Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement

Abstract

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

Figure 1

Schematic representation of TMV clones used in this study. MP, movement protein; CP, coat protein; MP sgp, MP subgenomic promoter; CP sgp, coat protein subgenomic promoter; GFP, green fluorescent protein.

Figure 2

Visualization of infection sites produced by TMV-CP:GFP (fluorescent disk) on inoculated N. tabaccum Xanthi-nn through fluorescence microscopy and compared with that produced by TMV-MP:GFP (fluorescent ring).

Figure 3

Patterns of accumulation of CP:GFP and CP^T42W:GFP in BY-2 protoplasts inoculated with TMV-CP:GFP or TMV-CP^T42W:GFP, respectively. Protoplasts were inoculated with transcribed RNAs, and the accumulation of CP:GFP or CP^T42W:GFP was analyzed by fluorescence microscopy. Pictures were taken at 12, 15, and 24 h.p.i.

Figure 4

Growth of infection sites on transgenic and nontransgenic tobacco plants. Growth of infection sites caused by TMV-MP:GFP-CP and TMV-CP:GFP on nontransgenic plants or transgenic plants expressing wt CP, CP^T42W, or TMV MP. The diameter of infection sites (in mm) was measured at 7 days after inoculation and averaged. (Bars = SE.)

Figure 5

Expansion of infection sites produced by TMV-CP:GFP and TMV-CP^T42W:GFP on nontransgenic N. tabaccum Xanthi-nn or transgenic plants expressing TMV MP (line 277). The diameter of infection sites (in mm) was measured at 4 dpi and averaged. For the mean size of infection sites, bars = SE. *, no infection sites detected.

Figure 6

Effects of CP^T42W on TMV MP accumulation during virus infection. Protoplasts of nontransgenic BY2 and transgenic BY2 expressing wt CP (BY-CP) or mutant CP^T42W (BY-CP^T42W) were inoculated with TMV-MP:GFP RNA. The patterns and cellular distribution of MP:GFP were followed in time by using fluorescence microscopy. The structures formed by MP:GFP were compared in protoplasts from nontransgenic BY-2, BY-CP, and BY-CP^T42W cells.

Figure 7

Effects of CP^T42W on TMV MP levels of accumulation during virus infection. Protoplasts of nontransgenic BY2 and transgenic BY2 expressing wt CP (BY-CP) or mutant CP^T42W (BY-CP^T42W) were inoculated with TMV RNA. The relative levels of accumulation of MP (A) and of CP (B) were measured and compared in protoplasts from nontransgenic BY-2, BY-CP, and BY-CP^T42W cells during infection in ELISA experiments.

Figure 8

Analyses of TMV and TMV-T42W RNA accumulation in BY-2 protoplasts. BY-2 protoplasts were inoculated with in vitro transcribed viral RNA, and samples were harvested from time 0 (time of initial contact of RNA with protoplasts) to 50 h after inoculation. Total RNA was purified and 2 μg was analyzed by Northern blot. The position of genomic RNA and subgenomic (sg) RNAs are indicated: sg-RNA I, sg-RNA II (MP subgenomic RNA), and sg-RNAIII (CP subgenomic RNA).