Nonsense mutations of replicase and movement protein genes contribute to the attenuation of an avirulent tomato mosaic virus.

Three recovery mutants of an avirulent Tomato mosaic virus genus: (Tobamovirus) (ToMV-K) with back mutations of the replicase and/or movement protein (MP) genes, have been constructed by site-directed mutagenesis, and infectious plasmids (pToMV-K) were obtained. The rescued phenotypes of the progeny viruses showed that the replicase and MP recovery mutant (ToMV-K(rase-mp)) induced severe symptoms on both systemic and necrotic plants similar to those induced by the virulent strain. The replicase back mutant (ToMV-K(rase)) produced chlorosis and mosaic symptoms on N. tabacum cv. Huangmiaoyu (systemic host), while the MP recovery mutant (ToMV-K(mp)) produced no systemic symptoms on Huangmiaoyu tobacco. Sequencing of the cDNA of progeny viruses revealed that the "back mutants" maintained these mutation sites during infection. Protein immunoblots indicated that the 98 and 126 kDa proteins were expressed in the plants systemically infected by ToMV-K and pToMV-K, whereas no 98 kDa protein was detected in the plants infected by ToMV. The MPs (27 kDa) of ToMV-K and pToMV-K in the plants were smaller in size than those (30 kDa) of ToMV and pToMVK(rase-mp). These data suggest that ToMV-K replicates and spreads by expressing the truncated 98 and 126 kDa replicases and 27 kDa MP in plants. The opal mutation at nucleotides (nt) 2670-2672 of the replicase gene mainly contributes to the attenuation of ToMV-K, whereas the mutations at nt 5632-5664 of the MP gene attenuate the induced symptoms.