Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus...Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus (CymMV), and/or Odontoglossum ring spot virus (ORSV), the total proteins were extracted from Phalaenopsis amabilis leaves infected with CymMV, ORSV, or both respectively. Differentially expressed proteins were identified by two-dimensional electrophoresis, and 27 of these proteins that had significant changes were further examined by mass spectrometry. Comparing CymMV-infected leaves with mock-inoculated ones, 2 proteins were significantly up-regulated, 9 were significantly down-regulated and 1 previously undetected protein was identified. 10 proteins were significantly up-regulated, 3 significantly down-regulated and 1 previously undetected protein was identified in ORSV-infected leaves. 6 proteins were significantly up-regulated and 9 significantly down-regulated proteins were found in co-infected leaves. These identified proteins are involved in disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification and the previously unknown proteins were not involved with known protein pathways. Proteins significantly up-regulated were ATP sulfurylase, down-regulated proteins included glutamate decarboxylase isozyme 2, RNA polymerase alpha subunit and chloroplastic peptide deformylase 1A were proteins with similar alteration trend after all infection treatments. Significantly up-regulated were Thioredoxin H-type and down-regulated Cytosolic phosphoglycerate kinase I which were proteins that have been shown to be specifically regulated by the infection with CymMV. Significantly up-regulated were proteins like Rubisco large subunit, Triosephosphate isomerase, NADP-specific isocitrate dehydrogenase and Cinnamoyl CoA reductase CCR2 by the infection of ORSV. Protein regulation in coinfected leaves followed a pattern similar to that of any of the single virus infection results.展开更多
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...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.展开更多
文摘Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus (CymMV), and/or Odontoglossum ring spot virus (ORSV), the total proteins were extracted from Phalaenopsis amabilis leaves infected with CymMV, ORSV, or both respectively. Differentially expressed proteins were identified by two-dimensional electrophoresis, and 27 of these proteins that had significant changes were further examined by mass spectrometry. Comparing CymMV-infected leaves with mock-inoculated ones, 2 proteins were significantly up-regulated, 9 were significantly down-regulated and 1 previously undetected protein was identified. 10 proteins were significantly up-regulated, 3 significantly down-regulated and 1 previously undetected protein was identified in ORSV-infected leaves. 6 proteins were significantly up-regulated and 9 significantly down-regulated proteins were found in co-infected leaves. These identified proteins are involved in disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification and the previously unknown proteins were not involved with known protein pathways. Proteins significantly up-regulated were ATP sulfurylase, down-regulated proteins included glutamate decarboxylase isozyme 2, RNA polymerase alpha subunit and chloroplastic peptide deformylase 1A were proteins with similar alteration trend after all infection treatments. Significantly up-regulated were Thioredoxin H-type and down-regulated Cytosolic phosphoglycerate kinase I which were proteins that have been shown to be specifically regulated by the infection with CymMV. Significantly up-regulated were proteins like Rubisco large subunit, Triosephosphate isomerase, NADP-specific isocitrate dehydrogenase and Cinnamoyl CoA reductase CCR2 by the infection of ORSV. Protein regulation in coinfected leaves followed a pattern similar to that of any of the single virus infection results.
文摘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.
