No abstract

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature‐sensitive mutant defective in cell‐to‐cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one‐base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame‐shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame‐shift mutants were replication‐competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell‐to‐cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis‐acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.

We have established an in vitro transcription system to produce infectious tobacco mosaic virus (TMV) RNA from a cloned cDNA copy. Using this system, several TMV mutants were transcribed in vitro from cDNA clones mutagenized at or near the leaky amber termination codon of the 130K protein gene, and their infectivity was assayed on tobacco plants.Three (two frame-shift and one non-sense) mutants with an intact 130K but a defective 180K protein gene were not infectious, while two mutants with a one-amino-acid insertion in the 180K protein gene were infectious. When the amber codon of the 130K protein gene was deleted, infectivity was lost. However, when the amber termination codon was replaced with ochre or tyrosine codon, infectivity was retained. Sequence analyses revealed that introduced mutations were retained in progeny viral sequences except in the progeny of the amber-to-tyrosine mutant, which was a mixture of the parental mutagenized virus and a pseudo-revertant with ochre codon.

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was -0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector. Key words: coatless TMV mutant/complementary DNA expression system/plant RNA vector/TMV-CAT chimera/tobacco mosaic virus tissue (Matthews, 1981)], when a foreign gene is introduced in place of the coat protein gene, the resultant chimeric TMV might direct high-level expression of the introduced foreign gene.The complementary DNA expression system by which infectious TMV RNA can be transcribed in vitro has recently been established (Dawson et al., 1986;Meshi et al., 1986). As the TMV genome can be manipulated at the level of DNA, reverse genetics can be applied to TMV and also TMV may be utilized as an expression vector in higher plants. To evaluate the availability of TMV for introducing foreign genes into plants and directing their expression, we constructed three TMV cDNA derivatives: one coatless TMV cDNA and two TMV-CAT chimera cDNAs. Here we describe the construction of the cDNA derivatives, and demonstrate that in vitro transcripts derived from these cDNAs were infectious for tobacco plants and that CAT was produced in the leaves inoculated with transcripts derived from the TMV-CAT chimera cDNAs.