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. 2007 Sep 15;366(1):107-16.
doi: 10.1016/j.virol.2007.03.052. Epub 2007 May 11.

Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein

Mohammed Bendahmane  1 , Iju Chen, Sebastian Asurmendi, Ariel Alejandro Bazzini, Judit Szecsi, Roger N Beachy
Affiliations

Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein

Mohammed Bendahmane et al. Virology. .

Abstract

Expression of tobacco mosaic virus (TMV) coat protein (CP) restricts virus disassembly and alters the accumulation of the movement protein (MP). To characterize the role of structure of transgenic CP in regulating virus disassembly and production of MP, we generated CPs with mutations at residues Glu50 and Asp77, located in the interface between juxtaposed CP subunits. In transgenic Nicotiana tabacum and BY-2 cells, three categories of coat protein-mediated resistance (CP-MR) levels were identified: wild-type CP-MR; elevated CP-MR; and no CP-MR. Mutant CPs that interfered with the accumulation of virus replication complexes conferred very high levels of protection to TMV, except by CP(E50D) which provided no protection in the systemic host (Xanthi-nn) but high CP-MR in the local lesion host (Xanthi-NN). In transgenic BY-2 cells CP(E50D) strongly reduced accumulation of MP:GFP. In general, there was a strong correlation between the capacity for CP to assemble to pseudovirions and CP-MR, while there was not strong correlation with packaging viral RNA and CP-MR. The data demonstrate that interference with one or more steps in virus infection and replication by wild type and mutant CP determine the degree of CP-MR.

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Figures

Figure 1
Figure 1
(A) Ribbon representation of the alpha-carbon backbone of two CP subunits belonging to two superimposed helical turns of TMV virion. The N and C termini located at the outer radius if the virus are indicated. The position of the interacting carboxylate residues Glu50 and Asp77 and of the Thr42 residue is shown. (B) Computer graphic representation of two and half virion helical turns of CP subunits. The N-terminus and the C-terminus of the CP molecules lie on the external surface of the assembled subunits.
Figure 2
Figure 2
Electron-microscopic analysis of assembled VLPs from N. tabacum plants infected with TMV mutated at amino acid E50 (TMV-CPE50Q, TMV-CPE50K, TMV-CPE50R, TMVCPE50D, TMV-CPE50M) or at amino acid D77 (TMV-CPD77N, TMV-CPD77K, TMV-CPD77R, TMV-CPD77E, TMV-CPD77A) or w.t. (TMV-U1). The samples were negatively stained and analyzed at a magnification of 39,000. The bar represents 100 nm.
Figure 3
Figure 3. Resistance of transgenic plant lines that produce mutant CPs to systemic infection by TMV
Protection assay for TMV infection in transgenic N. tabacum Xanthi-nn plants expressing wt CP (line 3646) or CP carrying mutations at amino acids Asp77 or Glu50. The plants were inoculated with the TMV-U1 virus (0.1 mg/ml), and disease development was scored as the percentage of plants that exhibit symptoms on upper leaves and accumulation of TMV in the systemic leaves was measured by ELISA using anti-CP antibody. ELISA was performed on samples from 10 plants for each line, as described (Bendahmane et al., 1997).
Figure 4
Figure 4. Accumulation of CP (A), MP (B) and replicase (C) proteins in BY-2 protoplasts non-transgenic or expressing wt CP or mutant CPs.
Protoplast were prepared from transgenic BY-2 cells expressing CPT42W, CPD77k, CPD77R, CPE50R or CPE50D, and then infected with TMV RNA. Protoplast samples were harvested at 11, 16, 24, and 36 hpi. The effect of the transgenic CP (mutant or wt) on the accumulation of the CP, the MR and the replicase of the infecting virus was assessed using antibodies directed against each of these viral proteins in ELISA experiments as described (Bendahmane et al., 1997).
Figure 5
Figure 5. Effect of mutant CPs expressed in transgenic BY2 cells on MP:GFP following TMV-MP:GFP infection.
(A) Patterns of accumulation of MP:GFP in BY-2 protoplasts inoculated with TMV-MP:GFP as described in Heinlein et al. (1995). Protoplasts were inoculated with TMV-MP:GFP RNA and the accumulation of MP:GFP was analyzed by fluorescence microscopy. Pictures were taken at 12, 15, and 24 hpi corresponding to early, mid and late stages of infection by TMV, respectively. MP:GFP patterns found at each stage are presented. (B) Quantification of the presence of each of the six paterns (above) of MP:GFP accumulation during TMV-MP:GFP infection in protoplast expressing mutant CPs at 10, 16, 24 and 36 hpi. The presence of each pattern was normalized for the total number of cells counted per point and expressed as a percent. The three groups of mutants CP with respect to their effect on MP accumulation are presented: group (1) CP mutants with postivie effects on MP accumulation similar to w.t. CP; group (2) CP mutants that lost the positive effect of w.t. CP on MP accumulation patterns; group (3) CP mutants that acquired negative effects on accumulation of MP.
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