Molecular-genetic analysis of N-gene mediated resistance to Tobacco Mosaic Virus in Solanaceae

A molecular genetic approach was used to study the signal transduction pathway of the N gene. The N gene is a dominant resistance gene in tobacco that confers disease resistance to Tobacco Mosaic Virus (TMV). In order to identify proteins that interact with the N protein to confer disease resistance, a yeast two-hybrid approach was used. Five candidate interactors were identified: two from A. thaliana, two from tomato, and one from both tomato and tobacco. To assess biological relevance of candidate interactors, transgenic and transient assays were used. Transgenic plants with perturbed expression levels of the candidate genes were made to determine if changes in expression levels would affect N-mediated disease resistance. Also, transient Virus-Induced-Gene-Silencing (VIGS) was used to determine if reduction of their expression levels would alter N-mediated resistance to TMV. From these assays, no evidence has been found that any of the interactors play a direct role in the N pathway.; In addition to searching for proteins that directly interact with the N protein, other possible signaling molecules were studied. Chapter three is presented in which the NPR1 gene from Arabidopsis is investigated as a possible downstream signal transduction component in the N pathway. The NPR1 gene has been shown to be a regulator of systemic acquired resistance (SAR). Plants with an npr1 mutation are unable to mount SAR in response to pathogens or SAR inducing chemicals. To assess if NPR1 is involved in N-mediated resistance to TMV in tobacco, we created transgenic tobacco with perturbed levels of NPR1 gene expression. Transgenic overexpressors showed high levels of transgene expression, but demonstrated no detectable change in resistance or susceptibility to TMV in the N and non-N tobacco lines. As a second approach, VIGS was used to transiently silence NPR1. Also, no change in resistance was observed in the transiently silenced plants. More sensitive techniques may be needed to confirm the role of NPR1 in the N-gene disease resistance pathway.