Articles
Transgenic Nicotiana benthamiana resistance to synergistic infection of two orchid viruses CymMV and ORSV
Article number
1193_10
Pages
69 – 76
Language
English
Abstract
Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) have been reported as co-infection (or synergistic interaction) resulting in more severe symptoms in orchids.
In this project, silencing dual RNA-dependent RNA polymerase (RdRp) transcripts of CymMV and ORSV based on RNA interference (RNAi), including amiRNA-CymMV-ORSV or antisense-CymMV-ORSV constructs were adopted to create transgenic plants that can resist to CymMV and ORSV at the same time.
A susceptible host model to both viruses, Nicotiana benthamiana, was generated first to test the feasibility of the RNAi strategy and gene stacking also was applied to increase copy number in transgenic N. benthamiana by cross pollination.
Successfully of viral resistant analyses were performed until F3 generation using Southern blot, real-time RT-PCR and western blot.
Resistant FF3 transgenic plants had a higher transgene expression as compared to non-resistant plants, coupled with the absence of CymMV and ORSV coat protein which suggests negligible CymMV and ORSV concentration in that resistant plant which allowed the plant to exhibit a normal phenotype after mixed inoculation for three weeks.
We believe this is the first report on the transgenic N. benthamiana plants that can resist against both CymMV and ORSV concurrently.
In this project, silencing dual RNA-dependent RNA polymerase (RdRp) transcripts of CymMV and ORSV based on RNA interference (RNAi), including amiRNA-CymMV-ORSV or antisense-CymMV-ORSV constructs were adopted to create transgenic plants that can resist to CymMV and ORSV at the same time.
A susceptible host model to both viruses, Nicotiana benthamiana, was generated first to test the feasibility of the RNAi strategy and gene stacking also was applied to increase copy number in transgenic N. benthamiana by cross pollination.
Successfully of viral resistant analyses were performed until F3 generation using Southern blot, real-time RT-PCR and western blot.
Resistant FF3 transgenic plants had a higher transgene expression as compared to non-resistant plants, coupled with the absence of CymMV and ORSV coat protein which suggests negligible CymMV and ORSV concentration in that resistant plant which allowed the plant to exhibit a normal phenotype after mixed inoculation for three weeks.
We believe this is the first report on the transgenic N. benthamiana plants that can resist against both CymMV and ORSV concurrently.
Authors
U. Petchthai, Z. Xie, S.M. Wong
Keywords
antisense, amiRNA, synergism, virus resistance, N. benthamiana
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