FIG 9.

TuMV acquires an envelope by hijacking the tonoplast. (A and B) N. benthamiana leaf midribs systemically infected with TuMV/6K₂:GFP were fixed by chemical fixation (A) or HPF (B), processed, and observed by TEM. A higher-magnification image of the area in the white rectangle is shown at the bottom. Monolayer dot-like structures are aligned along the tonoplast (A, panel I) and loaded into the vacuole from the cytoplasm (A, panel II, and B). (C) 3-D reconstruction of enveloped TuMV particles by ET. (Left) A higher-magnification image of the area in the white square in panel A (panel II) with a 180° rotation showing a single slice of the tomogram generated from the 90-nm-thick section and enveloped dot-like structures in the vacuole; (middle) the 3-D model generated from the whole tomogram of the left panel; (right) the 90° rotation of the dot-like structures in the middle panel. Yellow, tonoplast; red, dot-like structures. (D, E) CP and HCpro are present in purified vacuoles. (D) Vacuoles were isolated by Ficoll gradient centrifugation from N. benthamiana leaves systemically infected with TuMV. (E) Western blot analysis of viral proteins CP and HCpro in vacuoles purified from mock-infected N. benthamiana leaves and N. benthamiana leaves systemically infected with TuMV. The total proteins of N. benthamiana leaves systemically infected with TuMV were used as a positive control. V, vacuole; CW, cell wall; M, mitochondrion; rER, rough endoplasmic reticulum; G, Golgi apparatus; CL, chloroplast; P, particles.