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
Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of ea...Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.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
Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to...Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV-Cvrease-mp displayed on|y a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.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
Banana bunchy top virus(BBTV)poses a serious danger to banana crops worldwide.BBTV-encoded protein B4 is a determinant of pathogenicity.However,the relevant molecular mechanisms underlying its effects remain unknown.I...Banana bunchy top virus(BBTV)poses a serious danger to banana crops worldwide.BBTV-encoded protein B4 is a determinant of pathogenicity.However,the relevant molecular mechanisms underlying its effects remain unknown.In this study,we found that a functional peptide could be liberated from protein B4,likely via proteolytic processing.Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects,including dwarfism and sterility,in plants.The released protein fragment targets host proteins,such as the large subunit of RuBisCO(RbcL)and elongation factor 2(EF2),involved in protein synthesis.Therefore,the peptide released from B4(also a precursor)may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.展开更多
The realization of protein functional movement is usually accompanied by specific conformational changes,and there exist some key residues that mediate and control the functional motions of proteins in the allosteric ...The realization of protein functional movement is usually accompanied by specific conformational changes,and there exist some key residues that mediate and control the functional motions of proteins in the allosteric process.In the present work,the perturbation-response scanning method developed by our group was combined with the molecular dynamics(MD)simulation to identify the key residues controlling the functional movement of proteins.In our method,a physical quantity that is directly related to protein specific function was introduced,and then based on the MD simulation trajectories,the perturbation-response scanning method was used to identify the key residues for functional motions,in which the residues that highly correlated with the fluctuation of the function-related quantity were identified as the key residues controlling the specific functional motions of the protein.Two protein systems,i.e.,the heat shock protein 70 and glutamine binding protein,were selected as case studies to validate the effectiveness of our method.Our calculated results are in good agreement with the experimental results.The location of the key residues in the two proteins are similar,indicating the similar mechanisms behind the performance of their biological functions.展开更多
This study evaluated whether the administration of a NSAID, sodium diclofenac, can promote alterations in the expression of Fos protein in central amygdala (CEA) and the lateral hypothalamus (LH) after 6 h of experime...This study evaluated whether the administration of a NSAID, sodium diclofenac, can promote alterations in the expression of Fos protein in central amygdala (CEA) and the lateral hypothalamus (LH) after 6 h of experimental tooth movement with a controlled force of 70 g, applied to the superior central incisors of rats. Adult male rats were anesthetized and divided into four groups: Control, no orthodontic appliance (OA);OA activated with 70 g;OA activated with 70 g and pretreated with diclofenac sodium (5 mg/kg, intramuscular);and diclofenac sodium alone. Six hours after the onset of the experiment the rats were reanesthetized and perfused with 4% paraformaldehyde. The brains were removed and fixed, and sections containing the CEA and LH were processed for Fos protein immunohistochemistry. The results show that in the control group, intramuscular injection of a ketamine/xylazine mixture did not induce IR-Fos cells in the CEA or LH. However, in the 70 g group, IR-Fos was the strongest observed展开更多
Guided pesticide is an unique compound resulted from the conjugation with carrier （amino acid, protein, sugars, etc） and the active pesticide ingredient. One of the attributes of the guided pesticide is its potentia...Guided pesticide is an unique compound resulted from the conjugation with carrier （amino acid, protein, sugars, etc） and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein （MP） is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein （GFP） and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG （isopropyl-b-dthiogalactopyranoside） at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.展开更多
In this study, the expression of HSP70 during experimental tooth movement was dynamically observed and the relationship between HSP70 and orthodontic periodontal tissue remodeling were observed. The orthodontic applia...In this study, the expression of HSP70 during experimental tooth movement was dynamically observed and the relationship between HSP70 and orthodontic periodontal tissue remodeling were observed. The orthodontic appliance was placed between the right maxillary first molar and maxillary central incisors of adult SD rats to establish a rat molar movement model. Immunohistochemistry was performed 1, 3, 5, 7 and 14 day（s） after orthodontic force application to observe the expression and localization of inducible HSP70. The expression of HSP70 was strongly positive in the early stage of the tooth movement, became gradually less positive, and was weakly positive in the restoration stage. There was difference in staining pattern between different parts of PDL during the same period. These results suggest that the expression of HSP70 and difference in staining pattern among different parts of PDL during orthodontic tooth movement in rats may be implicated in stress response and remodeling of periodontal tissue.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.
基金supported by the grants from the National Natural Science Foundation of China(32072386 and 31801700)the Key Research and Development Project of Anhui Province,China(202004a06020013)the Postdoctoral Science Fund of Anhui Province,China(2019B360)。
文摘Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.
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the National Natural Science Foundation of China.
文摘Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV-Cvrease-mp displayed on|y a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.
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supported by grants from the Natural Science Foundation of China (No. 31301641 to J.Z.)the Program for Qualified Personnel of Taiwan Strait West Coast (No. K8812007 to L.H.X.)
文摘Banana bunchy top virus(BBTV)poses a serious danger to banana crops worldwide.BBTV-encoded protein B4 is a determinant of pathogenicity.However,the relevant molecular mechanisms underlying its effects remain unknown.In this study,we found that a functional peptide could be liberated from protein B4,likely via proteolytic processing.Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects,including dwarfism and sterility,in plants.The released protein fragment targets host proteins,such as the large subunit of RuBisCO(RbcL)and elongation factor 2(EF2),involved in protein synthesis.Therefore,the peptide released from B4(also a precursor)may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.
文摘The realization of protein functional movement is usually accompanied by specific conformational changes,and there exist some key residues that mediate and control the functional motions of proteins in the allosteric process.In the present work,the perturbation-response scanning method developed by our group was combined with the molecular dynamics(MD)simulation to identify the key residues controlling the functional movement of proteins.In our method,a physical quantity that is directly related to protein specific function was introduced,and then based on the MD simulation trajectories,the perturbation-response scanning method was used to identify the key residues for functional motions,in which the residues that highly correlated with the fluctuation of the function-related quantity were identified as the key residues controlling the specific functional motions of the protein.Two protein systems,i.e.,the heat shock protein 70 and glutamine binding protein,were selected as case studies to validate the effectiveness of our method.Our calculated results are in good agreement with the experimental results.The location of the key residues in the two proteins are similar,indicating the similar mechanisms behind the performance of their biological functions.
文摘This study evaluated whether the administration of a NSAID, sodium diclofenac, can promote alterations in the expression of Fos protein in central amygdala (CEA) and the lateral hypothalamus (LH) after 6 h of experimental tooth movement with a controlled force of 70 g, applied to the superior central incisors of rats. Adult male rats were anesthetized and divided into four groups: Control, no orthodontic appliance (OA);OA activated with 70 g;OA activated with 70 g and pretreated with diclofenac sodium (5 mg/kg, intramuscular);and diclofenac sodium alone. Six hours after the onset of the experiment the rats were reanesthetized and perfused with 4% paraformaldehyde. The brains were removed and fixed, and sections containing the CEA and LH were processed for Fos protein immunohistochemistry. The results show that in the control group, intramuscular injection of a ketamine/xylazine mixture did not induce IR-Fos cells in the CEA or LH. However, in the 70 g group, IR-Fos was the strongest observed
基金supported by the National Natural Science Foundation of China(30571235, 30840058)the Natural Science Fundamental Research Project of Jiangsu Colleges and Universities,China (10KJB210006)High-Level Talented Person Fund of Yangzhou University, China (2009-2010)
文摘Guided pesticide is an unique compound resulted from the conjugation with carrier （amino acid, protein, sugars, etc） and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein （MP） is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein （GFP） and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG （isopropyl-b-dthiogalactopyranoside） at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.
文摘In this study, the expression of HSP70 during experimental tooth movement was dynamically observed and the relationship between HSP70 and orthodontic periodontal tissue remodeling were observed. The orthodontic appliance was placed between the right maxillary first molar and maxillary central incisors of adult SD rats to establish a rat molar movement model. Immunohistochemistry was performed 1, 3, 5, 7 and 14 day（s） after orthodontic force application to observe the expression and localization of inducible HSP70. The expression of HSP70 was strongly positive in the early stage of the tooth movement, became gradually less positive, and was weakly positive in the restoration stage. There was difference in staining pattern between different parts of PDL during the same period. These results suggest that the expression of HSP70 and difference in staining pattern among different parts of PDL during orthodontic tooth movement in rats may be implicated in stress response and remodeling of periodontal tissue.
