以原核表达的番茄SlMC7重组蛋白为抗原,制备SlMC7多克隆抗体,采用ELISA和蛋白质点杂的方法,研究了番茄II型metacaspase蛋白(SlMC7)的抗体效价和表达特性,并对SlMC7蛋白及基因在番茄果实发育过程中的表达差异进行了研究,以期为后续的SlMC...以原核表达的番茄SlMC7重组蛋白为抗原,制备SlMC7多克隆抗体,采用ELISA和蛋白质点杂的方法,研究了番茄II型metacaspase蛋白(SlMC7)的抗体效价和表达特性,并对SlMC7蛋白及基因在番茄果实发育过程中的表达差异进行了研究,以期为后续的SlMC7在番茄果实中功能性研究提供材料基础和参考依据。结果表明:SlMC7多克隆抗体效价在256 000以上,在1∶10 000的稀释度下,可识别低至1.6ng的纯蛋白。对各时期番茄果实Western-blot和Real time PCR结果表明,相对于绿熟期,SlMC7蛋白及基因在破色期果实中表达量显著上升,表明SlMC7与番茄果实成熟存在一定相关性。展开更多
SlMC7 prokaryotic expression recombinant protein was used as test materials.SlMC7 polyclonal antiserum was obtained from immunized mice by using purified SlMC7 recombinant protein from Escherichia coli.ELISA and prote...SlMC7 prokaryotic expression recombinant protein was used as test materials.SlMC7 polyclonal antiserum was obtained from immunized mice by using purified SlMC7 recombinant protein from Escherichia coli.ELISA and protein dot plot methods were used to detect the antibody titer and expression characteristics and the expression difference at different stages,providing material foundation and reference basis for subsequent SlMC7 functional research in tomato fruit.The results showed that the titer of SlMC7 polyclonal antibodies was more than 1∶256000 and the sensitivity of SlMC7 polyclonal antibodies was 1.6 ng at the dilutability of 1∶10000.SlMC7 protein was detected at different stages,and SlMC7 had a higher expression level in color-break fruit,indicating that SlMC7 might be relevant to the fruit ripening of tomato.展开更多
Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cystei...Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cysteine protease metacaspase 1(MC1)functions as a new positive regulator of autophagy and cleaves Beclin 1 behind residues R97 and R99 in tobacco(Nicotiana benthamiana).Genetic analysis further demonstrated that the MC1-Beclin 1 cleavage module is both necessary and sufficient for the activation of plant autophagy.Mechanistically,this cleavage releases the N-terminal fragment of Beclin 1(aa 1-97),which exhibits intrinsic autophagy-inducing activity and is dependent on the phosphatidylinositol 3-kinase complex.Moreover,the activated autophagy boosts broad-spectrum antiviral responses,while the barley stripe mosaic virus-encodedγb protein targets MC1 and suppresses MC1-mediated Beclin 1 cleavage,thereby optimizing viral infection.The cleavage of Beclin 1 is thought to abolish its autophagic function in mammals;however,our findings unveil a distinctive plant autophagy mechanism whereby Beclin 1 activation necessitates MC1-mediated cleavage to drive antiviral autophagy.展开更多
Plants can produce animal cytokine-like immune peptides,among which plant elicitor peptides(Peps)derive from the C termini of their precursors(PROPEPs).Recently,the functions of Peps have been expanded beyond plant im...Plants can produce animal cytokine-like immune peptides,among which plant elicitor peptides(Peps)derive from the C termini of their precursors(PROPEPs).Recently,the functions of Peps have been expanded beyond plant immunity.However,a long-standing enigma is how PROPEPs are processed into Peps.Here,we report that the Ca2+-dependent type-ll metacaspases(MCs)constitute the proteolytic enzymes to mediate PROPEP processing in Arabidopsis.In protoplasts,co-expression of PROPEP1 with type-ll MCs,including MC4 to MC9,can promote the generation of processed Pep1.Destruction of the catalytic cysteine residue in MC4 or the conserved arginine residue preceding the Pep1 sequence blocks PROPEP1 cleavage,whereas the bacterial elicitor flg22 enhances the MC4-mediated PROPEP1 processing.MC4 cleaves PROPEP1 in vitro and also cleaves PROPEP2 to PROPEP8,but,surprisingly,not PROPEP6 in protoplasts.Domain swapping between PROPEP1 and PROPEP6 suggests a hidden role of the sequence context upstream of the Pep sequence for PROPEP processing.flg22-induced PROPEP1 processing and B otrytis cinerea resistance are severely impaired in the m c4/5/6/7 quadruple-mutant plants.Taken together,our study identifies the type-ll MCs as new players in Pep signaling,and lays the foundation for understanding the regulation of multifaceted functions of Peps in plant immunity and beyond.展开更多
文摘以原核表达的番茄SlMC7重组蛋白为抗原,制备SlMC7多克隆抗体,采用ELISA和蛋白质点杂的方法,研究了番茄II型metacaspase蛋白(SlMC7)的抗体效价和表达特性,并对SlMC7蛋白及基因在番茄果实发育过程中的表达差异进行了研究,以期为后续的SlMC7在番茄果实中功能性研究提供材料基础和参考依据。结果表明:SlMC7多克隆抗体效价在256 000以上,在1∶10 000的稀释度下,可识别低至1.6ng的纯蛋白。对各时期番茄果实Western-blot和Real time PCR结果表明,相对于绿熟期,SlMC7蛋白及基因在破色期果实中表达量显著上升,表明SlMC7与番茄果实成熟存在一定相关性。
基金Supported by National Natural Science Foundation of China(31672207)
文摘SlMC7 prokaryotic expression recombinant protein was used as test materials.SlMC7 polyclonal antiserum was obtained from immunized mice by using purified SlMC7 recombinant protein from Escherichia coli.ELISA and protein dot plot methods were used to detect the antibody titer and expression characteristics and the expression difference at different stages,providing material foundation and reference basis for subsequent SlMC7 functional research in tomato fruit.The results showed that the titer of SlMC7 polyclonal antibodies was more than 1∶256000 and the sensitivity of SlMC7 polyclonal antibodies was 1.6 ng at the dilutability of 1∶10000.SlMC7 protein was detected at different stages,and SlMC7 had a higher expression level in color-break fruit,indicating that SlMC7 might be relevant to the fruit ripening of tomato.
基金supported by the National Natural Science Foundation of China(32330086 and 32270168)to D.L.the Young Elite Scientists Sponsorship Program by CAST(2022QNRC001)to M.Y.the 2115 Talent Development Program of China Agricultural University.
文摘Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cysteine protease metacaspase 1(MC1)functions as a new positive regulator of autophagy and cleaves Beclin 1 behind residues R97 and R99 in tobacco(Nicotiana benthamiana).Genetic analysis further demonstrated that the MC1-Beclin 1 cleavage module is both necessary and sufficient for the activation of plant autophagy.Mechanistically,this cleavage releases the N-terminal fragment of Beclin 1(aa 1-97),which exhibits intrinsic autophagy-inducing activity and is dependent on the phosphatidylinositol 3-kinase complex.Moreover,the activated autophagy boosts broad-spectrum antiviral responses,while the barley stripe mosaic virus-encodedγb protein targets MC1 and suppresses MC1-mediated Beclin 1 cleavage,thereby optimizing viral infection.The cleavage of Beclin 1 is thought to abolish its autophagic function in mammals;however,our findings unveil a distinctive plant autophagy mechanism whereby Beclin 1 activation necessitates MC1-mediated cleavage to drive antiviral autophagy.
基金This work was supported by the Foundation of Guangzhou Science and Technology Key Project(201904020041)the National Natural Science Foundation of China(31770295)to J.-F.L.
文摘Plants can produce animal cytokine-like immune peptides,among which plant elicitor peptides(Peps)derive from the C termini of their precursors(PROPEPs).Recently,the functions of Peps have been expanded beyond plant immunity.However,a long-standing enigma is how PROPEPs are processed into Peps.Here,we report that the Ca2+-dependent type-ll metacaspases(MCs)constitute the proteolytic enzymes to mediate PROPEP processing in Arabidopsis.In protoplasts,co-expression of PROPEP1 with type-ll MCs,including MC4 to MC9,can promote the generation of processed Pep1.Destruction of the catalytic cysteine residue in MC4 or the conserved arginine residue preceding the Pep1 sequence blocks PROPEP1 cleavage,whereas the bacterial elicitor flg22 enhances the MC4-mediated PROPEP1 processing.MC4 cleaves PROPEP1 in vitro and also cleaves PROPEP2 to PROPEP8,but,surprisingly,not PROPEP6 in protoplasts.Domain swapping between PROPEP1 and PROPEP6 suggests a hidden role of the sequence context upstream of the Pep sequence for PROPEP processing.flg22-induced PROPEP1 processing and B otrytis cinerea resistance are severely impaired in the m c4/5/6/7 quadruple-mutant plants.Taken together,our study identifies the type-ll MCs as new players in Pep signaling,and lays the foundation for understanding the regulation of multifaceted functions of Peps in plant immunity and beyond.
