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Abstract Tomato mosaic virus (ToMV), a type species of the genus Tobamovirus, severely affects the yield and quality of crops. ToMV-N5 infection caused Lycopersicon esculentum Mill. main cultivar Hezuo 903 systemic necrosis. It is essential to obtain its viral infectious clone for analyzing the mechanism of ToMV-N5 pathogenesis. In this study, the full length of ToMV-N5 genomic RNA was cloned into pCB301 vector which carried duplicated 35S promoter and ribozyme and then pCB301-ToMV-N5 was transformed into Agrobacterium tumefaciens GV3101. Agroinfiltration results indicated that the seedlings of Nicotiana benthemiana and L.esculentum infected by pCB301-ToMV-N5 appreared the same phenotypic caused by ToMV-N5 viral particles. Vector pCB301-ToMV-N5CP45GFP was constructed by replacing the ORF of viral coat protein gene (CP) with that of green fluorescent protein gene (GFP) and the data showed that the vector could highly express GFP in leaves of N. benthemiana in the presence of gene silencing suppressor p19. Even in leaves infiltrated with A. tumefaciens cells diluted 1∶500 from a starting optical density (OD600) of 1.0, most cells of the infiltrated zone expressed GFP at 7 days post-infitation(dpi). Due to the lack of CP, pCB301-ToMV-N5CP45GFP could not move to upper leaves of host plants. pCB301-ToMV-N5CP45GFP-CPU5 was obtained by inserting CP (nt 5 498~6 145) of Tobacco mosaic virus (TMV) U5 strain and the phenotype of pCB301-ToMV-N5CP45GFP-CPU5 on N. benthamiana suggested that the recombinant ToMV-N5 could move to non-inoculated leaves and GFP the whole plant expressed in the whole plant at 10 dpi. While the results from the seedlings of L.esculentum inoculated with pCB301-ToMV-N5CP45GFP-CPU5 suggested that there was only some weak GFP expression on inoculated leaves under the microscope. The successful construct of Agrobacterium-mediated infectious clone and its expression vector provides basic data for dissecting the molecular mechanism of ToMV-N5 replication and movement.
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Received: 03 March 2014
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