Structural and functional alayses of the plant viral RNA polymerases for the breeding of virus-resistant transgenic plants.

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1. Active and template-specific tobacco mosaic virus (TMV) RNA polymerase was successfully purified from TMV-infected tobacco leaf extracts through an affinity chlomatography using anti-RNA polymerase domein of TMV 183K protein. The apoenzyme was presumed to be a heterodimer composed of each one molecules of 126K and 183K proteins. While, three different host proteins bound to helicase domein of TMV RNA polymerase were detected by the yeast two-hybrid system.2. The genome of both cucumber mosaic virus (CMV) and peanut stunt virus (PSV) consists of three single-stranded RNAs, RNA 1, 2 and 3. 1a protein and 2a protein encoded by RNA1 and RNA2, respectively, are necessary for the viral replication. Feasibility of exchanges of RNAs 1 and 2 was re-examined by using infectious RNA transcripts for CMV RNAs and PSV RNAs. When we coinoculated all the pseudorecombinants into tobacco protoplasts, pseodorecombinants with PSV RNA 1 and CMV RNA 2 were replicable, but those with CMV RNA 1 and PSV RNA … More 2 were not. Using yeast two-hybrid system, the C terminal region of PSV-1a protein was exhibited to interact with not only the N terminal region of PSV 2a protein but also that of CMV 2a protein. These results were correlated with those of replication in protoplasts.3. Using full-length infectious cDNA clones to RNA 1 and RNA2 of Soil-borne wheat mosaic virus (SBWMV), a series of RNA 1 mutants of 152K and 211K replicase proteins were constructed. Results showed that the 211K protein with methyltransferase, helicase and polymerase domains in the absence of the 152K protein possessing the former two domains is sufficient for virus replication at a low level. The 152K protein together with the 211K protein were shown to be required for the high-level RNA replication. Expression of a cysteine-rich protein encoded in the 3'-terminal region of RNA2 was absolutely required for virus replication. Infectious cDNA clones to a US strain were constructed. Reassortment experiments between US and Japanese strains showed that the combination of Japanese RNA1 and US RNA2 was systemically infectious to wheat plants whereas that of US RNA1 and Japanese RNA2 was not infectious even to Chenopodium quinoa, a local lesion host. Less