Analysis and application of CaMV 35S promotor-driven clones of tobacco mosaic virus
thesisposted on 15.12.2014, 10:33 by Emma Mary. Dagless
Recently, cDNA clones of RNA viruses have been fused to the 35S promoter from CaMV. Having received a 35S-cDNA clone of TMV-U1 we investigated its infectivity. Protoplast, microprojectile bombardment and plant transformation experiments were conducted. We were able to demonstrate that the clone was highly infectious to tissue known to host TMV. However, the 35S-TMV cDNA construct was unable to reliably induce an infection following the manual inoculation of leaves, possible reasons have been discussed. Using the 35S-TMV cDNA we generated four replication-marker constructs. Each construct contained the luciferase marker gene inserted in place of the TMV coat protein ORF. As a result, the constructs should have been capable of replication-dependent expression of luciferase. The constructs were tested using microprojectile bombardment and plant transformation experiments. Unfortunately the constructs did not appear to function in the manner intended. The design of each construct has been fully discussed and we conclude that further analysis of transgenic plants lines is required.;The application of TMV-based replication and replicase constructs for studies of Tm-1 and N gene mediated resistance has been considered. Resistance to the N gene has been investigated via microprojectile bombardment and plant transformation experiments. Evidence suggests that the formation of an active replicase-complex may be required to elicit the N gene-mediated hypersensitive response. The expression of TMV replicase proteins alone does not appear to elicit the response. Following the inoculation of TMV on to transgenic N. tabacum Samsun NN plants, expressing replication or replicase constructs, unusual phenotypes were observed. A number of interesting transgenic plant lines have been generated. Investigations have been conducted to determine whether they were resistant to TMV. Results suggest that N. tabacum SR1 plants expressing a TMV-based replicase construct exhibit a resistant phenotype.