Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein P3N-PIPO

Abstract

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.

Figure 1. Schematic representation of the TuMV genome.

The circle represents the genome-linked viral protein, VPg. Two short horizontal lines represent 5′ and 3′ untranslational region, respectively. The large box represents the long open reading frame (from nucleotides 131 to 9625). The mature proteins resulting from processing the large polyprotein are indicated as smaller boxes. PIPO (from nucleotides 3079 to 3258) derived from a frameshif on the P3 cistron is indicated as a shot grey bar. P3N-PIPO is indicated in green and CI in red. The poly(A) tail is shown as (A)n. For clarity, the relative sizes of the mature proteins are not drawn to scale.

Figure 2. Subcellular localization of TuMV CI in N. benthamiana leaf cells.

(A) Localization of CI-mRFP expressed alone in the leaf cells 48 hrs post-agroinfiltration (panel I) or in TuMV-infected leaf tissues 48 hrs (panel II) or 72 hrs (panel III) post-agroinfiltration. (B) Localization of CI-mRFP coexpressing with the plasma membrane marker GFP-REM (panels I, II) 48 hrs post-agroinfiltration. (C) Localization of CI-mRFP in the leaf cells coexpressing other viral proteins, i.e., HC-Pro (panel I), P3 (panel II), VPg (panel III) or CP (panel IV). Images were taken 48 hrs post-agroinfiltration. (D) Localization of CI-mRFP in the cells coexpressing P3N-PIPO. The ratio of agrobacterial culture mixtures containing plasmid CI to plasmid P3N-PIPO is indicated. Bars, 8 µm.

Figure 3. TuMV P3N-PIPO is a PD-localized protein and mediates the targeting of CI to PD in N. benthamiana.

(A, panels I, II) Localization of P3N-PIPO-GFP transiently expressed in the cell treated 48 hrs post-agroinfiltration. Paired P3N-PIPO structures under a higher magnification (panel II). (B) Colocalization of P3N-PIPO-GFP with the PD marker PDLP1-mRFP. Arrows point to PD costained by P3N-PIPO-GFP and PDLP1-mRFP. (C) Cells coexpressing P3N-PIPO-YFP and mTalin-CFP as a cell membrane marker (control). Fluorescence of P3N-PIPO-YFP in plasmolyzed leaf tissue containing mTalin-CFP (plasmolyzed). DIC, Differential interference contrast. (D) Colocalization of P3N-PIPO with CI-mRFP 48 hrs (panel I) and 72 hrs (panels II, III) post-agroinfiltration. Arrows point to the PD-localized P3N-PIPO-GFP and CI-mRFP. Insets are the enlarged images of the areas in white boxes in the corresponding panels. (E) Interactions of TuMV CI and P3N-PIPO proteins in vivo. BiFC analysis (48 hrs post-agroinfiltration) was used to assess interactions in cells coexpressing CI-YC and P3N-PIPO-YN (panel I), CI-YN and P3N-PIPO-YC (panel II). Arrows indicate the strong BiFC fluorescence at PD costained by the PD marker, PDLP1-CFP. Bars, 8 µm.

Figure 4. Targeting of P3N-PIPO and CI to PD requires the BFA-sensitive secretory pathway and is independent of the acto-myosin motility system.

PD marker PDLP-1-CFP (panels I), P3N-PIPO-YFP (panels II) and CI-mRFP in the presence of untagged P3N-PIPO (panels III) were transiently expressed in leaf cells treated with water (Control, A), 50 µg/mL BFA (BFA, B), co-agroinfiltrated with the untagged COPII mutant Sar1(H74L) [Sar1(H74L), C], co-agroinfiltrated with the untagged myosin XI-K tail (Myosin XI-K tail, D), co-agroinfiltrated with the untagged myosin VIII-1 tail (Myosin VIII-1 tail, E), and 25 µM Lat B (Lat B, F). Images were taken 48 hrs post-agroinfiltration. N, nucleus. Bars, 10 µm.

Figure 5. Association of TuMV CP with CI in TuMV-infected N. benthamiana leaf cells.

(Panels I, II) TuMV CP tagged by YFP (YFP-CP) in the cytoplasm when expressed alone (panel I) or during virus infection (panel II). (Panel III) When coexpressed with the recombinant TuMV::6K-GFP infectious clone, some mRFP-CP is also present in proximity to the 6K-GFP-labeled replication complex (arrow). (Panel IV) TuMV YFP-CP attachment to PD-associated CI structures.(arrows; Inset) in the cell periphery in the presence of P3N-PIPO during virus infection. All images are taken 48 hrs post-agroinfiltration. Chl, chloroplasts. Bars, 8 µm.

Figure 6. Subcellular localization of TEV P3N-PIPO and its interaction with TEV CI and the intercellular movement-defective mutant CI(AA3,4).

(A) Colocalization of TEV P3N-PIPO-GFP with the PD marker PDLP1-mRFP. Arrows point to the PD-located P3N-PIPO-GFP and PDLP1-mRFP. (B) Coexpression of TEV CI(AA3,4)-mRFP does not change PD-localization of TEV P3N-PIPO-GFP. Arrows indicate PD-located P3N-PIPO-GFP. Arrowheads indicate nucleus-localization of TEV CI(AA3,4)-mRFP. (C) BiFC analysis of interactions of TEV CI-YN and TEV P3N-PIPO-YC (panel I), TEV CI (AA3, 4)-YN and TEV P3N-PIPO-YC(panel II), TEV CI-YN and TEV CI-YC (panel III), and TEV CI (AA3, 4)-YN and TEV CI (AA 3, 4)-YC. N, nucleus. Bars, 10 µm.

Figure 7. Two TEV CI mutants defective in cell-to-cell movement fail to form either cytoplasmic inclusions (when expressed alone) or PD-associated structures (in the presence of P3N-PIPO).

(A) TEV CI-mRFP forms aggregates in the cytoplasm when expressed alone (panel I) and punctate spots along the cell walls when coexpressed with P3N-PIPO-GFP (panel II). (B) When expressed alone, TEV CI(AA3, 4)-mRFP (panel II) and TEV CI(AA100,101)-mRFP (panel III) are distributed in the nucleus and in periphery rather than forming typical inclusions in the cytoplasm (panel I). (C) In the presence of P3N-PIPO, TEV CI(AA3, 4)-mRFP (panel II) and TEV CI(AA100,101)-mRFP (panel III) are distributed in the nucleus and cell periphery rather than targeting PD (panel I). All images are taken 48 hrs post-agroinfiltration. Bars, 8 µm.

Figure 8. Model for potyvirus intercellular transport through PD.

The virion-CI movement complex is intracellularly transported to the modified PD where CI forms conical structures anchored by the PD-located P3N-PIPO. The virion is then fed through the CI structures and PD to enter the adjacent cell. CW, cell wall.