Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.How...Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.However,the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown.Here,we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H_(2)O_(2).Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28(CPK28)directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation.During pathogen infection,CPK28 dissociates from PIP2;7 and destabilizes,leading to PIP2;7 accumulation.As a countermeasure,oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation.Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28.Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection.Accordingly,we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant.The strategy only allows accumulation of PIP2;7^(S273/276A) during infection to limit potential side effects on normal plant growth.展开更多
作为钙离子的感受蛋白,钙依赖的蛋白激酶(calcium-dependent protein kinases,CDPKs)在拟南芥生长发育、应答逆境胁迫等方面具有重要作用.本研究探讨了拟南芥CDPK家族成员之一CPK28在应答渗透胁迫中的功能.结果表明,CPK28基因缺失导致...作为钙离子的感受蛋白,钙依赖的蛋白激酶(calcium-dependent protein kinases,CDPKs)在拟南芥生长发育、应答逆境胁迫等方面具有重要作用.本研究探讨了拟南芥CDPK家族成员之一CPK28在应答渗透胁迫中的功能.结果表明,CPK28基因缺失导致拟南芥幼苗对NaCl和甘露醇引起的渗透胁迫轻度敏感,而CPK28基因超表达增强了拟南芥对渗透胁迫的耐受性.拟南芥叶肉细胞原生质体瞬时表达结果和转化永久GFP(green fluorescent protein)融合蛋白结果均证明CPK28定位于细胞质膜上.RT-PCR(reverse transcription-polymerase chain reaction)和GUS(glucuronidase)化学染色结果表明,CPK28基因在拟南芥多种组织中均有表达,而且受NaCl和甘露醇胁迫诱导表达.本研究进一步检测了拟南芥幼苗受NaCl和甘露醇胁迫后体内的脯氨酸含量,发现CPK28基因超表达植株比野生型和功能缺失突变体体内积累更多的脯氨酸.利用半定量RT-PCR检测了已知的胁迫相关基因在野生型、CPK28功能缺失突变体和CPK28基因超表达植株中的表达模式,发现这些基因的表达在3种基因型材料中没有明显的差别,暗示CPK28可能不是通过调节基因的转录来参与胁迫应答过程.另外,本研究还发现CPK28可以催化自身磷酸化,这为解析CPK28的激酶活性调节提供了线索.上述研究结果表明CPK28可能参与了拟南芥应答NaCl和甘露醇胁迫反应,并正向调节这些反应过程.展开更多
As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the C...As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the CDPK family in Arabidopsis, in response to osmotic stress.The cpk28 mutant, a loss-of-function mutant, exhibited an NaCl- and mannitol-sensitive phenotype in green cotyledons, while CPK28-overexpressing plants displayed stronger tolerance to NaCl and mannitol stresses than wildtype plants. Reverse transcriptase-polymerase chain reaction and beta-glucuronidase staining assays showed that NaCl and mannitol stresses induced CPK28. CPK28-overexpressing lines accumulated significantly more proline relative to wild-type plants and mutant plants under NaCl and mannitol stresses. Transient expression of CPK28-GFP in mesophyll cell protoplasts, as well as stable transgenic lines expressing CPK28-GFP, showed that CPK28 was localized in the plasma membrane. Expression levels of known stress-responsive genes were not significantly altered in the null mutant and overexpression lines,suggesting that CPK28 possibly mediated the stress response via the regulation of target proteins rather than via regulation at the level of transcription. Meanwhile, CPK28could autophosphorylate. Taken together, these data demonstrated that CPK28, a potential positive regulator, is involved in the response to osmotic stress in Arabidopsis.展开更多
基金supported by the National Natural Science Foundation of China(32230089,32070139,32372493,32402315,and 32270208)the China Agricultural Research System(CARS-21)+1 种基金the China Postdoctoral Science Foundation(2022M721655)the Postdoctoral Innovation Talent Support Program(BX20220153).
文摘Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.However,the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown.Here,we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H_(2)O_(2).Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28(CPK28)directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation.During pathogen infection,CPK28 dissociates from PIP2;7 and destabilizes,leading to PIP2;7 accumulation.As a countermeasure,oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation.Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28.Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection.Accordingly,we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant.The strategy only allows accumulation of PIP2;7^(S273/276A) during infection to limit potential side effects on normal plant growth.
基金supported by the National Genetically Modified Organisms Breeding Major Projects(2011ZX08009-002)
文摘As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the CDPK family in Arabidopsis, in response to osmotic stress.The cpk28 mutant, a loss-of-function mutant, exhibited an NaCl- and mannitol-sensitive phenotype in green cotyledons, while CPK28-overexpressing plants displayed stronger tolerance to NaCl and mannitol stresses than wildtype plants. Reverse transcriptase-polymerase chain reaction and beta-glucuronidase staining assays showed that NaCl and mannitol stresses induced CPK28. CPK28-overexpressing lines accumulated significantly more proline relative to wild-type plants and mutant plants under NaCl and mannitol stresses. Transient expression of CPK28-GFP in mesophyll cell protoplasts, as well as stable transgenic lines expressing CPK28-GFP, showed that CPK28 was localized in the plasma membrane. Expression levels of known stress-responsive genes were not significantly altered in the null mutant and overexpression lines,suggesting that CPK28 possibly mediated the stress response via the regulation of target proteins rather than via regulation at the level of transcription. Meanwhile, CPK28could autophosphorylate. Taken together, these data demonstrated that CPK28, a potential positive regulator, is involved in the response to osmotic stress in Arabidopsis.
