MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco r...MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco rattle virus (TRV) vector to silence expression of the nuclear psbS gene in Nicotiana benthamiana. The 22-kiloDalton psbS protein is essential for xanthophyll- and H+-dependent thermal dissipation of excitation in higher plants widely known as nonphotochemical quenching (NPQ). Controls treated with the TRV-VIGS vector containing a bacterial chloramphenicol resistance gene as the silencing target were included to test for non-silencing effects of the viral vector system. PsbS protein was undetectable and both psbS mRNA transcript levels and NPQ capacity were dramatically reduced in new leaf tissue of VIGS-psbS plants only. Photosynthetic performance in TRV-VIGS-treated and uninfiltrated plants was assessed by application of CO2 exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a mild stress based on pigment content and light absorption characteristics in some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the transit sequence in the TRV vector followed by Agrobacterium-mediated transient expression of a modified gene consisting of an altered transit sequence fused to the native mature protein sequence. Nevertheless, NPQ in infused fully expanded leaves that expressed this re-introduced form was not fully restored indicating the possible importance of psbS incorporation prior to formation of grana stacks.展开更多
RNA-dependent protein kinase(PKR) is crucial for the innate immune response,cell growth,proliferation,signal transduction and apoptosis.The activation process of PKR has been studied for many years and is still under ...RNA-dependent protein kinase(PKR) is crucial for the innate immune response,cell growth,proliferation,signal transduction and apoptosis.The activation process of PKR has been studied for many years and is still under debate.To obtain new insight into the mechanism of PKR activation,we solved the crystal structure of a latent mutant of the PKR kinase domain(PKR-KD) in the apo form at a resolution of 2.9.The overall structure of PKR-KD is similar to previously reported structures.Structural analysis revealed a classical back-to-back dimer and a newly defined face-to-face dimer.Analytical ultracentrifugation experiments,electrostatic surface maps and the model of PKR-KD in complex with the eIF2 substrate all support that the face-to-face dimer is more reflective of PKR in solution.Our results provide new information on PKR dimerization and its activation mechanism.展开更多
文摘MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco rattle virus (TRV) vector to silence expression of the nuclear psbS gene in Nicotiana benthamiana. The 22-kiloDalton psbS protein is essential for xanthophyll- and H+-dependent thermal dissipation of excitation in higher plants widely known as nonphotochemical quenching (NPQ). Controls treated with the TRV-VIGS vector containing a bacterial chloramphenicol resistance gene as the silencing target were included to test for non-silencing effects of the viral vector system. PsbS protein was undetectable and both psbS mRNA transcript levels and NPQ capacity were dramatically reduced in new leaf tissue of VIGS-psbS plants only. Photosynthetic performance in TRV-VIGS-treated and uninfiltrated plants was assessed by application of CO2 exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a mild stress based on pigment content and light absorption characteristics in some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the transit sequence in the TRV vector followed by Agrobacterium-mediated transient expression of a modified gene consisting of an altered transit sequence fused to the native mature protein sequence. Nevertheless, NPQ in infused fully expanded leaves that expressed this re-introduced form was not fully restored indicating the possible importance of psbS incorporation prior to formation of grana stacks.
基金supported by the National Key Basic Program of China (2012CB917201 and 2009CB825504)the National Natural Science Foundation of China (31170684)the Fundamental Research Funds forthe Central Universities (65020241)
文摘RNA-dependent protein kinase(PKR) is crucial for the innate immune response,cell growth,proliferation,signal transduction and apoptosis.The activation process of PKR has been studied for many years and is still under debate.To obtain new insight into the mechanism of PKR activation,we solved the crystal structure of a latent mutant of the PKR kinase domain(PKR-KD) in the apo form at a resolution of 2.9.The overall structure of PKR-KD is similar to previously reported structures.Structural analysis revealed a classical back-to-back dimer and a newly defined face-to-face dimer.Analytical ultracentrifugation experiments,electrostatic surface maps and the model of PKR-KD in complex with the eIF2 substrate all support that the face-to-face dimer is more reflective of PKR in solution.Our results provide new information on PKR dimerization and its activation mechanism.
