Ringspot Virus Coat Protein Gene Research Articles

Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene

Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.

Post-transcriptional gene silencing is involved in resistance of transgenic papayas to papaya ringspot virus

Transgenic papayas carrying the papaya ringspot virus coat protein gene were inoculated with Papaya ringspot virus. Infection was monitored by evaluating symptoms and by reverse transcription polymerase chain reaction (RT-PCR). Among eight tested transgenic lines, clone G2 was found highly resistant to virus infection during 3 years of testing. Further analysis of this clone revealed complex multicopy transgene insertion with aberrant copies. The suspected post transcriptional gene silencing was confirmed by siRNA detection. While the R0 generation of G2 transgenic papaya was found to be fully resistant to the infection, Papaya ringspot virus was able to break this resistance in subsequent generations by suppressing post-transcriptional gene silencing (PTGS).

Introduction of Odontoglossum ringspot virus Coat Protein Gene into Cymbidium niveo-marginatum mediated by Agrobacterium tumefaciens to Produce Transgenic Plants

We cloned the Odontoglossum ringspot virus (ORSV) coat protein (CP) gene from ORSV-infected Epidendrum and constructed a pHPUNOJE-1 vector by ligating this CP gene, green fluorescence protein (GFP) gene, and hygromycin resistant (Hygr) gene into the plasmid pGreenII. This chimeric vector was mediated into A. tumefaciens LBA 4404. By infecting and co-culturing the rhizomes of Cym. niveo-marginatum with A. tumefaciens LBA 4404, ORSV CP gene transgenic shoots were obtained. GFP was observed in 64% of the 81 surviving rhizomes and in 39% of 215 regenerated shoots. The presence of ORSV CP, GFP, and Hygr genes was confirmed in the transgenic shoots by using polymerase chain reaction (PCR) analysis, whereas that of ORSV CP was confirmed by enzyme-linked immunosorbent assay (ELISA).

Transformation of Plum with the Papaya Ringspot Virus Coat Protein Gene and Reaction of Transgenic Plants to Plum Pox Virus

Transgenic plum plants expressing the papaya ringspot virus (PRV) coat protein (CP) were produced by Agrobacterium-mediated transformation of hypocotyl slices. Hypocotyl slices were cocultivated with Agrobacterium tumefaciens strain C58/Z707 containing the plasmid pGA482GG/CPPRV-4. This plasmid carries the PRVCP gene construct and chimeric NPTII and GUS genes. Shoots were regenerated on Murashige and Skoog salts, vitamins, 2% sucrose, 2.5 μm indolebutyric acid, 7.5 μm thidiazuron, and appropriate antibiotics for selection. Integration of the foreign genes was verified through kanamycin resistance, GUS assays, polymerase chain reaction (PCR), and Southern blot analyses. Four transgenic clones were identified. Three were vegetatively propagated and graft-inoculated with plum pox virus (PPV)-infected budwood in a quarantine, containment greenhouse. PPV infection was evaluated over a 2- to 4-year period through visual symptoms, enzyme-linked immunosorbent assay, and reverse transcriptase PCR assays. While most plants showed signs of infection and systemic spread of PPV within l-6 months, one plant appeared to delay the spread of virus and the appearance of disease symptoms. Virus spread was limited to basal portions of this plant up to 19 months postinoculation, but, after 32 months symptoms were evident and virus was detected throughout the plant. Our results suggest that heterologous protection with PRVCP, while having the potential to delay PPV symptoms and spread throughout plum plants, may not provide an adequate level of long-term resistance.

Transformation of Grape (Vitis vinifera L.)

Transgenic grapevines were regenerated from somatic embryos produced from immature zygotic embryos of two seedless grape selections and from leaves of in vitro-grown plants of `Thompson Seedless'. Somatic embryos were bombarded with gold microparticles using the Biolistic PDS-1000/He device (Bio-Rad Labs) and then exposed to engineered A. tumefaciens EHA101 (E. Hood, WSU). Alternately, somatic embryos were exposed to A. tumefaciens without bombardment. Following cocultivation, secondary embryos multiplied on Emershad and Ramming proliferation medium under kan selection. Transgenic embryos were identified after 3 to 5 months and developed into rooted plants on woody plant medium with 1 mM N6-benzyladenine, 1.5% sucrose, and 0.3% activated charcoal. Seedless selections were transformed with plasmids pGA482GG (J. Slightom, Upjohn) and pCGN7314 (Calgene), which carry GUS and NPTII genes. `Thompson Seedless' was transformed with pGA482GG and pGA482GG/TomRSVcp-15 (D. Gonsalves, Cornell Univ.) containing the tomato ringspot virus coat protein gene. Integration of foreign genes into grapevines was verified by growth on kan, GUS, and PCR assays, and Southern analyses.

Molecular cloning, sequencing and expression in Escherichia coli of the odontoglossum ringspot virus coat protein gene

The sequence of the 3'-terminal 1865 nucleotides of the genome of the tobamovirus odontoglossum ringspot virus (ORSV) was determined. This sequence contained two open reading frames (ORFs), 912 and 477 nucleotides long. The 912 nucleotide ORF has been identified as the cell-to-cell transport protein gene. The 477 nucleotide ORF was expressed in Escherichia coli, and the product was detected by antibodies specific for the coat protein of ORSV. The amino acid sequence of protein encoded by this ORF shares 84% similarity with the tobacco mosaic virus (TMV) (vulgare) coat protein. The 3'-terminal untranslated region of ORSV comprises 414 nucleotides, 210 nucleotides more than that of TMV (vulgare) RNA.