Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.
Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.
Vazquez Rovere
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Instituto de Biotecnología, CICVyA CNIA, Instituto Nacional de Tecnología Agropecuaria, Buenos Aires, Argentina.
Asurmendi
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Hopp
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D003001:Cloning, Molecular; D020868:Gene Silencing; D017793:Luteovirus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D030821:Plants, Genetically Modified / Q000821:virology*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000235:genetics / Q000821:virology*; D014170:Transformation, Genetic
D012324:RNA-Dependent RNA Polymerase