Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA

Abstract

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.

Cell-to-cell Transport of Cowpea Mosaic Virus Requires Both the 58K/48K Proteins and the Capsid Proteins

“…At first sight this result seems contradictory with our results because the B RNA of RCMV will probably not be encapsidated by the SHMV coat protein and therefore must be transported as naked RNA in this mixed infection. However a simple explanation would be to assume that the CPMV transport protein(s) transports (or facilitates the transportation of) encapsidated RNA only, whereas the tobarnovirus transport protein can transport (or perhaps can transport only) naked RNA; tobacco mosaic tobamovirus coat protein is indeed dispensable for transport from cell to cell (Takamatsu et al, 1987).…”

Replication of tobamovirus RNA

“…However, due to the lack of the CP, the virus could not spread systemically. 82) In order to create a vector that was capable of systemic infection, Dawson et al constructed a chimeric TMV, in which the coat protein-coding region was replaced with a foreign gene, and a second CP mRNA promoter, followed by a CP gene, was inserted behind the foreign gene sequence. However, upon infection of plants, homologous recombination deleted the region between the twice-repeated subgenomic RNA promoter sequences, which included the foreign gene.…”

Long-distance movement of cherry leaf roll virus in infected tobacco plants

“…In contrast to cell-to-cell movement, many factors are involved in long-distance movement; the requirements are different depending on the virus group. In addition to the MP, a functional coat protein (CP) is required for long-distance movement by some viruses belonging to the tobamo- (Takamatsu et al, 1987), bromo- (Allison et al, 1990;Flasinski et al, 1995), carmo- (Heaton et al, 1991), cucumo-(Suzuki et * Author for correspondence. Fax +34 68 266613. e-mail EBSPB10@CEBAS.…”