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. 1998 Sep 15;95(19):11169-74.
doi: 10.1073/pnas.95.19.11169.

Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum

C Reichel  1 , R N Beachy
Affiliations

Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum

C Reichel et al. Proc Natl Acad Sci U S A. .

Abstract

The tobacco mosaic virus (TMV) movement protein (MP) facilitates transport of virus infection between adjacent cells by modifying plasmodesmata. Previous studies suggested that the cytoskeleton and the endomembrane system are involved in this transport. We examined the effects of TMV infection on the endoplasmic reticulum (ER) in transgenic Nicotiana benthamiana that accumulate the green fluorescent protein (GFP) in the ER. Fluorescence microscopy was used to show that early in infection the ER undergoes dramatic morphological changes that include the conversion of tubular ER into large aggregates that revert to tubular ER in later stages of infection. These changes parallel MP accumulation and degradation. Furthermore, a fusion protein comprising MP fused to GFP accumulates in or on these large aggregates of ER. Expression of MP-GFP in the absence of virus infection led to the production of fluorescent aggregates of the same apparent form and size. Microsomes isolated from infected leaves contain MP. We show that the MP appears to behave as an integral ER membrane protein and is exposed on the cytosolic face of the ER. The importance of the association of MP with ER and its possible role in intracellular and intercellular spread of infection is discussed.

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Figures

Figure 1
Figure 1
Fluorescence micrographs of epidermal leaf cells of N. benthamiana (A) or N. benthamiana erGFP (B–H) infected with TMV or TMV-MP:GFP. (A1) Fluorescence surrounding the nucleus (n) due to MP-GFP early in infection (arrow). (A2) Fluorescent structures containing MP-GFP in cells early in infection. (A3) Cortical fluorescent bodies in early to mid stages of infection. (B) Noninfected epidermal cells of N. benthamiana erGFP. (B1) Perinuclear fluorescent halo of erGFP (arrow). (B2) Normal reticulate pattern of cortical ER. (B3) Fast-moving tracks of erGFP fluorescence (arrowheads). (C–F) Left panels excited with blue light, right panels excited with UV. Note: UV excitation exclusively excites erGFP, while excitation with blue light excites both MP-GFP and erGFP. (C, C′) Early stage of infection, showing small cortical aggregates formed at vortexes of ER network; much of the fluorescence is contributed by MP-GFP (arrows). (D, D′) Large cortical aggregates and disrupted cortical ER in mid-stages of infection. (E, E′) Recovery of tubular ER and dissociation of large cortical aggregates and development of filamentous fluorescence of MP-GFP colocalized with microtubules (arrows) as well as a punctate fluorescence on the cell surface (arrowheads) in mid to late stages of infection. (F, F′) Cortical aggregates decrease and filaments comprising MP-GFP on microtubules (arrows) are apparent in late stages of infection; punctate fluorescent structures often aligned with microtubules (arrows); ER has recovered to preinfection state. (G) Large cortical ER aggregates induced in N. benthamiana erGFP during infection by wild-type TMV. (H) Tubular cisternae of the cortical ER are disrupted and converted to lamellar aggregates. (Bars = 0.5 μm.)
Figure 2
Figure 2
Fluorescence micrographs of N. benthamiana leaves after particle bombardment with pe35S-MP:GFP. Detached leaves were investigated by fluorescence microscopy 15–20 hr after bombardment. In epidermal cells MP-GFP localized to large fluorescent aggregates (A), which often were associated with filamentous structures (B and C) and presumably correspond to MP-GFP coalignment with microtubules. (Bars = 0.5 μm.)
Figure 3
Figure 3
Western blot analyses of fractions from sucrose density gradients. Membrane preparations with bound (5 mM MgCl2) or displaced (0.1 mM MgCl2) ribosomes were separated on an 18–55% sucrose gradient. Fractions were analyzed with indicated antibodies. Sedimentation was from left to right. (A) Anti-GFP. (B) Anti-MP. (C) Anti-ATPase. (D) Anti-BiP.
Figure 4
Figure 4
Proteinase K treatment of fractions from sucrose gradients. Microsomal membranes were collected from fractions of the sucrose gradients (see Fig. 3) and subjected to proteinase K digestion in the absence (−) or presence (+) of Triton X-100 and analyzed by Western blotting with anti-GFP (Upper) or anti-MP (Lower) antibodies.
Figure 5
Figure 5
Effects of membrane washes on association of MP with ER. Microsomal membranes were collected as in Fig. 4 and incubated 30 min on ice with 2.5 M urea, 0.5 M NaCl, 1% Triton X-100, or 1% CHAPS. After samples were collected by centrifugation, they were subjected to Western blot analysis with anti-MP, anti-GFP, anti-ATPase, and anti-BiP antibodies.
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