Abstract
Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.
Highlights
N. benthamiana co-infiltrated with Agrobacterium harboring pXGY1-Odontoglossum ringspot virus (ORSV) and ORSV inoculum exhibited different phenotypes: the majority of plants showed resistance or latency, with mild or no apparent symptoms, the virus could be detected in vivo; some plants were immune, with no viral symptoms or virus detected (Figure 2(a)); and a few plants were susceptible, with both viral symptoms and virus detected
PXGY1-ORSV-mediated agroinfiltration could partially, but not completely reduce the ORSV infection rate. These data demonstrate that our ORSV cp-mediated RNA silencing vector can successfully degrade invading virus RNA
The results indicated that chlorotic spot, irregular twisting, and dwarfing symptoms developed in leaves inoculated with the pCAMBIA2300-ORSV-FL clone and systemically infected new leaves; these symptoms were comparable to those in N. benthamiana plants inoculated with ORSV sap, while no symptoms of viral infection were observed in empty vector-inoculated or untreated leaves (Figure 2(b))
Summary
Orchids (Orchidaceae) are a family of diverse and widespread flowering plants with high ornamental and economic value; during the cultivation. ORSV causes the most widespread viral disease in perennial orchids and is responsible for large economic losses in the orchid cultivation industry [1]. Infections with ORSV frequently cause leaf chlorosis and twisting and, when the condition becomes more serious, numerous symptoms appear, including necrotic lesions and ringspots, dwarfed plants, changes in the color and size of flowers, and even death of the entire plant [2]. Viruses generally alter the metabolic pathways of host cells and destroy their normal physiological functions. They utilize the host replication system, leading to the gradual emergence of obvious symptoms. The conventional method for elimination of orchid virus is the devastating and non-preventive process of isolating, burning, and discarding the remnants of symptomatic plants in landfill sites
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