A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of ...A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.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
RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability ...RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability to restrict the RNAi machinery to ensure successful systemic invasion. The B2 protein of insect Flock House Virus (FHV-B2) and AC2 protein of Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are two well-characterized suppressors of RNAi, capable of reversing reporter gene silencing. In this study, we compared the strength of the two suppressors by assaying for the degree of RNAi reversion and the duration of sustaining the reversal in planta. The suppression activity was observed by assaying for GFP fluorescence at 3 dpi, 7 dpi and 14 dpi. The phenotypic observations were corroborated with small RNA Northern Blotting and semi-quantitative RT-PCR. The results indicate that suppressor strength of FHVB2 is comparable to MYMIV-AC2, although they are encoded by virus infecting host from two different eukaryotic kingdoms. This study will provide new insights to dissect the conservation in the RNAi pathways during the host-virus interactions.展开更多
文摘A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
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.
文摘RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability to restrict the RNAi machinery to ensure successful systemic invasion. The B2 protein of insect Flock House Virus (FHV-B2) and AC2 protein of Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are two well-characterized suppressors of RNAi, capable of reversing reporter gene silencing. In this study, we compared the strength of the two suppressors by assaying for the degree of RNAi reversion and the duration of sustaining the reversal in planta. The suppression activity was observed by assaying for GFP fluorescence at 3 dpi, 7 dpi and 14 dpi. The phenotypic observations were corroborated with small RNA Northern Blotting and semi-quantitative RT-PCR. The results indicate that suppressor strength of FHVB2 is comparable to MYMIV-AC2, although they are encoded by virus infecting host from two different eukaryotic kingdoms. This study will provide new insights to dissect the conservation in the RNAi pathways during the host-virus interactions.
