Anther dehiscence controls optimal interaction between pollen and stigma,thereby determining the successful sexual reproduction.The regulators or mechanisms of this process remain elusive.Here,two CRISPR/Cas9 mutants ...Anther dehiscence controls optimal interaction between pollen and stigma,thereby determining the successful sexual reproduction.The regulators or mechanisms of this process remain elusive.Here,two CRISPR/Cas9 mutants of a rice exocyst subunit gene SEC3A,sec3a-1 and sec3a-2,showed anther indehiscence at anthesis and male sterility at maturity.Pollen viability and germination in the mutants were partly defective,whereas their female gametes undergone a normal development.Hybrid or self-pollinated seeds could be produced by artificial pollination,suggesting potential use of a weak sec3a mutant as a female line during hybrid breeding.SEC3A is widely expressed in various tissues,including anther walls.Further results showed an excessive IAA accumulation and no endothecium lignification in sec3a-1/2 anthers.Our findings suggest that SEC3A appears to regulate anther dehiscence by modulating auxin signaling,providing insights into regulation of anther dehiscence and function of exocyst in plants.展开更多
Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade f...Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane.However,it is unclear whether this is the case in plants.In this study,we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion.Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays,we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a.Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex.Intriguingly,we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments.Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly,suggesting that the RABA2a-SNARE-and exocyst-mediated secretory pathways are largely independent.Consistently,our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes,and genetic and cell biologyanalyses indicate that the two pathways select different cargos.Finally,we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings.Collectively,our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.展开更多
The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, w...The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, we found that EXO7oA1 interacted with the GOLD domain of Patellin3 （PATL3）. The direct EXO7OA1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO7oA1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo7oa1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo7oe2, PRsec6 or exo84b mutants. These data suggested that EXO7oA1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicletethering/fusion. Both EXO7oA1 and PATL3 were shown to bind PI4P and PI（4,5）P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO7oA1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunitspecific function, and provided data and tools for further characterization of PATL family proteins.展开更多
基金supported by the Science Fund for Creative Research Groups of the National Natural Science Foundation of China(32171970)the Chongqing Outstanding Scientists Project(cstc2022ycjh-bgzxm0073)the Natural Science Foundation of Chongqing,China(cstc2021jcyj-cxttX0004)。
文摘Anther dehiscence controls optimal interaction between pollen and stigma,thereby determining the successful sexual reproduction.The regulators or mechanisms of this process remain elusive.Here,two CRISPR/Cas9 mutants of a rice exocyst subunit gene SEC3A,sec3a-1 and sec3a-2,showed anther indehiscence at anthesis and male sterility at maturity.Pollen viability and germination in the mutants were partly defective,whereas their female gametes undergone a normal development.Hybrid or self-pollinated seeds could be produced by artificial pollination,suggesting potential use of a weak sec3a mutant as a female line during hybrid breeding.SEC3A is widely expressed in various tissues,including anther walls.Further results showed an excessive IAA accumulation and no endothecium lignification in sec3a-1/2 anthers.Our findings suggest that SEC3A appears to regulate anther dehiscence by modulating auxin signaling,providing insights into regulation of anther dehiscence and function of exocyst in plants.
基金This work was supported by the Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes(2019KSYS006)was also financially supported by grants from the Natural Science Foundation of China(31770306)+7 种基金the Natural Science Foundation of Guangdong Province(2020A1515010966)the Guangdong Innovation Research Team Fund(2016ZT06S172)the Shenzhen Sci-Tech Fund(KYTDPT20181011104005)Z.M.and Y.M.were supported financially by the Singapore Ministry of Education(MOE)Tier 1(RG32/20)and Tier 3(MOE2019-T3-1-012)X.Z.was supported financially by the National Science Foundation for Young Scientists of China(32000558)the China Postdoctoral Science Foundation(grant no.2019M660494)R.L.L.was supported financially by the Natural Science Foundation of China(31970182,31670182)the Fundamental Research Funds for the Central Universities(2019ZY29).
文摘Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane.However,it is unclear whether this is the case in plants.In this study,we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion.Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays,we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a.Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex.Intriguingly,we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments.Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly,suggesting that the RABA2a-SNARE-and exocyst-mediated secretory pathways are largely independent.Consistently,our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes,and genetic and cell biologyanalyses indicate that the two pathways select different cargos.Finally,we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings.Collectively,our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.
基金supported by grant 31200236 from the National Natural Science Foundation of China(NSFC)Grants KYTZ201402 and KJQN201534 from the Fundamental Research Funds for the Central Universities in China+1 种基金Grant 130809001 from the Jiangsu University Superiority Discipline Construction ProjectThe laboratory of T.M.is cofunded by the Netherlands Organization for Scientific Research(NWO 867.15.020)
文摘The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, we found that EXO7oA1 interacted with the GOLD domain of Patellin3 （PATL3）. The direct EXO7OA1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO7oA1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo7oa1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo7oe2, PRsec6 or exo84b mutants. These data suggested that EXO7oA1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicletethering/fusion. Both EXO7oA1 and PATL3 were shown to bind PI4P and PI（4,5）P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO7oA1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunitspecific function, and provided data and tools for further characterization of PATL family proteins.
