Production of reactive oxygen species(ROS)is a conserved immune response primarily mediated by NADPH oxidases(NOXs),also known in plants as respiratory burst oxidase homologs(RBOHs).Most microbe-associated molecular p...Production of reactive oxygen species(ROS)is a conserved immune response primarily mediated by NADPH oxidases(NOXs),also known in plants as respiratory burst oxidase homologs(RBOHs).Most microbe-associated molecular patterns(MAMPs)trigger a very fast and transient ROS burst in plants.However,recently,we found that lipopolysaccharides(LPS),a typical bacterial MAMP,triggered a biphasic ROS burst.In this study,we isolated mutants defective in LPS-triggered biphasic ROS burst(delt)in Arabidopsis,and cloned the DELT1 gene that was shown to encode RBOHD.In the delt1-2 allele,the antepenultimate residue,glutamic acid(E919),at the C-terminus of RBOHD was mutated to lysine(K).E919 is a highly conserved residue in NADPH oxidases,and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease.Consistently,we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure.It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly.However,we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association,suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs.Taken together,our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.展开更多
Reactive oxygen species(ROS)produced by respiratory burst oxidase homologs(RBOHs)are critical for plant immunity.Despite transcriptional and post-translational regulation of RBOHD activity,the dynamic control of the R...Reactive oxygen species(ROS)produced by respiratory burst oxidase homologs(RBOHs)are critical for plant immunity.Despite transcriptional and post-translational regulation of RBOHD activity,the dynamic control of the Ros burst during plant immune responses remains unclear.Here,we demonstrate that upon infection with avirulent Puccinia striformis f.sp.tritici(Pst),the wheat transcription factor TaWRKY40 is activated,driving an extracellular ROS burst by binding to the TaNOX10(RBOHD)promoter and inducing its activation.Furthermore,TaWRKY40 undergoes phosphorylation by the brassinolide signaling kinase TaBSK3,promoting TaWRKY40 nuclear translocation and enhancing TaNOX10 transactivation.This cascade increases extracellular Ros levels,conferring resistance to stripe rust.When encountering viru-lent Pst races,anotherwheat WRKY transcription factor,TaWRKY19,is upregulated,transcriptionallysup-pressing TaWRKY40 and binding to the same cis element in the TaNOX1O promoter.This inhibits TaNOX10 expression,suppresses Ros accumulation,and renders wheat susceptible.These findings reveal a tran-scriptional activation module comprising TaBSK3-TaWRKY40-TaNOX10 that governs ROS production and establish a TaWRKY19-TaWRKY40 dual-regulatory module that fine-tunes ROS burst during wheat-Pst interactions.Importantly,this coordinated regulation of ROS by TaWRKY19 and TaWRKY40 enables wheat to have differential resistance against Pst races with diverse virulence levels.展开更多
Volatile organic compounds(VOCs)play key roles in plant–plant communication,especially in response to pest attack.E-2-hexenal is an important component of VOCs,but it is unclear whether it can induce endog-enous plan...Volatile organic compounds(VOCs)play key roles in plant–plant communication,especially in response to pest attack.E-2-hexenal is an important component of VOCs,but it is unclear whether it can induce endog-enous plant resistance to insects.Here,we show that E-2-hexenal activates early signaling events in Ara-bidopsis(Arabidopsis thaliana)mesophyll cells,including an H2O2 burst at the plasma membrane,the directedflow of calcium ions,and an increase in cytosolic calcium concentration.Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamond-back moth Plutella xylostella L.,and this phenomenon is largely lost in the wrky46 mutant.Mechanistically,E-2-hexenal induces the expression of WRKY46 and MYC2,and the physical interaction of their encoded proteins was verified by yeast two-hybrid,firefly luciferase complementation imaging,and in vitro pull-down assays.The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expres-sion,as demonstrated by luciferase reporter,yeast one-hybrid,chromatin immunoprecipitation,and electrophoretic mobility shift assays.This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes(TT4 and CHIL)and the accumulation of totalflavonoids,thereby modulating plant tolerance to insects.Together,our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis,providing new in-sights into the mechanisms by which VOCs trigger plant defense responses.展开更多
Production of reactive oxygen species(ROS)via the activity of respiratory burst oxidase homologs(RBOHs)plays a vital role in multiple layers of the plant immune system,including pathogen-associated molecular pattern-t...Production of reactive oxygen species(ROS)via the activity of respiratory burst oxidase homologs(RBOHs)plays a vital role in multiple layers of the plant immune system,including pathogen-associated molecular pattern-triggered immunity(PTI),damage-associated molecular pattern-triggered immunity(DTI),effector-triggered immunity(ETI),and systemic acquired resistance(SAR).It is generally established that RBOHD is activated by different receptor-like cytoplasmic kinases(RLCKs)in response to various immune elicitors.In this study,we showed that RPM1-INDUCED PROTEIN KINASE(RIPK),an RLCK VII subfamily member,contributes to ROS production in multiple layers of plant immune system.The ripk mutants showed reduced ROS production in response to treatment with all examined immune elicitors that trigger PTI,DTI,ETI,and SAR.We found that RIPK can directly phosphorylate the N-terminal region of RBOHD in vitro,and the levels of phosphorylated S343/S347 residues of RBOHD are sigfniciantly lower in ripk mutants compared with the wild type upon treatment with all tested immune elicitors.We further demonstrated that phosphorylation of RIPK is required for its function in regulating RBOHD-mediated ROS production.Similar to rbohd,ripk mutants showed reduced stomatal closure and impaired SAR,and were susceptible to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our results indicate that RIPK regulates broad-spectrum RBOHD-mediated ROS signaling during PTI,DTI,ETI,and SAR,leading to subsequent RBOHD-dependent immune responses.展开更多
Reactive oxygen species(ROS)production is a conserved immune response in Arabidopsis primarily medi-ated by respiratory burst oxidase homolog D(RBOHD),a nicotinamide adenine dinucleotide phosphate(NADPH)oxidase associ...Reactive oxygen species(ROS)production is a conserved immune response in Arabidopsis primarily medi-ated by respiratory burst oxidase homolog D(RBOHD),a nicotinamide adenine dinucleotide phosphate(NADPH)oxidase associated with the plasma membrane.A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production.However,the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear.In this study,we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4(FIN4),which encodes the first enzyme in de novo NAD biosynthesis.fin4 mu-tants show reduced NADPH levels and impaired ROS production.However,FIN4 and other genes involved in NAD-and NADPH-generating pathways are not highly upregulated upon elicitor treatment,raising a pos-sibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD.To verify this possibility,we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE(RIPK),a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity,and identified NADP-malic enzyme 2(NADP-ME2),an NADPH-generating enzyme.Compared with wild-type plants,nadp-me2 mutants display decreased NADP-ME activity,lower NADPH levels,and reduced ROS production in response to immune elicitors.Furthermore,we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro.The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH,thus fueling RBOHD to sustain ROS production in plant immunity.展开更多
Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translo...Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translocation and subcellular accumulation are unknown.Here,we used a mobilizable,highly responsive genetically encoded fluorescent indicator,green fluorescent protein(GFP)-N160RbohD,to monitor PA dynamics in living cells.The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels.Cellular,pharmacological,and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane,which mainly depended on hydrolysis of phospholipase D.By contrast,heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity,as well as secretory and endocytic trafficking.Strikingly,we showed that gravity triggers asymmetric PA distribution at the root apex,a response that is suppressed by PLDz2 knockout.The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that the synthesis,transport,and metabolism of PA.展开更多
基金the National Natural Science Foundation of China(No.31622006)the Postdoctoral Science Foundation of China(Nos.2018M630683 and 2018T110601)
文摘Production of reactive oxygen species(ROS)is a conserved immune response primarily mediated by NADPH oxidases(NOXs),also known in plants as respiratory burst oxidase homologs(RBOHs).Most microbe-associated molecular patterns(MAMPs)trigger a very fast and transient ROS burst in plants.However,recently,we found that lipopolysaccharides(LPS),a typical bacterial MAMP,triggered a biphasic ROS burst.In this study,we isolated mutants defective in LPS-triggered biphasic ROS burst(delt)in Arabidopsis,and cloned the DELT1 gene that was shown to encode RBOHD.In the delt1-2 allele,the antepenultimate residue,glutamic acid(E919),at the C-terminus of RBOHD was mutated to lysine(K).E919 is a highly conserved residue in NADPH oxidases,and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease.Consistently,we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure.It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly.However,we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association,suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs.Taken together,our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.
基金supported by the National Natural Science Foundation of China(32225041,32421004,and 32293243)the China Agricultural Research System(CARS-3),Science Foundation for Distinguished Young Scholars of Shaanxi Province(2024JC-JCQN-22)+1 种基金Shaanxi Provincial Sci-Tech Development Project(Central Government Guided Local Fund)(2024ZY-JCYJ-02-43)the Cyrus Tang Foundation.
文摘Reactive oxygen species(ROS)produced by respiratory burst oxidase homologs(RBOHs)are critical for plant immunity.Despite transcriptional and post-translational regulation of RBOHD activity,the dynamic control of the Ros burst during plant immune responses remains unclear.Here,we demonstrate that upon infection with avirulent Puccinia striformis f.sp.tritici(Pst),the wheat transcription factor TaWRKY40 is activated,driving an extracellular ROS burst by binding to the TaNOX10(RBOHD)promoter and inducing its activation.Furthermore,TaWRKY40 undergoes phosphorylation by the brassinolide signaling kinase TaBSK3,promoting TaWRKY40 nuclear translocation and enhancing TaNOX10 transactivation.This cascade increases extracellular Ros levels,conferring resistance to stripe rust.When encountering viru-lent Pst races,anotherwheat WRKY transcription factor,TaWRKY19,is upregulated,transcriptionallysup-pressing TaWRKY40 and binding to the same cis element in the TaNOX1O promoter.This inhibits TaNOX10 expression,suppresses Ros accumulation,and renders wheat susceptible.These findings reveal a tran-scriptional activation module comprising TaBSK3-TaWRKY40-TaNOX10 that governs ROS production and establish a TaWRKY19-TaWRKY40 dual-regulatory module that fine-tunes ROS burst during wheat-Pst interactions.Importantly,this coordinated regulation of ROS by TaWRKY19 and TaWRKY40 enables wheat to have differential resistance against Pst races with diverse virulence levels.
基金supported by the National Natural Science Foundation of China (31270655).
文摘Volatile organic compounds(VOCs)play key roles in plant–plant communication,especially in response to pest attack.E-2-hexenal is an important component of VOCs,but it is unclear whether it can induce endog-enous plant resistance to insects.Here,we show that E-2-hexenal activates early signaling events in Ara-bidopsis(Arabidopsis thaliana)mesophyll cells,including an H2O2 burst at the plasma membrane,the directedflow of calcium ions,and an increase in cytosolic calcium concentration.Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamond-back moth Plutella xylostella L.,and this phenomenon is largely lost in the wrky46 mutant.Mechanistically,E-2-hexenal induces the expression of WRKY46 and MYC2,and the physical interaction of their encoded proteins was verified by yeast two-hybrid,firefly luciferase complementation imaging,and in vitro pull-down assays.The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expres-sion,as demonstrated by luciferase reporter,yeast one-hybrid,chromatin immunoprecipitation,and electrophoretic mobility shift assays.This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes(TT4 and CHIL)and the accumulation of totalflavonoids,thereby modulating plant tolerance to insects.Together,our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis,providing new in-sights into the mechanisms by which VOCs trigger plant defense responses.
基金Financial support for this study was provided by the National Key Research and Development Program of China(2018YFD1000800)National Natural Science Foundation of China(31970279 and 31801015)China Postdoctoral Science Foundation(2019T120522 and 2019M652101).
文摘Production of reactive oxygen species(ROS)via the activity of respiratory burst oxidase homologs(RBOHs)plays a vital role in multiple layers of the plant immune system,including pathogen-associated molecular pattern-triggered immunity(PTI),damage-associated molecular pattern-triggered immunity(DTI),effector-triggered immunity(ETI),and systemic acquired resistance(SAR).It is generally established that RBOHD is activated by different receptor-like cytoplasmic kinases(RLCKs)in response to various immune elicitors.In this study,we showed that RPM1-INDUCED PROTEIN KINASE(RIPK),an RLCK VII subfamily member,contributes to ROS production in multiple layers of plant immune system.The ripk mutants showed reduced ROS production in response to treatment with all examined immune elicitors that trigger PTI,DTI,ETI,and SAR.We found that RIPK can directly phosphorylate the N-terminal region of RBOHD in vitro,and the levels of phosphorylated S343/S347 residues of RBOHD are sigfniciantly lower in ripk mutants compared with the wild type upon treatment with all tested immune elicitors.We further demonstrated that phosphorylation of RIPK is required for its function in regulating RBOHD-mediated ROS production.Similar to rbohd,ripk mutants showed reduced stomatal closure and impaired SAR,and were susceptible to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our results indicate that RIPK regulates broad-spectrum RBOHD-mediated ROS signaling during PTI,DTI,ETI,and SAR,leading to subsequent RBOHD-dependent immune responses.
基金supported by the National Natural Science Foundation of China(31970279 and 31622006)the Zhejiang Science and Technology Major Program(2021C02064-7 and 2021C02009).
文摘Reactive oxygen species(ROS)production is a conserved immune response in Arabidopsis primarily medi-ated by respiratory burst oxidase homolog D(RBOHD),a nicotinamide adenine dinucleotide phosphate(NADPH)oxidase associated with the plasma membrane.A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production.However,the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear.In this study,we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4(FIN4),which encodes the first enzyme in de novo NAD biosynthesis.fin4 mu-tants show reduced NADPH levels and impaired ROS production.However,FIN4 and other genes involved in NAD-and NADPH-generating pathways are not highly upregulated upon elicitor treatment,raising a pos-sibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD.To verify this possibility,we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE(RIPK),a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity,and identified NADP-malic enzyme 2(NADP-ME2),an NADPH-generating enzyme.Compared with wild-type plants,nadp-me2 mutants display decreased NADP-ME activity,lower NADPH levels,and reduced ROS production in response to immune elicitors.Furthermore,we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro.The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH,thus fueling RBOHD to sustain ROS production in plant immunity.
基金supported by the National Key R&D Program of China(2022YFA1303400 and 2022YFD1201700)the National Natural Science Foundation of China(32270301 and 31970300)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund(CX-20-2007)the Fundamental Research Funds for the Central Universities(XUEKEN2022002).
文摘Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translocation and subcellular accumulation are unknown.Here,we used a mobilizable,highly responsive genetically encoded fluorescent indicator,green fluorescent protein(GFP)-N160RbohD,to monitor PA dynamics in living cells.The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels.Cellular,pharmacological,and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane,which mainly depended on hydrolysis of phospholipase D.By contrast,heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity,as well as secretory and endocytic trafficking.Strikingly,we showed that gravity triggers asymmetric PA distribution at the root apex,a response that is suppressed by PLDz2 knockout.The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that the synthesis,transport,and metabolism of PA.
