Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants

Abstract

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

Figure 1

(A) Electron micrograph of a damaged cell close to a functional uninfected sieve element (SE) in a pPVXSma‐p18‐inoculated Nicotiana benthamiana leaf, containing overproliferated endoplasmic reticulum (ER) (arrow). (B) Coat protein immunogold labelling (CP‐IGL) of a pPVXSma‐p18‐inoculated leaf, showing a vascular parenchyma cell with laminated inclusion components (LICs) surrounded by amorphous CP aggregates (enlarged in C). (D) CP‐IGL of a pPVXSma‐p18DD‐inoculated leaf, showing the cytoplasm of a xylem parenchyma cell containing LICs, proteinaceous material and virions (enlarged in E). (F) CP–IGL of a PVXSma‐p18DD infected systemic leaf, showing a mesophyll cell containing LICs surrounded by CP (arrow). (G) PVXSma‐p18DD virions (arrows) in a mesophyll cell in a systemic infected leaf. (H) Light microscopy of an Evans blue‐stained pPVXSma‐Z13‐inoculated leaf at 4 days post‐inoculation (dpi). (I) Evans blue‐stained leaf inoculated with phosphate‐buffered saline as a control. (J) Portion of a leaf treated as in (H) at 10 dpi (enlargement in K). (L) Section of a pGR106‐Z13 agroinfiltrated leaf showing LICs. (M) CP‐IGL of a pGR106‐Z13 infected cell with large amounts of CP accumulated in the cytoplasm among the ER endomembrane system (arrow). Bars: (A, D, F, L, M) 500 nm; (B) 1 µm; (C, E, G) 250 nm; (J) 100 µm; (H, I) 50 µm; (K) 20 µm.

Figure 2

(A) Evans blue‐stained PVXSma‐infected systemic leaf, showing infected cells around class IV and V (minor) veins (arrow). (B) Evans blue staining of a cluster of PVXSma‐infected cells at the end of a class V vein (the arrow indicates the terminal part of a xylem element, visible by merging two images at different focus planes). (C) Evans blue staining of a cluster of PVXSma‐infected cells at the end of a class V vein. (D) Coat protein immunogold labelling (CP‐IGL) of a wild‐type virus‐infected systemic leaf, showing virus accumulation (V) in a vascular parenchyma cell (frame enlarged in E) (X, mature xylem; Ch, chloroplasts). (F) CP‐IGL of a developing xylem element in a systemic leaf, showing wild‐type virus accumulation in the residual cytoplasm (C) (M, mitochondria) (frame enlarged in G). (H) CP‐IGL of a differentiated xylem element containing wild‐type virus (frame enlarged in I). Bars: (A) 200 µm; (B, C) 50 µm; (D) 1 µm; (E, F, H) 500 nm; (G, I) 100 nm.

Figure 3

(A) Coat protein immunogold labelling (CP‐IGL) of the longitudinal section of a class V vein in a PVXSma‐infected systemic leaf at 10 days post‐inoculation (dpi) (X, mature xylem; XP, xylem parenchyma; SE, sieve element) (the cross‐section of a similar vein is shown in inset B). (C) CP‐IGL of the xylem framed in (A) showing PVXSma accumulation. (D) CP‐IGL of the PVXSma‐infected xylem parenchyma cell framed in (A) containing disordered masses of virus particles. (E) CP‐IGL of the PVXSma‐infected immature SE framed in (A). (F) Light microscopy of a longitudinal leaf section after CP‐IGL and silver enhancement showing the CP (dark spots indicated by arrows) in the terminal part of class III veins and xylem elements (inset: negative control not labelled with anti‐CP). (G) Section in (F) under polarized epifluorescence with CP evidenced as bright green spots (arrows) (inset: negative control under polarized epifluorescence). Bars: (A) 5 µm; (B) 10 µm; (C–E) 250 nm; (F, G insets) 20 µm.