A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins

Abstract

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Figure 1. PDLP1 interacts with GFLV 2B at the base of the tubules.

(A) RFP:2B tubule formation occurs on cell walls (dotted lines) at PDLP1:GFP-labeled foci. Colocalisation between PDLP1:GFP and RFP:2B is restricted to the base of the tubules (arrows; Inset, 4X mag.). (B to F) FRET-FLIM analyses. (B) Mean fluorescence lifetime (τ, ns) of GFP:2B over the length of the tubule when expressed alone or together with PDLP1:RFP (left bars), of GFP:2B over the length of the tubule when expressed alone or together with RFP:2B (central bars) and of 30K:GFP alone or together with PDLP1:RFP (right bars). Mean FRET values (percentage) are given in red. Significant differences (Student's t tests; P<0.05) are indicated with asterisks. Error bars  =  standard deviation. n is the number of measurements and N the number of independent experiments. (C, D) Fluorescence intensity (left) and lifetime images (right) of GFP:2B in the presence of PDLP1:RFP (C) and of GFP:2B alone (D). Note the change in lifetime restricted to the base of the tubule (C). (E, F) Fluorescence lifetime images of GFP:2B in the presence of RFP:2B (E) and of 30K:GFP in the presence of PDLP1:RFP (F). Note the color shift between (D) and (E). Fluorescence lifetime measurements are illustrated using the false color code shown in (C, right) ranging from 1 ns (blue) to 3 ns (orange). Donor and acceptor combinations and maximum FRET values were measured at sites identified by arrowheads. Bars  = 5 µm.

Figure 2. PDLPs collectively interact with GFLV 2B.

Fluorescence intensity (left) and lifetime images (right) of GFP:2B in the presence of PDLP2:RFP (A), PDLP3:RFP (B), PDLP4:RFP (C), PDLP5:RFP (D), PDLP6:RFP (E), PDLP7:RFP (F) and PDLP8:RFP (G). Fluorescence intensity images are shown as grey scale pictures and lifetime images are represented using the false color code shown in the bottom right panel ranging from 1 ns (blue) to 3 ns (orange). Maximum FRET values (percentages) were measured at sites identified by arrowheads. Bars  = 5 µm.

Figure 3. GFLV 2B is not a secretory cargo but inhibition of ER-export prevents tubule formation.

Localization patterns and expression analyses related to GFP:2B (A to E), 30K:GFP (F to J) and PDLP1:GFP (K to O). Tagged proteins were coexpressed with either Sar1:RFP (A, F, K) or the secretion inhibitory mutant Sar1[H74L]:RFP (B, C, G, H, L, M), and location observed in median (left and central panels) or cortical (right panels) sections of leaf epidermal cells. Statistical analyses related to tubule formation efficiency by GFP:2B (D), plasmodesmal targeting of 30K:GFP (I) and ER-retention of PDLP1:GFP (N) upon coexpression with Sar1:RFP (white bars) or Sar1[H74L]:RFP (grey bars). Tubule formation efficiency is calculated as the ratio of cells containing tubules over the total number of fluorescent cells. Mean values are indicated in the histograms. Error bars, standard deviation. (E, J, O) Anti-GFP immunoblot (top) and Coomassie blue–stained gels (bottom) analyses of cells expressing GFP:2B (E), 30K:GFP (J) and PDLP1:GFP (O) together with Sar1:RFP or Sar1[H74L]:RFP. WT  =  wild type. Asterisks mark statistically significant differences between treated and mock-treated samples (t-test, P<0.01). N  =  nucleus. Arrowheads point at proteins of expected molecular mass. Scale bars  = 10 µm.

Figure 4. Genetic disruption of PDLP genes inhibits 2B tubule formation.

(A to C) Tubule formation in WT and pdlp1/2/3 mutants measured upon ectopic expression of GFP:2B alone (A) or together with PDLP1 (B) or PDLP3 (C). Statistically significant differences (Fisher exact test, P<0.001) are marked with asterisks. Error bars  =  standard errors. (D to F) Intracellular localisation of ectopically expressed GFP:2B in epidermal cells of the pdlp1/2/3 mutant line (D and E show two cells from the same treatment, one without tubules (D) and one with reduced tubules (E)) and in cells of WT plants (F). D is a maximum intensity projection from 15 consecutive sections representing 6.75 µm; E and F are single optical sections. Bars  = 20 µm.

Figure 5. GFLV cell-to-cell and long distance movement is altered in pdlp1/2/3 mutants.

(A) Infection foci generated by GFLV:RFP on WT (top) and pdlp1/2/3 (bottom) plants at 3 dpi. Scale bar  = 1 mm. (B) Corresponding box plot with whiskers from minimum to maximum of fluorescent foci size distribution. Calculated mean values are given for each graph. Significant differences (ANOVA; P<0.05) are indicated with asterisks. (C) Analysis of GFLV (left chart) and ORMV (right chart) long distance movement. Inoculated leaves were removed at 2.5 and 3 dpi and plants tested for systemic infection at 14 dpi. Light grey, healthy plants. Dark grey, infected plants. Significant differences (ANOVA, P<0.05) are indicated with asterisks. N  =  number of independent experiments. n  =  number of samples.

Figure 6. PDLPs are important contributors to CaMV movement in planta.

(A) Analysis of CaMV long distance movement in WT and pdlp1/2/3 plants. Inoculated leaves were removed at 6 dpi and plants tested for infection at 21 dpi. Light grey, healthy plants. Dark grey, infected plants. The treatments were significantly different at P<0.05 (ANOVA); N  =  number of independent experiments. (B) Symptoms observed on pdlp1/2/3 (upper) and WT (lower) plants at 21 dpi. Plants were sorted by symptom severity from top left to bottom right (C) Fluorescence lifetime (τ, ns) measured for P1:GFP when expressed alone (left bar) or together with PDLP1:RFP (right bar). Mean FRET values (percentage) are given in red. The treatments were significantly different at P<0.05 (Student's t-test). (D) Fluorescence intensity (left) and lifetime images (right) of P1:GFP alone (top) and P1:GFP in the presence of PDLP1:RFP (bottom). Fluorescence intensity images are shown as grey scale pictures and lifetime images are represented using the false color code shown in the bottom panel ranging from 1 ns (blue) to 3 ns (orange). Donor and acceptor combinations are given in all panels. FRET value (percentage) was measured at site identified by arrowhead. Note: Although P1:GFP is targeted to PD, unlike GFLV GFP:2B, it is unable to form tubules unless supplemented with unfused P1 . Error bars  =  standard deviation. n  =  number of measurements. N  =  number of independent experiments. Bars  = 5 µm.

Figure 7. Genetic disruption of multiple PDLP genes does not affect GFLV and CaMV replication.

(A) Detection of progeny GFLV RNA1 and RNA2 (top), 18S ribosomal RNA (middle) and total rRNA (bottom) from WT (left) and pdlp1/2/3 (right) protoplasts, 72 hours post-transfection. GFLV vRNAs and 18S ribosomal RNA were detected by northern blot using radiolabeled probes. rRNA were detected after ethidium bromide staining. Ratios of RNA2 over 18S signal intensities are indicated below the respective lanes. (B) Detection of CaMV P4 capsid protein (top), GFP (transfection control, middle) and Coomassie blue stained proteins (bottom) from CaMV-infected WT and pdlp1/2/3 protoplasts. (-) refers to healthy Arabidopsis protoplasts. Ratios of P4 over GFP signal intensities are given below the respective lanes.