Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can direct...Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.展开更多
Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided int...Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.展开更多
To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plan...To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plants carry an early immune responsive gene FRK1(flg22-induced receptor-like kinase 1)promoter fused with a luciferase(LUC)reporter.A series of mutants with altered pFRK1::LUC activity were identified and named as Arabidopsis genes governing immune gene expression(aggie).The aggie5 mutant showed reduced pFRK1::LUC induction in response to multiple MAMPs treatments.Moreover,the activation of the endogenous FRK1 gene by MAMPs was also compromised in aggie5.Significantly,the bacterial flagellin epitope flg22-induced MAPK activation,ROS production,and immunerelated gene expression were reduced in aggie5.Map-based cloning coupled with next-generation sequencing revealed that aggie5 encodes a kinase-inactive BAK1,which is involved in brassinosteroid responses,innate immunity,and cell death control.Consistently,the aggie5/bak1-16 mutant displayed compromised responses to BR treatment.Furthermore,the aggie5/bak1-16 mutant exhibited seedling lethality when combined with the mutation of its closest homolog BKK1/SERK4.The data suggest that BAK1 kinase activity is required for its multiple functions in plant immunity,development,and cell death control.The isolation and characterization of these Aggie genes will contribute to a better understanding of host immune signaling and provide genetic resources to improve crop resistance.展开更多
Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting ...Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid （BR） signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRII. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.展开更多
Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-...Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.展开更多
文摘目的 探讨miR-125b通过靶向抑制Bak1表达影响人髓系白血病(AML)细胞增殖的机制及意义。方法选择miR-125b mimics、miR-125b inhibitor、miR-125b NC分别转染人AML细胞株THP-1(miR-125b mimics组、miR-125b inhibitor组、NC组)。采用荧光定量PCR检测miR-125b mRNA相对表达量, MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western-blot法检测蛋白表达水平,利用生物信息学分析和荧光素酶双报告系统明确miR-125b的下游靶基因。结果 miR-125b mimics转染后THP-1细胞中miR-125b表达水平显著上调( P <0.05),miR-125b inhibitor转染后细胞中miR-125b水平则显著降低( P <0.05)。转染24 h、48 h后,MTT法检测显示miR-125b mimics组的细胞增殖活性显著低于miR-125b inhibitor组与NC组( P <0.05),细胞凋亡率显著高于miR-125b inhibitor组与NC组( P <0.05),后两组对比差异无统计学意义( P >0.05)。转染48 h后,Western-blot法检测显示miR-125b mimics组的 Bak1蛋白表达水平显著高于miR-125b inhibitor组与NC组( P <0.05),三组PI3K、AKT蛋白表达水平对比差异无统计学意义( P >0.05)。在HEK293细胞株中,miR-125b mimic转染48 h后可以显著降低Bak1萤火虫荧光素酶活性( P <0.05),而对于含有突变型载体的Bak1萤火虫荧光素酶活性无显著影响( P >0.05)。结论 过表达miR-125b可通过靶向抑制Bak1的活性,抑制人AML细胞株的增殖活性,促进细胞凋亡。
基金Acknowledgments This study was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China （Grants 30425029, 30421001, 90717001）. We greatly thank Prof Hong Ma （Penn. State University, USA） for critical reading and writing improvement and Prof Nam-Hai Chua （The Rockefeller University, USA） for helpful comments. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Prof Sheng Luan （University of California, Berkeley, USA） for providing the construct pATC940. We thank Prof Hong-Quan Yang （SIPPE, CAS） for providing LexA yeast two-hybrid system and Prof Zhi-Yong Wang （The Stanford University, USA） for providing the BRI1 antibody. We thank Mr Xiao-Shu Gao for the help on Confocal Laser Scanning Microscopy.
文摘Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.
基金Science and technology introduction and innovation project of Xinjiang uygur autonomous region people's hospital(No.20170305).
文摘Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.
基金supported by the National Natural Science Foundation of China(32272504)Chinese Universities Scientific Fund(2024TC187)to F.C.
文摘To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plants carry an early immune responsive gene FRK1(flg22-induced receptor-like kinase 1)promoter fused with a luciferase(LUC)reporter.A series of mutants with altered pFRK1::LUC activity were identified and named as Arabidopsis genes governing immune gene expression(aggie).The aggie5 mutant showed reduced pFRK1::LUC induction in response to multiple MAMPs treatments.Moreover,the activation of the endogenous FRK1 gene by MAMPs was also compromised in aggie5.Significantly,the bacterial flagellin epitope flg22-induced MAPK activation,ROS production,and immunerelated gene expression were reduced in aggie5.Map-based cloning coupled with next-generation sequencing revealed that aggie5 encodes a kinase-inactive BAK1,which is involved in brassinosteroid responses,innate immunity,and cell death control.Consistently,the aggie5/bak1-16 mutant displayed compromised responses to BR treatment.Furthermore,the aggie5/bak1-16 mutant exhibited seedling lethality when combined with the mutation of its closest homolog BKK1/SERK4.The data suggest that BAK1 kinase activity is required for its multiple functions in plant immunity,development,and cell death control.The isolation and characterization of these Aggie genes will contribute to a better understanding of host immune signaling and provide genetic resources to improve crop resistance.
基金supported by the grants from the National Natural Science Foundation of China to J.L.(91117008 and 90917019)National Basic Research Program of China to J.L.(2011CB915401)Fundamental Research Funds for the Central Universities to S.X.(lzujbky-2009-35)
文摘Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid （BR） signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRII. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.
基金supported by the National Natural Science Foundation of China(32100155 and 32072507)the Natural Science Foundation of Jiangsu Province(BK20221000)+1 种基金the fellowship of China Postdoctoral Science Foundation(2021M701740)the Jiangsu Funding Program for Excellent Postdoctoral Talent(2022ZB343 and 2022ZB768)。
文摘Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.
