Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants...Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.展开更多
Plasmodesmata (PDs) are cytoplasmic structures that link adjacent cells to form the symplast of a plant. PDs are involved extensively in a plant's life by mediating symplastic transport of a wide range of ions and...Plasmodesmata (PDs) are cytoplasmic structures that link adjacent cells to form the symplast of a plant. PDs are involved extensively in a plant's life by mediating symplastic transport of a wide range of ions and molecules. Major components of a plasmodesma (PD) include a plasma membrane, a desmotubule, and a cytoplasmic annulus, all of which are readily detectable by electron microscopy. Both the plasma membrane and the desmotubule contain proteinaceous particles, thought to be involved in altering the size of the cytoplasmic annulus. Cytoskeleton elements (actin and myosin) are essential for maintaining the integrity of PDs. Together with these elements, calcium_binding proteins probably play a significant role in regulating PD function. Symplastic transport occurs through the cytoplasmic annulus for the great majority of solutes, while other substances may traverse through the desmotubule internal compartment, the desmotubule shell, or the plasma membrane. The symplast is subdivided into several domains with varying molecular size exclusion limits (ranging from <1 kD to >10 kD). Plasmodesmata can be either primary or secondary; the former are developed during new wall formation and the latter are made in existing walls. The dynamic nature of plasmodesmata is also reflected by their changing frequencies, which, in turn, depend on the developmental and physiological status of the tissue or the entire plant. While diffusion is the major mechanism of symplastic transport, plasmodesmata are selective for certain ions and molecules. Upon viral infection, viral movement proteins interact with PD receptor proteins and, as a result of yet unknown mechanisms, the plasmodesmata are remarkably dilated to allow viral movement proteins and the bound viral genome to enter healthy cells. Some proteins of plant origin are also able to traverse plasmodesmata, presumably in ways similar to viral movement proteins. Some of these plant proteins are probably signal molecules contributing to cell differentiation and other acti展开更多
Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene...Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was re- placed by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in to- bacco plants containing the N gene results from the functional difference of their MP genes.展开更多
Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of...Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the dete展开更多
The infection of host plants by many different viruses causes reactive oxygen species(Ros)accumulation and yellowing symptoms,but the mechanisms through which plant viruses counteract RoS-mediated immunity to facilita...The infection of host plants by many different viruses causes reactive oxygen species(Ros)accumulation and yellowing symptoms,but the mechanisms through which plant viruses counteract RoS-mediated immunity to facilitate infection and symptom development have not been fully elucidated.Most plant viruses are transmitted by insect vectors in the field,but the molecular mechanisms underlying virus-host-insect interactions are unclear.In this study,we investigated the interactions among wheat,barley yellow dwarf virus(BYDV),and its aphid vector and found that the BYDV movement protein(MP)interacts with both wheat catalases(CATs)and the 26S proteasomeubiquitin receptor non-ATPase regulatorysubunit2homolog(PSMD2)to facilitate the 26S proteasome-mediateddegradation of CATs,promotingviral infection,disease symptom development,and aphid transmission.Overexpression of the BYDV MP gene in wheat enhanced the degradation of CATs,which leading to increased accumulation of ROS and thereby enhanced viral infection.Interestingly,transgenic wheat lines overexpressing BYDV MP showed significantly reduced proliferation of wingless aphids and an increased number of winged aphids.Consistent with this observation,silencing of CAT genes also enhanced viral accumulation and reduced the proliferation of wingless aphids but increased the occurrence of winged aphids.In contrast,transgenic wheat plants overexpressing TaCAT1 exhibited the opposite changes and showed increases in grain size and weight upon infection with BYDV.Biochemical assays demonstrated that BYDV MP interacts with PSMD2 and promotes 26S proteasome-mediated degradation of TaCAT1 likely in a ubiquitination-independent manner.Collectively,our study reveals a molecular mechanism by which a plant virus manipulates the Ros production system of host plants to facilitate viral infection and transmission,shedding new light on the sophisticated interactions among viruses,host plants,and insect vectors.展开更多
[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movemen...[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.展开更多
文摘Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.
文摘Plasmodesmata (PDs) are cytoplasmic structures that link adjacent cells to form the symplast of a plant. PDs are involved extensively in a plant's life by mediating symplastic transport of a wide range of ions and molecules. Major components of a plasmodesma (PD) include a plasma membrane, a desmotubule, and a cytoplasmic annulus, all of which are readily detectable by electron microscopy. Both the plasma membrane and the desmotubule contain proteinaceous particles, thought to be involved in altering the size of the cytoplasmic annulus. Cytoskeleton elements (actin and myosin) are essential for maintaining the integrity of PDs. Together with these elements, calcium_binding proteins probably play a significant role in regulating PD function. Symplastic transport occurs through the cytoplasmic annulus for the great majority of solutes, while other substances may traverse through the desmotubule internal compartment, the desmotubule shell, or the plasma membrane. The symplast is subdivided into several domains with varying molecular size exclusion limits (ranging from <1 kD to >10 kD). Plasmodesmata can be either primary or secondary; the former are developed during new wall formation and the latter are made in existing walls. The dynamic nature of plasmodesmata is also reflected by their changing frequencies, which, in turn, depend on the developmental and physiological status of the tissue or the entire plant. While diffusion is the major mechanism of symplastic transport, plasmodesmata are selective for certain ions and molecules. Upon viral infection, viral movement proteins interact with PD receptor proteins and, as a result of yet unknown mechanisms, the plasmodesmata are remarkably dilated to allow viral movement proteins and the bound viral genome to enter healthy cells. Some proteins of plant origin are also able to traverse plasmodesmata, presumably in ways similar to viral movement proteins. Some of these plant proteins are probably signal molecules contributing to cell differentiation and other acti
基金This work was supported by China National Key Basic ResearchDevelopment Program(Grant No.G2000016204)the National Outstanding Youth Foundation from National Natural Science Foundation of China(Grant No.30125032).
文摘Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was re- placed by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in to- bacco plants containing the N gene results from the functional difference of their MP genes.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30300151 ）.
文摘Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the dete
基金supported by grants to Y.Wu and L.Z.from the Shaanxi Key Research and Development Program(No.2022KWZ-11)the Ministry of Science and Technology Plans to Introduce High-End Foreign Experts(G2022172015L)the National Natural Science Foundation of China(Nos.32372501 and 31701761).
文摘The infection of host plants by many different viruses causes reactive oxygen species(Ros)accumulation and yellowing symptoms,but the mechanisms through which plant viruses counteract RoS-mediated immunity to facilitate infection and symptom development have not been fully elucidated.Most plant viruses are transmitted by insect vectors in the field,but the molecular mechanisms underlying virus-host-insect interactions are unclear.In this study,we investigated the interactions among wheat,barley yellow dwarf virus(BYDV),and its aphid vector and found that the BYDV movement protein(MP)interacts with both wheat catalases(CATs)and the 26S proteasomeubiquitin receptor non-ATPase regulatorysubunit2homolog(PSMD2)to facilitate the 26S proteasome-mediateddegradation of CATs,promotingviral infection,disease symptom development,and aphid transmission.Overexpression of the BYDV MP gene in wheat enhanced the degradation of CATs,which leading to increased accumulation of ROS and thereby enhanced viral infection.Interestingly,transgenic wheat lines overexpressing BYDV MP showed significantly reduced proliferation of wingless aphids and an increased number of winged aphids.Consistent with this observation,silencing of CAT genes also enhanced viral accumulation and reduced the proliferation of wingless aphids but increased the occurrence of winged aphids.In contrast,transgenic wheat plants overexpressing TaCAT1 exhibited the opposite changes and showed increases in grain size and weight upon infection with BYDV.Biochemical assays demonstrated that BYDV MP interacts with PSMD2 and promotes 26S proteasome-mediated degradation of TaCAT1 likely in a ubiquitination-independent manner.Collectively,our study reveals a molecular mechanism by which a plant virus manipulates the Ros production system of host plants to facilitate viral infection and transmission,shedding new light on the sophisticated interactions among viruses,host plants,and insect vectors.
基金Supported by National Natural Science Foundation of China(30870109)~~
文摘[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.
