig. 6C). This evidence supports the idea that P3N-PIPOplays a role in the targeting of 6K2 vesicles to PD.

6K2 Interacts Physically with CI But Not P3N-PIPO

Since P3N-PIPO and CI are necessary and sufficientto support the PD targeting and intercellularmovement

Figure 5. Intracellular motility of 6K2 does notrequire functional post-Golgi trafficking. A andB, Colocalization of 6K2-mCherry relative tothe ER marker GFP-HDEL when coexpressedwith either RabE1d (A) or the dominant nega-tive RabE1d(NI) (B). Bars = 10 mm. C and D,Localization of 6K2-mCherry relative to theGolgi marker ST-GFP when coexpressed witheither RabE1d (C) or the dominant negativeRabE1d(NI) (D). Bars = 5 mm. E to G, Quanti-fication of 6K2-mCherry-formed vesicle abun-dance per single cell (E; Student’s t test, n = 10,P . 0.05), average vesicle size (F; Student’s ttest, n = 10, P . 0.05), and average movementspeed of the vesicles (G; Student’s t test, n = 5,P . 0.05). The unit au/frame refers to the lineprofiles of fluorescence intensity in arbitraryunits per frame. WT, Wild type.

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of 6K2 vesicles, wewondered if 6K2 interacts physicallywith either of these proteins. The interactions between6K2 and either P3N-PIPO or CI were first tested in asplit ubiquitin-based yeast two-hybrid system (Y2H-SUS).PLV-Cub-6K2 was found to interact with CI-NubG butnot with P3N-PIPO-NubG (Fig. 7A).

The interaction of 6K2 with CI and P3N-PIPO wasfurther assessed with bimolecular fluorescence com-plementation (BiFC) in plant cells.When theN-terminalhalf of YFP (YFPn)-fused 6K2 (YFPn-6K2) was coex-pressedwith the C-terminal half of YFP (YFPc)-fused CI(YFPc-CI), YFP fluorescencewas reestablished (Fig. 7E),while no YFP fluorescence was discerned when YFPn-6K2was coexpressedwith YFPc-fused P3N-PIPO (P3N-PIPO-YFPc; Fig. 7F). When YFPn-6K2 was coexpressedwith unfused YFPc (Fig. 7D) or P3N-PIPO-YFPc wascoexpressed with YFPn (Fig. 7C), no YFP signals weredetected. Interestingly, coexpression of YFPn-6K2 andYFPc-CI used for BiFCwith CFP-6K2 (red) revealed thatYFP fluorescent punctae largely colocalized with the CFPfluorescence of 6K2 vesicles (Fig. 7E). This suggests that6K2 and CI interact mainly at the sites of 6K2 vesicles. Todemonstrate that the proteins shownnot to interact (YFPn-6K2 and P3N-PIPO-YFPc) were expressed at similar levelsto proteins that are shown to interact (YFPn-6K2 andYFPc-CI), a western-blot assay was conducted with anti-EYFPnand anti-EYFPc sera. As can be seen in Figure 7, G to I, theproteins YFPn-6K2 and P3N-PIPO-YFPc and the proteinsYFPn-6K2 and YFPc-CI were expressed at similar levels.

Interaction between CI and 6K2 Is Required for theIntercellular Movement of 6K2 Vesicles

In a previously reported screening, several mutantsof the CI helicase from the potyvirus Tobacco etch virusthat delayed virus intercellular movement were identi-fied. One of the identified mutants, CI(RE-122,124-AA),presented severely delayed intercellular movement butshowed no defect in the viral replication (Carrington et al.,1998). The corresponding mutation (QS-124,126-AA) was

introduced in the CI-NubG fusion plasmid, and its inter-actionwithPLV-Cub-6K2was tested.CI(QS-124,126-AA)-NubG failed to interact with PLV-Cub-6K2 (Fig. 7A, row5). The QS-124,126-AA mutation was then introducedin thefluorescently labeledCI-YFPprotein tomakeCI(QS-124,126-AA)-YFP, and its localization was examinedwhen coexpressed with P3N-PIPO-CFP. Interestingly,as CI-YFP (Fig. 8A), the mutated CI(QS-124,126-AA)-YFP still colocalized with P3N-PIPO-CFP (Fig. 8B).The localization of CI to PD is mediated by P3N-PIPOthrough the physical interaction between the two proteins(Wei et al., 2010b). By using fluorescence resonance en-ergy transfer (FRET), we revealed that, just like wild-typeCI-YFP (Supplemental Fig. S1D), CI(QS-124,126-AA)-YFPcould still interact with P3N-PIPO-CFP (SupplementalFig. S1E).

We then tested if theCI(QS-124,126-AA)mutant togetherwith P3N-PIPO can facilitate the intercellular movement of6K2 vesicles. For this, the dual construct pCambia/6K2-mCherry/GFP-HDEL was coexpressed with P3N-PIPO-YFP and either CI-YFP or CI(QS-124,126-AA)-YFP, and thecell-to-cell movement of 6K2-mCherry was evaluated asdescribed (Agbeci et al., 2013). 6K2-mCherry was only ableto move intercellularly when coexpressed with P3N-PIPO-YFP andCI-YFP (Fig. 8C) but not with P3N-PIPO-YFP andCI(QS-124,126-AA)-YFP (Fig. 8D). This result indicates thatthe interaction between 6K2 and CI is important for theintercellular movement of 6K2.

The Subcellular Basis of the Roles of CI and P3N-PIPO inthe Intercellular Movement of TuMV 6K2 Vesicles

Since P3N-PIPO and CI form a minimal viral com-plex that supports the movement of 6K2 vesicles, andthe interaction between 6K2 and CI is important for theintercellular movement of 6K2 vesicles, we next in-vestigated the subcellular basis of how P3N-PIPO andCI could support the intercellular movement of 6K2vesicles by transmission electron microscopy (TEM).In noninfected cells, simple, linear PDs (Fig. 9A, P and

Figure 6. PD targeting of 6K2 is affectedwhen a functional post-Golgi trafficking pathway is impaired. A and B, Colocalization of6K2-mCherry with PDLP1-GFP-labeled PD during TuMV infection when coexpressed with either RabE1d (A) or the dominantnegative RabE1d(NI) (B). Bars on the left = 5 mm; bars on the right (high-magnification images) = 1 mm. C, Quantification of thecolocalization between 6K2-mCherry and PDLP1-GFP during infection when coexpressed with either RabE1d or the dominantnegative RabE1d(NI). The y axis shows the Pearson’s correlation coefficient (PCC; Student’s t test, n = 20, P, 0.05). Columnwitha single asterisk shows that the t test for that row is statistically significant. WT, Wild type.

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arrows) were seen and no vesicles and cylindrical in-clusion bodies were revealed (Fig. 9A). TEM imagingof the ectopic coexpression of 6K2 with CI and P3N-PIPO in Nicotiana benthamiana leaf cells showed thatthere was a severe alteration in the PD structure, with theformation of branched or structurally modified PDs (Fig.9B, P and arrows). Cylindrical inclusion bodies (Fig. 9B,CI and arrows) that linked a group of vesicular structures(Fig. 9B, V and arrows) and branched PDs also were seen(Fig. 9B). Interestingly, in N. benthamiana leaf cells withthe ectopic coexpression of 6K2 with CI(QS-124,126-AA)and P3N-PIPO, although there was a formation ofbranched or structurally modified PDs (Fig. 9C, P andarrow), no cylindrical inclusion bodies were observed.Vesicular structureswere seen away fromPDs (Fig. 9C, Vand arrows). On the other hand, in N. benthamiana leafcells with the ectopic coexpression of 6K2 with CI butwithout P3N-PIPO, although cylindrical inclusion bodies(Fig. 9D, CI and arrows) and vesicular structures (Fig. 9D,V and arrows) were observed, theywere not close to PDsbut inside the cytoplasm instead (Fig. 9D). Moreover,

consistent with the proposed role of P3N-PIPO in themodification of PD (Vijayapalani et al., 2012), no struc-tural change of PDs was revealed in these cells (Fig. 9D,P and arrows). These TEM images indicated that, whileP3N-PIPO is involved in the structural change of PDs, CIcould serve as a connection between P3N-PIPO-modifiedPD and 6K2-tagged replication vesicles for the correct PDtargeting and intercellular transport of 6K2-coated vesi-cles through PDs.

DISCUSSION

CI and P3N-PIPO Compose a Minimal Complex Where CIServes as a Docking Point for the Intercellular Movementof 6K2 Vesicles

It is generally accepted that plant viruses move inter-cellularly through the modification of PDs. Most plantviruses encode for at least one protein that is dedicated tothe intercellular movement of viral particles (Ueki andCitovsky, 2011). In the case of TuMV, there are at least

Figure 7. 6K2 interacts with CI but not P3N-PIPO. A and B, Y2H-SUS assay of PLV-Cub-RHD3 andNubG-RHD3 (used as a positivecontrol), PLV-Cub-6K2 andCI-NubG, PLV-Cub-6K2 and freeNubG (used as a negative control), PLV-Cub-6K2 andP3N-PIPO-NubG,and PLV-Cub-6K2 and CI(QS-124,126-AA)-NubG. In A, the mated cells were grown in SC-Leu-Trp-His + 3-amino-1,2,4-triazole(3AT), and in B, the mated cells were grown in SC-Leu-Trp as mating controls. C to F, BiFC analysis of unfused YFPn and P3N-PIPO-YFPc (yellow) with ectopically expressed P3N-PIPO-CFP (red; C), YFPn-6K2 and unfused YFPc (yellow) with ectopically expressed6K2-mCherry (red;D), YFPn-6K2 andCI-YFPc (yellow)with coexpression of ectopic 6K2-CFP (red; E), and YFPn-6K2and P3N-PIPO-YFPc (yellow)with coexpressionof ectopic 6K2-CFP (red; F). Bars = 10mm.G to J, Immunoblots of leaves expressing either YFPn-6K2and YFPc-CI or YFPn-6K2 and P3N-PIPO-YFPc using anti-EYFPn rabbit serum (G) and anti-EYFPc rabbit serum (I). The lower gels (Hfor G and J for I) indicate protein loading by Coomassie Blue staining.

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three proteins involved in the cell-to-cellmovement of thevirus: P3N-PIPO, CI, and CP. Among these three viralproteins, P3N-PIPO, targeted to PDs, is known to be in-volved in the increase of the SEL of PDs (Vijayapalaniet al., 2012). P3N-PIPO also is involved in the targeting ofCI to PD through physical interaction between thesetwo proteins (Wei et al., 2010a). It has been proposedthat CI and P3N-PIPO form a complex that could fa-cilitate the intercellular movement of potyviruses ininfected plants (Wei et al., 2010a); however, it is notclear how this complex is involved in the intercellularmovement of potyviruses.

TuMV 6K2-induced replication vesicles are capable ofmoving intercellularly through PDs during TuMV in-fection. However, when expressed ectopically, eventhough 6K2 can induce the formation of these vesicles,these 6K2 vesicles are incapable of moving from cell tocell (Grangeon et al., 2013). In this study, we found that

the viral proteins P3N-PIPO and CI are necessary andsufficient for the targeting of 6K2 vesicles to PD andalso are able to facilitate the intercellular movementof 6K2 vesicles during ectopic expression. Thus, wepropose that CI and P3N-PIPO compose a minimalcomplex required for the intercellular movement of6K2 vesicles. The transport of P3N-PIPO to PDs re-quires a classic secretory pathway (Wei et al., 2010b;this study), while the targeting of 6K2 to PD takes adifferent route other than the classic secretory pathway(this study). We found that disrupting the PD localiza-tion of P3N-PIPO through the expression of RabE1d(NI)impairs the PD targeting of the 6K2 vesicles. This indi-cates that P3N-PIPO is one of the key factors involved inthe PD targeting of 6K2 vesicles. P3N-PIPO is capable ofinteracting with CI for the PD localization of CI (Weiet al., 2010a). We found that 6K2 is capable of interactingphysically with the CI protein but not P3N-PIPO. We

Figure 8. Although still localized on PD,mutated CI(QS-124,126-AA)-YFP doesnot support the intercellular movement of6K2-mCherry. A and B, Colocalization ofP3N-PIPO-CFP with CI-YFP (A) and CI(QS-124,126-AA)-YFP (B). Bars = 5 mm. Cand D, Intercellular movement of 6K2-mCherry vesicles by coexpression of thedual construct pCambia/6K2-mCherry/GFP-HDEL with P3N-PIPO-YFPand eitherCI-YFP (C) or CI(QS-124,126-AA)-YFP (D).Bars = 10 mm.

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thus propose that, during the intercellular transport of6K2 vesicles though PDs, CI could serve as a connectionbetween P3N-PIPO and the 6K2-induced replicationvesicles. This notion is further supported by the fact thatthe CI(QS-124,126-AA) mutant, which disrupts its in-teraction with 6K2, fails to support the intercellularmovement of the 6K2 vesicles, although the mutated CIcan still maintain its interaction with P3N-PIPO and belocalized to the PDs. It is interesting that the mutated CIalso fails to form cylindrical inclusions. However, we donot knowwhether this is because of failed CI interactionwith 6K2 or that the amino acids mutated per se arerequired for the formation of cylindrical inclusions.P3N-PIPO, localized to the PD, is known to be able tomodify the SEL of PDs (Vijayapalani et al., 2012).During infection, CI localizes to PD through its inter-action with P3N-PIPO (Wei et al., 2010b). It is highlylikely that 6K2 vesicles, once they are transported tothe vicinity of PDs, are docked to PDs via its interac-tion with CI. Indeed, in our TEM analysis, cylindricalinclusion bodies that link a group of vesicular struc-tures and structurally modified PDs are frequentlyseen in cells ectopically expressing P3N-PIPO, CI, and6K2, but no such cylindrical inclusion bodies are seenin cells expressing P3N-PIPO, the CI(QS-124,126-AA)mutant, and 6K2, despite the structurally modifiedPDs and vesicular structures away from the modifiedPDs.Based on the results discussed above, we propose the

following model for the intercellular movement ofTuMV 6K2 replication vesicles. In a TuMV-infected cell,upon successful replication, the P3N-PIPO protein is

transported to PD, through classic ER-to-Golgi and post-Golgi transport pathways. The SEL of the PD is thenincreased by P3N-PIPO (Vijayapalani et al., 2012). CIpresumably localizes to the modified PD through itsinteraction with P3N-PIPO (Wei et al., 2010b). The 6K2-formed replication vesicles traffic along the actomyosinsystem toward the cell periphery, eventually tether toPD by the interaction between the 6K2 on the repli-cation vesicle and the inclusion bodies formed by CIbound to P3N-PIPO, then the vesicles are docked tothe P3N-PIPO-modified PD. The PD-docked replica-tion vesicles then go through the modified PD into theneighboring cell (Grangeon et al., 2013).

An Infection-Independent Intercellular 6K2 VesicleMovement Assay

Previous studies on the regulation of the intercellularmovement of potyvirusesused infection assays (Carringtonet al., 1998; Gómez de Cedrón et al., 2006;Wei et al., 2010b;Vijayapalani et al., 2012; Deng et al., 2015). These studiesprovide valuable information about the function of bothviral and host factors involved in the intercellular move-ment of the viruses; however, the viral replication aswell asintracellular and intercellular movement are linked duringpotyviral infection (Blanc et al., 1997; Carrington et al., 1998;Deng et al., 2015). This makes the precise identification ofthe mechanism of action of host and viral proteins in theintercellular spread of the virus difficult.

In this study, an infection-free intercellular 6K2 vesiclemovement assay is developed. Since there is no repli-cation, the only process being observed is the cell-to-cell

Figure 9. Subcellular basis of the roles of CIand P3N-PIPO in the intercellular movement ofTuMV 6K2 vesicles. TEM images show a cellfrom noninfected N. benthamiana leaf (A), acell with ectopic coexpression of 6K2 with CIand P3N-PIPO inN. benthamiana leaf (B), a cellwith ectopic coexpression of 6K2 with CI(QS-124,126-AA) and P3N-PIPO inN. benthamianaleaf (C), and a cell with ectopic coexpression of6K2 with CI inN. benthamiana leaf (D). ArrowP shows plasmodesmata, arrow V shows 6K2-induced vesicles, and arrow CI shows the cy-lindrical inclusion bodies. Bars = 500 nm (A,C, and D) and 100 nm (B).

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spread of the 6K2 vesicles. When combined with the ex-pression of the dual construct pCambia/6K2-mCherry/GFP-HDEL (Grangeon et al., 2013), this system can beused to quantify the intercellular movement of 6K2vesicles by measuring the number of cells showinggreen and red fluorescence in comparison with thenumber of cells showing red fluorescence only. Withthis system, the efficiency of this process could bemeasured accurately under different experimentalconditions. This also could prove to be a valuable toolin future studies about the precise mode of action ofadditional viral and host proteins involved in the in-tercellular spread of TuMV to form a more compre-hensive model of the intracellular and intercellularmovement of this virus. One of the proteins of interestto be studied further is CP. This protein is located tothe PD together with P3N-PIPO and CI (Wei et al.,2010b), but its involvement in the cell-to-cell move-ment of replication vesicles is still not clear.

MATERIALS AND METHODS

Plasmid Construction and Site-Directed Mutagenesis

The coding sequence of CI was amplified by PCR using the TuMV infectiousclone (Cotton et al., 2009) as template and cloned into the pCR8 entry vectorfollowing the instructions from the manufacturer (ThermoFisher Scientific).P3N-PIPO results from the translation of an RNA template containing an Ainsertion in the conserved GA6 (fromGGAAAAAA to GGAAAAAAA; Olspertet al., 2016). To mimic this, we inserted a G between A3 and A4 in the GA6sequence (GGAAAAAA to GGAAAGAAA) by PCR (Chung et al., 2008). Thisinsertion does not result in any changes in the amino acid sequence. The am-plified P3N-PIPO was then cloned into the pCR8 entry vector following theinstructions from the manufacturer (ThermoFisher Scientific).

TheCI andP3N-PIPOgenes cloned intopCR8were cloned intopXN22-DEST(Grefen et al., 2007) using LR Clonase II plus enzyme mix (ThermoFisher Sci-entific) following the manufacturer’s instructions to obtain the CI-NubG andP3N-PIPO-NubG fusions. Construction of the PLV-Cub-6K2 plasmid was asdescribed (Jiang et al., 2015). The construction of PLV-Cub-RHD3 and NubG-RHD3 was described by Chen et al. (2011). For the fluorescently labeled ver-sions of CI and P3N-PIPO proteins, the CI and P3N-PIPO genes were clonedinto the Gateway-compatible vector pEarleyGate102 (X-CFP) to obtain thefluorescent fusion proteins CI-CFP and P3N-PIPO-CFP. CI and P3N-PIPOwereadditionally cloned into pEarleyGate101 (X-YFP) (Earley et al., 2006) using LRClonase II plus enzyme mix (ThermoFisher Scientific) to obtain CI-YFP andP3N-PIPO-YFP. The pCambia/6K2-mCherry and pCambia/6K2-CFP (Jianget al., 2015), the dual pCambia/6K2-mCherry/GFP-HDEL (Grangeon et al.,2013), and the PDmarkers PDLP1-GFP (Amari et al., 2010, 2011) were providedby Jean-François Laliberté. The point mutations of CI were generated with theQuikChange Lightning kit (Agilent) using the pEarleyGate101/CI-YFP andpXN22/CI-NubG plasmids as template to obtain the CI(QS-124,126-AA)-YFPand CI(QS-124,126-AA)-NubG protein fusions, following the manufacturer’sinstructions. The pVKHEn6-RabE1d and pVKHEn6-RabE1d(NI) plasmidswere used for the expression of these proteins. The construction of these plas-mids was described by Zheng et al. (2005).

Transient Protein Expression in Nicotiana benthamiana

The pCambia/6K2-mCherry, pCambia/6K2-mCherry/GFP-HDEL, pVKH-RabE1d, and pVKH-RabE1d(NI) plasmids were provided in Agrobacteriumtumefaciens. The P3N-PIPO-YFP, P3N-PIPO-CFP, CI-YFP, and CI-CFP plasmidswere transformed into A. tumefaciens by using a modified freeze-thaw proce-dure (Höfgen and Willmitzer, 1988). A. tumefaciens containing the corre-sponding plasmids was grown in Luria-Bertani broth supplemented withkanamycin alone [for pCambia/6K2-mCherry, pCambia/6K2-mCherry/GFP-HDEL, pVKH-RabE1d, and pVKH-RabE1d(NI)] or kanamycin, gentamycin,and rifampicin overnight at 28°C with shaking. The cells were centrifuged at

2,000g for 10 min and resuspended in infiltration buffer (10 mM MgCl2 and150 mM acetosyringone). The cell suspension was incubated for 4 h at room tem-perature before infiltration. The OD600 was adjusted to 0.05 for the RabE1d- andRabE1d(NI)-containing strains and to 0.1 for the rest. The agroinfiltrationwasdone in4-week-old N. benthamiana plants as described previously (Sparkes et al., 2006).

Confocal Microscopy

Agroinfiltrated leaf sections were imaged using a Leica SP8 and/or LeicaD6000 with a 403 immersion objective at 3 dpi. Lasers of 448, 488, and 561 nmwere used to excite CFP, GFP, and mCherry, respectively. The capture wasdone at 460 to 480, 500 to 540, and 580 to 620 nm for CFP, GFP, and mCherry,respectively. Image processing was done in ImageJ (http://rsbweb.nih.gov/ij/).The Pearson’s correlation coefficient between the fluorescent signals wasmeasured using the JACoP plug-in (Bolte and Cordelières, 2006) in ImageJ. Todetermine the abundance and size of the small vesicles, a stack was taken ofindividual cells expressing 6K2-mCherry, then the vesicles were counted usingthe 3D Object Counter plug-in in ImageJ using a size window of 10 to 1,000voxels. To measure the average speed of the 6K2 vesicles, a time-lapse film wastaken and the speed was measured with the MTrackJ plug-in (Meijering et al.,2012) in ImageJ.

Y2H-SUS Assay

The Y2H-SUS experiments were carried out as described by Grefen et al.(2007). The THY-AP4 (MATa ura3 leu2 lexA: lacZ: trp1 lexA: HIS3 lexA:ADE2)strain containing PLV-Cub-6K2 was used. CI-NubG and its mutant version CI(QS-124,126-AA)-NubG were transformed into the THY-AP5 (MAT55 URA3leu2 trp1 his3 loxP: ade2) yeast strain. The transformation of the yeast strainswas performed using a lithium acetate-based protocol (Grefen et al., 2007).Following transformation, the THY-AP4 and THY-AP5 strains were mated andplated on SC-Leu-Trp plates (Grefen et al., 2007). Mated cells were then platedon SC-Leu-Trp-His plates supplemented with X-Gal and 1 mM of the HIS3competitive inhibitor 3-amino-1,2,4-triazole (Brennan and Struhl, 1980).

BiFC Analysis

For BiFC analysis, YFPn-6K2, YFPc-CI, and P3N-PIPO-YFPc were made byfusion of either residues 1 to 174 of YFP (termed YFPn) or residues 175 to239 (termed YFPc) with 6K2 and CI or P3N-PIPO in the corresponding Gatewaydestination vectors pMDC32-YFPn-C, pMDC32-YFPc-C, and pMDC32-N-YFPc.These Gateway-compatible destination vectors were created by insertingHindIII-SstI fragments of pUGW2-nEYFP, pUGW2-cEYFP, and pUGW0-cEYFP (Nakagawa et al., 2007) into the same cutting site of the binary vec-tor pMDC32 (Curtis and Grossniklaus, 2003). The YFPn-6K2, YFPc-CI, andP3N-PIPO-YFPc plasmids were then transformed into A. tumefaciens by usinga modified freeze-thaw procedure (Höfgen and Willmitzer, 1988). YFPn-6K2was then coexpressed with unfused YFPc, or YFPc-CI or P3N-PIPO-YFPc, andunfused YFPn with P3N-PIPO-YFPc with A. tumefaciens-mediated transientexpression (Sparkes et al., 2006) at OD600 = 0.03 in N. benthamiana leaf lowerepidermal cells. The fluorescence of YFP was then assessed 96 h postinfiltrationusing a Leica SP8 microscope with a 403 immersion objective. Lasers of 488 nmwere used to excite YFP, and the emission was captured at 510 to 560 nm.

Western-Blot Analyses

The leaves of 6-week-old N. benthamiana plants agroinfiltrated with eitherYFPn-6K2 and P3N-PIPO-YFPc or YFPn-6K2 and YFPc-CI for BiFC analysis wereground in liquid nitrogen, and 1 g of the powder was mixed with 1 mL of SDS-PAGE loading buffer in 1.5-mL tubes. The proteins were extracted by boilingthemixture for 10min at 100°C. After centrifugation for 1min at 13,000 rpm, thesupernatant was used for western blotting. Proteins were loaded on a 12% SDS-PAGE gel. SDS-PAGEwas performed on a Protean III apparatus (Bio-Rad), andseparated proteins were transferred onto a polyvinylidene difluoride mem-brane. Immunoblotting was carried out once with anti-EYFPn rabbit serum asthe primary antibody at a 1:10,000 dilution and, the other time, with anti-EYFPc

rabbit serum at a 1:5,000 dilution. The secondary antibody was anti-rabbit IgG-peroxidase (Sigma-Aldrich) at a 1:5,000 dilution. Signals were detected usingInvitrogen Novex ECL (HRP Chemiluminescent Substrate Reagent Kit)according to the manufacturer’s recommendations. The protein loading wasverified by Coomassie Blue staining.

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TEM

Small pieces (1.5 mm 3 2 mm) of mock- or agro-infiltrated leaf (5 dpi) orupper TuMV systemically infected leaf were cut and fixed in 2.5% (w/v) glu-taraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 24 h at 4°C. Afterrinsing three times for 10 min each in washing buffer at room temperature, thesamples were postfixed in 1% (w/v) osmium tetroxide with 1.5% (w/v) potas-sium ferrocyanide in sodium cacodylate buffer for 2 h at 4°C. The samples werethen rinsed in washing buffer at room temperature (three times for 15 min each)and stained with 1% (w/v) tannic acid for 1 h at 4°C. After rinsing in water (threetimes for 10 min each) at room temperature, the samples were dehydrated in agraded acetone series (30%, 50%, 70%, 80%, 90%, and 100%) for 20 min at eachstep at room temperature. The 100% acetone rinse was repeated two more times.The samples were then gradually infiltrated with increasing concentrations ofEpon 812 resin (50%, 66%, 75%, and 100%) mixed with acetone for a minimum of8 h for each step. A 25-p.s.i. vacuumwas applied, when the samples were in pureEpon 812 resin. Sampleswere finally embedded in pure, fresh Epon 812 resin andpolymerized at 60°C for 48 h. After polymerization, the 100-nm ultrathin sectionswere obtained and stained with 4% (w/v) uranyl acetate for 8 min and Reynoldslead citrate for 5 min. Then, the sections were examined in a Tecnai T12 trans-mission electron microscope (FEI) operating at 120 kV. Images were recordedusing an AMT XR80C CCD camera system (FEI).

FRET Microscopy

For FRET microscopy analysis, a spectral imaging method was performed.Agroinfiltrated N. benthamiana leaf sections expressing CFP and YFP alone,coexpression of 6K2-CFP and P3N-PIPO-YFP, coexpression of CI-CFP andP3N-PIPO-YFP, and coexpression of CI(QS-124,126-AA)-CFP and P3N-PIPO-YFP were imaged at 5 dpi using a Zeiss LSM780 Laser Scanning ConfocalMicroscope with spectral detector and a 633/1.4 immersion Oil DICIII PlanApochromat objective. The excitation was from a 405-nm/30-mW laser. Thespectrumwas measured from 455 to 560 nm in 10-nm increments. The CFP andYFP (a result of FRET from CFP and YFP) signal was separated from the FRETimage through the spectral unmixing process. Image processing was done inImageJ (http://rsbweb.nih.gov/ij/).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The mutated CI(QS-124,126-AA) still interactswith P3N-PIPO.

ACKNOWLEDGMENTS

We thank the research group of the Facility for Electron MicroscopyResearch at McGill University, where we conducted all the TEM experiments.

Received October 13, 2017; accepted October 27, 2017; published October 31,2017.

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