The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1992.t01-24-00999.x

Potato virus X as a vector for gene expression in plants

https://mmbr.asm.org/content/mmbr/44/2/175.full.pdf

5'-terminal cap structure in eucaryotic messenger ribonucleic acids.

Summary
Double-stranded cDNA of potato virus X (PVX) genomic RNA has been cloned and sequenced. The sequence [6435 nucleotides excluding the poly(A) tract] revealed five open reading frames (ORFs) which were numbered one to five starting at the 5′ terminus of the RNA. They encoded proteins of M
r 165588 (166K), 24622 (25K), 12324 (12K), 7595 (8K) and 25080 (coat protein), respectively. ORFs 1 and 2 were inphase coding regions. The ORF 1 product contained domains of homology with the tobacco mosaic virus 126K and 183K products. The ORF 2 and 3 products showed homologies with the barley stripe mosaic virus 58K and 14K proteins, the beet necrotic yellow vein virus 42K and 13K products and the white clover mosaic virus 26K and 13K products, respectively. The significance of these homologies with respect to putative functions of the PVX-encoded proteins are discussed.

The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses.

We efficiently transformed the commercial cultivars of potato (Solanum tuberosum) Bintje, Desiree and Escort after optimizing the conditions for regeneration from potato tuber discs. For transformation, tuber discs were cocultivated with Agrobacterium tumefaciens using a disarmed binary vector system. This system allowed the introduction of a chimaeric gene encoding the coat protein (CP) of potato virus X (PVX) into two cultivars most susceptible to this virus, Bintje and Escort. Five transgenic plant lines with expression levels of CP higher than 0.1% of soluble leaf protein were analyzed for resistance to a challenging inoculation with PVX (1 μg/ml). We observed a delay in symptom development as well as a drastic reduction in the accumulation of virus. Furthermore, we found a correlation between the expression level of the CP–gene and the reduction in virus accumulation. Cytogenetic analysis of 62 independently obtained transgenic lines showed the normal tetraploid number of chromosomes (2n = 4x = 48) in 97 percent of the examined plants. Phenotypically all these plants appeared normal. One plant line exhibited an abnormal phenotype and contained about the octaploid number of chromosomes (4n ≈ 8x = 96). These results and preliminary data on morphological characteristics, as these are determined in the official variety registration procedure, indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.

The Genetic Engineering of Two Commercial Potato Cultivars for Resistance to Potato Virus X

The relationships of genome organization among elongated (rod-shaped and filamentous) plant viruses have been analyzed Sequences in coding and noncoding regions of barley stripe mosaic virus (BSMV) RNAs 1, 2, and 3 were compared with those of the monopartite RNA genomes of potato virus X (PVX), white clover mosaic virus (WClMV), and tobacco mosaic virus, the bipartite genome of tobacco rattle virus (TRV), the quadripartite genome of beet necrotic yellow vein virus (BNYVV), and icosahedral tricornaviruses These plant viruses belong to a supergroup having 5′-capped genomic RNAs The results suggest that the genomic elements in each BSMV RNA are phylogenetically related to those of different plant RNA viruses RNA 1 resembles the corresponding RNA 1 of tricornaviruses The putative proteins encoded in BSMV RNA 2 are related to the products of BNYVV RNA 2, PVX RNA, and WClMV RNA Amino acid sequence comparisons suggest that BSMV RNA 3 resembles TRV RNA 1 Also, it can be proposed that in the case of monopartite genomes, as a rule, every gene or block of genes retains phylogenetic relationships that are independent of adjacent genomic elements of the same RNA Such differential evolution of individual elements of one and the same viral genome implies a prominent role for gene reassortment in the formation of viral genetic systems

/pdf/probable-reassortment-of-genomic-elements-among-elongated-15ru795bms.pdf

Probable reassortment of genomic elements among elongated RNA-containing plant viruses

The 5'-end structure of potato virus X RNA was determined following enzymatic methylation in vitro. A single 3H-methyl group was introduced into the 2'-position of the 5'-penultimate residue and the end structure was determined as m7GpppG(m)pAp(Xp)3G. This part of the RNA apparently is involved in binding to ribosomes since it can be partially protected against RNase digestion by wheat germ 40S ribosomes. PVX RNA was not retained by poly(U)-sepharose, indicating that it does not contain a 3'-terminal poly(A) tract.

/pdf/analysis-of-terminal-structures-of-rna-from-potato-virus-x-2ymrs6jgkl.pdf

Michal Rubin

Papers

Analysis of terminal structures of RNA from potato virus X.