Transfer of Plum Pox Virus Coat Protein Genes from a Plum Pox-resistant Transgenic Clone of Prunus domestica Plum to Its Progeny through Hybridization

Sharka or plum pox virus (PPV) is a major disease of stone fruit and causes severe economic losses in Europe. There is little resistance to PPV in most Prunus species, thus genetic engineering represents a potentially useful approach to obtain resistant germplasm. Transgenic plums containing the PPV coat protein (CP) or the related papaya ringspot virus (PRV)-CP gene were produced through Agrobacterium tumefaciens-mediated transformation. These transgenic plum clones were then evaluated for resistance to PPV infection in the greenhouse by graft or aphid inoculation with PPV. While symptoms of PPV appeared in most transgenic clones, all plants of PPV-CP transgenic clone C5 were symptomless and ELISA and immunocapture-reverse transcriptase PCR negative for over three years following inoculation with two strains of PPV (Ravelonandro et al., Plant Dis. 81:1231-1235, 1997). Clone C5, which contains multiple copies of the PPV-CP gene, was hybridized with PRV-CP transgenic plants or untransformed plum cultivars. Progeny were obtained containing no transgenes, only the PPV-CP, only the PRV-CP, or both the PRV-CP and PPV-CP transgenes. Seedlings were inoculated with PPV. At 5 and 11 months post-inoculation, seedlings containing the PPV-CP genes from C5 were symptomless and ELISA negative. Seedlings containing only PRV-CP transgenes or non-transformed controls showed symptoms of PPV infection and were ELISA positive. These results indicate that the PPV-CP transgenes can be transferred to progeny through hybridization and that these genes can impart resistance to PPV in transgenic seedlings. The inheritance of the multicopy inserts of the PPV-CP and PRV-CP transgenes is being analyzed. The combined effects of both transgenes on resistance to PPV and the stability of PPV resistance in the progeny of the resistant C5 transgenic line are currently under evaluation.

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Field Trials of Plum Clones Transformed with the Plum pox virus Coat Protein (PPV-CP) Gene

Plant Disease ◽

10.1094/pd-90-1012 ◽

2006 ◽

Vol 90 (8) ◽

pp. 1012-1018 ◽

Cited By ~ 52

Author(s):

T. Malinowski ◽

M. Cambra ◽

N. Capote ◽

B. Zawadzka ◽

M. T. Gorris ◽

...

Keyword(s):

Coat Protein ◽

Field Trials ◽

Climatic Conditions ◽

Genetically Engineered ◽

Marker Genes ◽

Plum Pox Virus ◽

Post Inoculation ◽

Cp Gene ◽

Infection Pressure ◽

High Level

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance.

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Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.132.6.850 ◽

2007 ◽

Vol 132 (6) ◽

pp. 850-858 ◽

Cited By ~ 54

Author(s):

Jean-Michel Hily ◽

Michel Ravelonandro ◽

Vern Damsteegt ◽

Carole Bassett ◽

Cesar Petri ◽

...

Keyword(s):

Coat Protein ◽

Gene Silencing ◽

Nicotiana Benthamiana ◽

High Capacity ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Hairpin Rna ◽

Woody Perennial ◽

Virus Coat ◽

Interfering Rna

Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.

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EXPRESSION OF THE PLUM POX VIRUS COAT PROTEIN REGION IN ESCHERICHIA COLI

Acta Horticulturae ◽

10.17660/actahortic.1989.235.17 ◽

1989 ◽

pp. 131-132

Author(s):

D. Mattanovich ◽

G. Himmler ◽

M. Laimer ◽

A. da Camara Machado ◽

V. Hanzer ◽

...

Keyword(s):

Escherichia Coli ◽

Coat Protein ◽

Plum Pox Virus ◽

Coat Protein Region ◽

Virus Coat Protein ◽

Virus Coat

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EXPRESSION OF THE PLUM POX VIRUS COAT PROTEIN GENE IN NICOTIANA CLEVELANDII

Acta Horticulturae ◽

10.17660/actahortic.1990.280.94 ◽

1990 ◽

pp. 577-580 ◽

Cited By ~ 5

Author(s):

M. Laimer da Câmara Machado ◽

A. da Câmara Machado ◽

D. Mattanovich ◽

F. Regner ◽

V. Hanzer ◽

...

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Plum Pox Virus ◽

Virus Coat Protein ◽

Virus Coat ◽

Nicotiana Clevelandii

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Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection

Plant Disease ◽

10.1094/pdis.1997.81.11.1231 ◽

1997 ◽

Vol 81 (11) ◽

pp. 1231-1235 ◽

Cited By ~ 64

Author(s):

M. Ravelonandro ◽

R. Scorza ◽

J. C. Bachelier ◽

G. Labonne ◽

L. Levy ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Coat Protein ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Chain Reaction ◽

Cp Gene ◽

Aphid Feeding ◽

Polymerase Chain ◽

Dormancy Cycles ◽

Das Elisa

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

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TRANSGENIC PLUMS (PRUNUS DOMESTICA L.) OF 'STARTOVAYA' EXPRESS THE PLUM POX VIRUS COAT PROTEIN GENE

Acta Horticulturae ◽

10.17660/actahortic.2012.929.64 ◽

2012 ◽

pp. 445-450 ◽

Cited By ~ 1

Author(s):

R. Mikhailov ◽

A. Firsov ◽

O. Shulga ◽

S. Dolgov

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Virus Coat Protein ◽

Virus Coat

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

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.120.6.943 ◽

1995 ◽

Vol 120 (6) ◽

pp. 943-952 ◽

Cited By ~ 36

Author(s):

Ralph Scorza ◽

Laurene Levy ◽

Vern Damsteegt ◽

Luz Marcel Yepes ◽

John Cordts ◽

...

Keyword(s):

Coat Protein ◽

Kanamycin Resistance ◽

Ringspot Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Papaya Ringspot Virus ◽

Indolebutyric Acid ◽

Systemic Spread ◽

Foreign Genes ◽

Virus Coat

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.

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Resistance to Passion fruit woodiness virus in Transgenic Passionflower Expressing the Virus Coat Protein Gene

Plant Disease ◽

10.1094/pd-90-1026 ◽

2006 ◽

Vol 90 (8) ◽

pp. 1026-1030 ◽

Cited By ~ 33

Author(s):

F. Trevisan ◽

B. M. J. Mendes ◽

S. C. Maciel ◽

M. L. C. Vieira ◽

L. M. M. Meletti ◽

...

Keyword(s):

Coat Protein ◽

Transgenic Plants ◽

In Vitro Selection ◽

Passion Fruit ◽

Cp Gene ◽

Virus Coat ◽

Passion Fruit Woodiness Virus ◽

Leaf Disks ◽

Yellow Passion Fruit

We report the use of the coat protein (CP) gene from Passion fruit woodiness virus (PWV) to produce resistant transgenic plants of yellow passion fruit. A full-length CP gene from a severe PWV isolate from the state of São Paulo, Brazil (PWV-SP) was cloned into pCAMBIA 2300 binary vector, which was further introduced into Agrobacterium tumefaciens strain EHA 105. Leaf disks were used as explants for transformation assays, e.g., 2,700 and 2,730 disks excised from plants from the Brazilian cultivars IAC-275 and IAC-277, respectively. In vitro selection was performed in kanamycin. After transferring to the elongation medium, 119 and 109 plantlets of IAC-275 and IAC-277, respectively, were recovered. Integration of the PWV CP gene was confirmed in seven of eight plants evaluated by Southern blot analysis, showing different numbers of insertional events for the CP gene. Three transgenic plants (T3, T4, and T7) expressed the expected transcript, but the 32 kDa PWV CP was detected by Western blot in only two plants (T3 and T4). The results of three successive mechanical inoculations against the transgenic plants using three PWV isolates showed that the primary transformant T2 of IAC-277 was immune to all isolates.

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