|Zaitlin, Milton - CORNELL UNIVERSITY|
Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 24, 1999
Publication Date: N/A
Interpretive Summary: Since the advent of plant biotechnology in the mid 1980s, a number of methods have been developed to create resistance to plant viruses through transformation with genes of viral origin. The use of viral replicase genes has become one of the most effective and preferred methods for virus resistance, however, the mechanism responsible for this resistance varies. Sometimes viral RNA is responsible for resistance, in other cases expression of the viral protein is necessary to obtain resistant plants. Cucumber mosaic virus (CMV) is a serious problem to agriculture worldwide. We developed viral replicase gene-mediated resistance against CMV a number of years ago, by inserting a modified CMV replicase gene into the tobacco genome. Plants containing this gene are often unable to be infected by CMV. The resistance interferes with both the ability of the virus to replicate in the plant, as well as its ability to move through plant tissues. Determining how such genes create resistance when expressed by the plant genome is important for expanding this resistance to more distantly related strains of CMV, as well as for applicability of the technique to related plant viruses. This manuscript demonstrates that the ability of the plant to produce a transgene protein is important for developing replicase gene-mediated resistance against CMV.
Technical Abstract: Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted viral movement when inoculated mechanically or by aphid vectors (Anderson et al., Proc. Natl. Acad. Sci., USA 89: 8759-8763, 1992). Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full length or truncated replicase proteins. In contrast, among plants which contained nontranslatable transgene constructs, only one of sixty-one lines examined exhibited delays or resistance. Transgenic plants exhibiting a range of resistance levels were examined for transgene copy number, mRNA and protein levels. Although ribonuclease protection assays demonstrated that transgene mRNA levels were very low, resistant lines had consistently more steady-state transgene mRNA than susceptible lines. Furthermore, chlorotic or necrotic local lesions developed on the inoculated leaves of transgenic lines containing translatable transgenes, but not on inoculated leaves of lines containing nontranslatable transgenes. These results do not rule out the possibility of a role for the transgene mRNA, but demonstrate that expression of the transgene protein facilitates replicase-mediated resistance to CMV in tobacco.