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, 166 (3), 1345-58

Association of the P6 Protein of Cauliflower Mosaic Virus With Plasmodesmata and Plasmodesmal Proteins

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Association of the P6 Protein of Cauliflower Mosaic Virus With Plasmodesmata and Plasmodesmal Proteins

Andres Rodriguez et al. Plant Physiol.

Abstract

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.

Figures

Figure 1.
Figure 1.
CaMV and host constructs used for confocal microscopy or coimmunoprecipitation (co-IP). A, Structure of CaMV P6 and Arabidopsis (Arabidopsis thaliana) Soybean Response to Cold (AtSRC2.2) proteins. The functions of P6 domains D1 to D4 tested for interaction with AtSRC2.2 are indicated by the shaded boxes. The Mini TAV is the minimal region for the translational transactivation function. The NLSa sequence corresponds to the nuclear localization signal of influenza virus. The NLS sequence corresponds to the nuclear localization signal of human ribosomal protein L22. B, Structure of P6 (Angel et al., 2013), AtSRC2.2, PDLP (Thomas et al., 2008), and CaMV MP fusions developed for confocal microscopy and/or co-IP. aa, Amino acid.
Figure 2.
Figure 2.
AtSRC2.2 preferentially interacts with D2 of P6 in a yeast two-hybrid analysis. A, Interaction of AtSRC2.2 with the full-length CaMV P6. B, Interaction of AtSRC2.2 with CaMV P6 domains. Numbers in the x axis of each bar graph represent the different transformant combinations illustrated in the schematic diagrams at the left. In the schematic diagrams, the striped box represents the transcriptional activator domain and black boxes show the DNA-binding domain.
Figure 3.
Figure 3.
AtSRC2.2-RFP is partially colocalized with P6-GFP upon coagroinfiltration into N. benthamiana leaves. A, Expression of AtSRC2.2-RFP alone. B, Expression of P6-GFP alone. C to E, Coagroinfiltration of AtSRC2.2-RFP with P6-GFP. C, Expression of AtSRC2.2-RFP. D, Expression of P6-GFP. E, Overlay of photos C and D. Picture was taken 3 d post inoculation (dpi). White arrows indicate AtSRC2.2-RFP aggregates of a size that only exist when coagroinfiltrated with P6-GFP.
Figure 4.
Figure 4.
co-IP studies with AtSRC2.2-RFP with P6-GFP and subcellular localization of P6ΔD1D2-GFP. A, co-IP of AtSRC2.2-RFP with P6-GFP after coagroinfiltration of N. benthamiana leaves. Lane 1, Mock-inoculated control leaf. Lane 2, Expression of AtSRC2.2-RFP. Lane 3, Expression of P6∆D1D2-GFP. Lane 4, Expression of P6-GFP. Lane 5, Coexpression of P6∆D1D2-GFP with AtSRC2.2-RFP. Lane 6, Coexpression of P6-GFP with AtSRC2.2-RFP. Aa, Western blot for total proteins probed with RFP antibodies. Ab, Western blot for total protein probed with GFP antibodies. Ac, co-IP of proteins using GFP antibodies and probed in a western blot with RFP antibodies. B, Localization of P6ΔD1D2-GFP expressed in N. benthamiana leaves at 3 dpi.
Figure 5.
Figure 5.
co-IP and colocalization of P6D1D2-GFP with AtSRC2.2-RFP. co-IP of P6D1D2-GFP with AtSRC2.2-RFP after coagroinfiltration of N. benthamiana leaves. Lane 1, Mock-inoculated control leaf. Lane 2, Expression of AtSRC2.2-RFP. Lane 3, Expression of P6D1D2-GFP. Lane 4, Coexpression of AtSRC2.2-RFP and P6D1D2-GFP. Aa, Western blot for total proteins probed with RFP antibodies. Ab, Western blot for total proteins probed with GFP antibodies. Ac, co-IP of proteins using GFP antibodies and probed in a western blot with RFP antibodies. Ad, co-IP of proteins using GFP antibodies and probed in a western blot with GFP antibodies. B to D, Photos illustrate relocalization of AtSRC2.2 with P6D1D2-GFP. B, AtSRC2.2-RFP. C, P6D1D2-GFP. D, Overlay of B and C.
Figure 6.
Figure 6.
Association of AtSRC2.2 with the membrane marker protein AtPIP2A and with plasmodesmal marker proteins PDLP1 and CaMV MP. A to C, Photos show that AtSRC2.2-RFP does not colocalize with free GFP in a plasmolyzed cell. A, AtSRC2.2-RFP. B, Free GFP. C, Overlay of A and B. D to F, Photos illustrate colocalization of AtSRC2.2 with the plasma membrane marker protein aquaporin AtPIP2A-GFP in a plasmolyzed cell. D, SRC2.2-RFP. E, AtPIP2A-GFP. F, Overlay of D and E. G to I, Photos illustrate colocalization of CaMV MP-GFP with AtSRC2.2-RFP in N. benthamiana leaf tissue. G, MP-GFP. H, AtSRC2.2-RFP. I, Overlay of G and H. J to L, Photos illustrate that CaMV AtSRC2.2-RFP is colocalized with PDLP1-GFP in both the membrane and cell wall of plasmolyzed N. benthamiana cells. J, PDLP1-GFP. K, AtSRC2.2-RFP. L, Overlay of J and K. N. benthamiana cells were plasmolyzed by infiltration of 30% glycerol. The white arrows illustrate colocalization of AtSRC2.2-RFP and PDLP1-GFP in the plasmolyzed membrane, whereas the yellow arrows illustrate colocalization in the cell wall. The higher magnifications of the colocalized signals in the insets are highlighted by the purple arrows in I and L.
Figure 7.
Figure 7.
AtSRC2.2 and PDLP1 colocalize with CaMV P1 at the base and tip of tubule structures in N. benthamiana leaf cells. A, P1-GFP forms punctate spots that are associated with plasmodesmata. B, Coagroinfiltration of unmodified P1 with P1-GFP leads to the formation and labeling of tubule structures. C to E, Coagroinfiltration of unmodified P1, P1-GFP, and AtSRC2.2-RFP illustrates that AtSRC2.2-RFP colocalizes with P1-GFP at the base of tubule structures. C, P1-GFP. D, AtSRC2.2-RFP. E, Overlay of C and D. F to H, Coagroinfiltration of unmodified P1, P1-GFP, and AtSRC2.2-RFP shows that AtSRC2.2-RFP also colocalized with P1-GFP at the tip of tubule structures. F, P1-GFP. G, AtSRC2.2-RFP. H, Overlay of F and G. I to K, Coagroinfiltration of unmodified P1, P1-RFP, and PDLP1-GFP shows that PDLP1-GFP may also be colocalized with P1-RFP at the tip and base of tubule structures. I, P1-RFP. J, PDLP1-GFP. K, Overlay of I and J.
Figure 8.
Figure 8.
Association of P6-RFP with the plasmodesmal protein PDLP1-GFP and with aniline blue. A to C, Photos illustrate the association of P6-RFP with PDLP1 in N. benthamiana leaf tissue after coagroinfiltration. A, PDLP1-GFP. B, P6-RFP. C, Overlay of A and B. D to F, Photos illustrate colocalization of P6-RFP with PDLP1-GFP plasmolyzed cells of N. benthamiana. D, PDLP1-GFP. E, P6-RFP. F, Overlay of D and E. G to I, Photos illustrate the association of P6-RFP with aniline blue in N. benthamiana leaf tissue after agroinfiltration. G, Cell walls stained with aniline blue. H, P6-RFP. I, Overlay of G and H.
Figure 9.
Figure 9.
co-IP of PDLP1-GFP with P6-RFP after coagroinfiltration of N. benthamiana leaves. Lane 1, Mock-inoculated control leaf. Lane 2, Expression of P6-RFP. Lane 3, Expression of PDLP1-GFP. Lane 4, Coexpression of P6-RFP and PDLP1-GFP. a, Western blot for total proteins probed with RFP antibodies. b, Western blot for total proteins probed with GFP antibodies. c, co-IP of proteins using GFP antibodies and probed in a western blot with RFP antibodies. d, co-IP of proteins using GFP antibodies and probed in a western blot with GFP antibodies.

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