The Construction and Complementary Expression of two TMV Defective Particles in Tobacco

Two defective mutants of the tobacco mosaic virus (TMV), TMVRP and TMVCP were constructed and assembled in vitro. In TMVRP, the 3'-end and part of the TMV coat protein (CP) gene was deleted; in TMRCP, most of the replicase genes were deleted. These mutant particles were separately or complementally inoculated into the tobacco protoplasts by electroporation. The synthesis of TMV coat protein was deteted by immuno dot-blot technique at 2 h after inoculation only in those complementally infected protoplasts. In addition, using the RT/PCR technique, the minus-strand viral RNA of the TMV 3'-end including CP gene was also only detected in those protoplasts which were complementally infected with both of the mutant particles. The synthesis of the minus-strand RNA started to be detectable at 1h after inoculation and it was confirmed by Southern blot analysis.