1.12.24
A CMV RNA 1 TRANSGENE IN TOBACCO CONFERS SYSTEMIC RESISTANCE TO VIRAL INFECTION WHILE COMPLEMENTING REPLICATION OF VIRAL RNAS 2 AND 3

T CANTO and P PALUKAITIS

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Background and objectives
Two viral proteins are known to be viral components of the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV); the 1a and 2a proteins, encoded by viral RNAs 1 and 2, respectively. We have transformed tobacco to express biologically active 1a protein in order to use it in studies of the viral RdRp. Here we report that as well as constitutively expressing functional 1a protein, some of the transgenic lines simultaneously and unexpectedly displayed a high resistance to systemic infection by the homologous virus. We present our results on the nature of this resistance in relation with the existing models [1].

Results and conclusions
Tobacco plants transformed with a cDNA corresponding to a modilied full-length RNA 1 of the Fny strain of CMV were able to sustain replication of viral RNAs 2 and 3 when inoculated in absence of RNA 1. Initially, these plants remained asymptomatic and the levels of viral RNA in the inoculated leaf were 50 to 100 times lower than in plants that had been inoculated with the three viral RNAs. Furthermore, RNAs 2 and 3 were detected in non-inoculated leaves, suggesting that the transgene not only sustained viral replication but also long distance movement. No RNA 1 could be detected by Northern blot analysis. However, with a delay of several days relative to control plants, symptoms of infection appeared in the nascent leaves that correlated with the detection of viral RNA 1 in these tissues. This regenerated viral RNA I contained a marker specific for the transgenic messenger RNA. Although the FO-generation plants were susceptible to CMV, selection in one line that showed variable susceptibility to CMV through Fl - and F2-generation plants led to F3-generation lines with systemic resistance to Fny-CMV. Northern blot analysis of viral RNA accumulation in the inoculated leaves of these resistant plants showed a dramatic decrease in the levels of viral RNA 1 (ranging from twenty-fold to undetectable) relative to the levels found in both non-transformed and susceptible transgenic tobacco plants. A proportional, but less pronounced, two- to twelve-fold decrease was also observed in the levels of viral RNAs 2-4. The latter probably was caused by the very low amounts of la protein, affecting the rate of viral replication in a feedback effect. No trace of viral RNAs was found in non-inoculated leaves. In spite of the systemic resistance, these plants could complement local replication of inoculated CMV RNAs 2 and 3 even in plants previously inoculated with virus, indicating that neither a constitutive nor an induced complete transgene silencing occurs. The nature of this resistance is most likely to be homology-dependent since it is not effecfive against the LS strain of CMV which belongs to a dilferent CMV subgroup than the Fny strain. Although the data show that a transgene-mediated gene-silencing process takes place, the absence of systemic subliminal infection by viral RNAs 2 and 3 suggests the existence of additional interference with viral long-distance movement.

References
1. Baulcombe, D. (1996). Plant Cell 8, 1833-1844.