The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous repfication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccesfl in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamna, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on pgin to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.The expression of foreign genes in plants has proven advantageous to the study of molecular biology (1,2). Stable gene transfer to whole plants can be achieved by a combination of DNA transformation and tissue culture techniques or by virus transfection. With viral-based vectors the ease of infection avoids the time-consuming procedures, "position" effects, and "somaclonal" variation seen when foreign genes are integrated into the plant genome (1). The merits of using a systemically expressing plant RNA virus-based vector have been extensively reviewed (3-7). Several features of tobacco mosaic virus (TMV) (8,9) suggest that it might be usefully adapted for such a purpose: (i) tobamoviruses have a wide host range; (it) they can move cell-to-cell mediated by a virus-encoded peptide; (iii) they exhibit rapid systemic spread in plants; (iv) TMV infections are maintained for the lifetime ofthe plant; (v) TMV RNA is replicated to high levels as autonomous sequences; (vi) this replication results in rapid and productive cytoplasmic gene expression; (vii) temperature-sensitive mutations of RNA synthesis are available to modulate expression of foreign genes; (viii) TMV also lacks the packaging constraints found with nonhelical viruses, including existing DNA plant virus vectors; and (ix) the TMV genome can now be manipulated as a DNA copy and then transcribed in vitro to produce infectious RNA molecules.The TMV genome consists of one 6395-nucleotide (nt) molecule of messenger-sense, single-stranded RNA, encoding at least four proteins (10; for review see ref. 9; Fig. 1). It has similarities of sequence and replication strategy with a series of plant and animal RNA viruses (11). The 126-and 183-kDa replicase proteins are translated directly from the genomic RNA, whereas the 30-kDa cell-to-cell movement protein and 17.5-kDa coat protein are translated from two 3'-coterminal su...

The nucleotide sequence of the DNA of maize streak virus (MSV) has been determined. The data were accommodated into one DNA circle of 2687 nucleotides, in contrast to previously characterised geminiviruses which have been shown to possess two circles of DNA. Comparison of the nucleotide sequences of the DNA of MSV with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) showed no detectable homology. Analysis of open reading frames revealed seven potential coding regions for proteins of mol. wt. greater than or equal to 10 000, three in the viral (+) sense and four in the complementary (‐) sense. The position of likely transcription signals on the MSV DNA sequence would suggest a bidirectional strategy of transcription as proposed for CLV and TGMV. Nine inverted repeat sequences which have a potential of forming hairpin structures of delta G greater than or equal to ‐14 kcal/mol have been detected. Three of these hairpin structures are in non‐coding regions and could be involved in the regulation of transcription and/or replication.

SUMMARYInsertion and deletion mutagenesis of the two virion-sense genes, V1 and V2, of maize streak virus (MSV) prevents symptomatic infections following Agrobacteriummediated 'agroinoculation' of maize seedlings. These genes code for an Mr 10900 protein and for coat protein, respectively. Mutants containing insertions or deletions in the coat protein gene, V2, were able to replicate to low levels, producing dsDNA although virion ssDNA was not detected and symptoms were not observed. Hence, unlike the bipartite geminiviruses, MSV requires coat protein to produce symptomatic systemic infection. Mutations in gene V1 which considerably shortened the Mr 10900 protein (V1 gen¢) resulted either in low levels of replication, in which all the DNA forms associated with a wild-type infection were produced, or in no infection, in which case coat protein production may also have been affected. A V1 mutant generated in vivo with 11 of the 14 N-terminal amino acids altered, was viable and produced symptoms typical of a wild-type infection. Infectivity, assessed by replication and symptom expression, was restored by co-inoculating constructs containing single mutations in different open reading frames, thus rescue can occur by transcomplementation of gene products. The experiments showed that the mutations did not affect the nucleotide sequence requirements for replication and that in all cases intermolecular recombination eventually resulted in dominant wild-type virus.