华癸中慢生根瘤菌7653R MCHK-3535基因在自生和与紫云英共生中的功能
|
摘要
【目的】探究Mesorhizobium huakuii 7653R MCHK-3535基因在共生固氮中的功能。【方法】构建MCHK-3535插入失活突变体、超表达和互补菌株,对其进行共生表型鉴定;并测定各菌株的生长曲线、运动性及生物膜形成;利用qRT-PCR方法和组织表达定位分析MCHK-3535在共生过程中的时空表达特征。【结果】与野生型7653R相比,突变体Δ3535达到稳定期的生物量增加,运动性下降,生物膜形成增加。此外,MCHK-3535基因的失活会显著提高紫云英固氮能力和植物地上部分鲜重,但不影响根瘤数量及重量;且互补菌株C3535能部分回补到野生型性状,超表达菌株OV3535均无显著性差异;qRT-PCR和启动子组织表达定位发现,MCHK-3535基因主要在根瘤的侵染区、过渡区及固氮区表达,并且表达持续整个固氮期。【结论】MCHK-3535基因在自生及与紫云英共生过程中发挥重要功能,参与根瘤的正常发育并负向调控固氮酶活性。
[Objective] To study the function of MCHK-3535 gene in symbiotic nitrogen fixation between Mesorhizobium huakuii 7653 R and Astragalus sinicus. [Methods] We constructed the deletion mutant Δ3535,overexpression strains OV3535 and complementary strains C3535, followed by identifying symbiotic phenotypes,detecting the growth curve, swimming motility and biofilm formation Besides, we used qRT-PCR and promoter-GUS fusion vector to detect the expression characteristics of MCHK-3535 in the symbiotic process.[Results] Compared with the wild type 7653 R, mutant Δ3535 grew faster, decreased motility, and increased biofilm formation, moreover, Δ3535 significantly increased the ability of nitrogen fixation and the fresh weight of plants,while the number and weight of root nodules were not affected, in addition, the complementary strain C3535 partially compensated to the wild-type phenotype, whereas OV3535 had no significant difference in these aspects.The results of qRT-PCR and the promoter-GUS reporter system showed that MCHK-3535 expressed in the infection zone, transitional zone and nitrogen fixation zone of the nodules, and the gene expressed lasted for the entire period of nitrogen fixation. [Conclusion] MCHK-3535 functions in the symbiotic nitrogen fixation process and may participates in the normal development of nodules and negatively regulates nitrogenase activity.
[Objective] To study the function of MCHK-3535 gene in symbiotic nitrogen fixation between Mesorhizobium huakuii 7653 R and Astragalus sinicus. [Methods] We constructed the deletion mutant Δ3535,overexpression strains OV3535 and complementary strains C3535, followed by identifying symbiotic phenotypes,detecting the growth curve, swimming motility and biofilm formation Besides, we used qRT-PCR and promoter-GUS fusion vector to detect the expression characteristics of MCHK-3535 in the symbiotic process.[Results] Compared with the wild type 7653 R, mutant Δ3535 grew faster, decreased motility, and increased biofilm formation, moreover, Δ3535 significantly increased the ability of nitrogen fixation and the fresh weight of plants,while the number and weight of root nodules were not affected, in addition, the complementary strain C3535 partially compensated to the wild-type phenotype, whereas OV3535 had no significant difference in these aspects.The results of qRT-PCR and the promoter-GUS reporter system showed that MCHK-3535 expressed in the infection zone, transitional zone and nitrogen fixation zone of the nodules, and the gene expressed lasted for the entire period of nitrogen fixation. [Conclusion] MCHK-3535 functions in the symbiotic nitrogen fixation process and may participates in the normal development of nodules and negatively regulates nitrogenase activity.
引文
[1]Mylona P,Pawlowski K,Bisseling T.Symbiotic nitrogen fixation.The Plant Cell,1995,7(7):869-885.
[2]Desbrosses GJ,Stougaard J.Root nodulation:a paradigm for how plant-microbe symbiosis influences host developmental pathways.Cell Host&Microbe,2011,10(4):348-358.
[3]Ferguson BJ,Indrasumunar A,Hayashi S,Lin MH,Lin YH,Reid DE,Gresshoff PM.Molecular analysis of legume nodule development and autoregulation.Journal of Integrative Plant Biology,2010,52(1):61-76.
[4]Gupta S,Gajbhiye VT,Gupta RK.Effect of light on the degradation of two neonicotinoids viz acetamiprid and thiacloprid in soil.Bulletin of Environmental Contamination and Toxicology,2008,81(2):185-189.
[5]Zipfel C,Oldroyd GED.Plant signalling in symbiosis and immunity.Nature,2017,543(7645):328-336.
[6]Müller DB,Vogel C,Bai Y,Vorholt JA.The plant microbiota:systems-level insights and perspectives.Annual Review of Genetics,2016,50:211-234.
[7]Cook DE,Mesarich CH,Thomma BPHJ.Understanding plant immunity as a surveillance system to detect invasion.Annual Review of Phytopathology,2015,53:541-563.
[8]Hengge R.Principles of c-di-GMP signalling in bacteria.Nature Reviews Microbiology,2009,7(4):263-273.
[9]Pérez-Mendoza D,Aragón IM,Prada-Ramírez HA,Romero-Jiménez L,Ramos C,Gallegos MT,Sanjuán J.Responses to elevated c-di-GMP levels in mutualistic and pathogenic plant-interacting bacteria.PLoS ONE,2014,9(3):e91645.
[10]Wang YW,Xu J,Chen AM,Wang YZ,Zhu JB,Yu GQ,Xu L,Luo L.GGDEF and EAL proteins play different roles in the control of Sinorhizobium meliloti growth,motility,exopolysaccharide production,and competitive nodulation on host alfalfa.Acta Biochimica et Biophysica Sinica,2010,42(6):410-417.
[11]Hengge R,Grundling A,Jenal U,Ryan R,Yildiz F.Bacterial signal transduction by cyclic di-GMP and other nucleotide second messengers.Journal of Bacteriology,2016,198(1):15-26.
[12]Newell PD,Monds RD,O'Toole GA.LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1.Proceedings of the National Academy of Sciences of the United States of America,2009,106(9):3461-3466.
[13]Amikam D,Galperin MY.PilZ domain is part of the bacterial c-di-GMP binding protein.Bioinformatics,2005,22(1):3-6.
[14]Sudarsan N,Lee ER,Weinberg Z,Moy RH,Kim JN,Link KH,Breaker RR.Riboswitches in eubacteria sense the second messenger cyclic di-GMP.Science,2008,321(5887):411-413.
[15]Fang X,Ahmad I,Blanka A,Schottkowski M,Cimdins A,Galperin MY,Romling U,Gomelsky M.GIL,a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.Molecular Microbiology,2014,93(3):439-452.
[16]Duerig A,Abel S,Folcher M,Nicollier M,Schwede T,Amiot N,Giese B,Jenal U.Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression.Genes&Development,2009,23(1):93-104.
[17]Conner JG,Zamorano-Sánchez D,Park JH,Sondermann H,Yildiz FH.The ins and outs of cyclic di-GMP signaling in Vibrio cholerae.Current Opinion in Microbiology,2017,36:20-29.
[18]Chou SH,Galperin MY.Diversity of cyclic Di-GMP-binding proteins and mechanisms.Journal of Bacteriology,2016,198(1):32-46.
[19]Peng JL,Hao BH,Liu L,Wang SM,Ma BG,Yang Y,Xie FL,Li YG.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells.PLoS One,2014,9(4):e93626.
[20]Abd-El-Karem Y,Elbers T,Reichelt R,Steinbüchel A.Heterologous expression of Anabaena sp.PCC7120cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium(Ensifer)meliloti 1021.Applied Microbiology and Biotechnology,2010,89(4):1177-1192.
[21]Sch?per S,Krol E,Skotnicka D,Kaever V,Hilker R,S?gaard-Andersen L,Becker A.Cyclic di-GMP regulates multiple cellular functions in the symbiotic Alphaproteobacterium Sinorhizobium meliloti.Journal of Bacteriology,2015,198(3):521-535.
[22]Robledo M,Rivera L,Jiménez-Zurdo JI,Rivas R,Dazzo F,Velázquez E,Martínez-Molina,Hirsch AM,Mateos PF.Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces.Microbial Cell Factories,2012,11:125.
[23]Tian CF,Garnerone AM,Mathieu-Demazière C,Masson-Boivin C,Batut J.Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.Proceedings of the National Academy of Sciences of the United States of America,2012,109(17):6751-6756.
[24]Zhang YB,Wei C,Jiang WD,Wang L,Li CR,Wang YY,Dow JM,Sun WX.The HD-GYP domain protein RpfG of Xanthomonas oryzae pv.oryzicola regulates synthesis of extracellular polysaccharides that contribute to biofilm formation and virulence on rice.PLoS ONE,2013,8(3):e59428.
[25]Lovering AL,Capeness MJ,Lambert C,Hobley L,Sockett RE.The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.mBio,2011,2(5):e00163-11.
[26]Zhang Y,Kitajima M,Whittle AJ,Liu WT.Benefits of genomic insights and CRISPR-Cas signatures to monitor potential pathogens across drinking water production and distribution systems.Frontiers in Microbiology,2017,8:2036.
[27]Gao T,Meng Q,Gao HC.Thioesterase YbgC affects motility by modulating c-di-GMP levels in Shewanella oneidensis.Scientific Reports,2017,7:3932.
[2]Desbrosses GJ,Stougaard J.Root nodulation:a paradigm for how plant-microbe symbiosis influences host developmental pathways.Cell Host&Microbe,2011,10(4):348-358.
[3]Ferguson BJ,Indrasumunar A,Hayashi S,Lin MH,Lin YH,Reid DE,Gresshoff PM.Molecular analysis of legume nodule development and autoregulation.Journal of Integrative Plant Biology,2010,52(1):61-76.
[4]Gupta S,Gajbhiye VT,Gupta RK.Effect of light on the degradation of two neonicotinoids viz acetamiprid and thiacloprid in soil.Bulletin of Environmental Contamination and Toxicology,2008,81(2):185-189.
[5]Zipfel C,Oldroyd GED.Plant signalling in symbiosis and immunity.Nature,2017,543(7645):328-336.
[6]Müller DB,Vogel C,Bai Y,Vorholt JA.The plant microbiota:systems-level insights and perspectives.Annual Review of Genetics,2016,50:211-234.
[7]Cook DE,Mesarich CH,Thomma BPHJ.Understanding plant immunity as a surveillance system to detect invasion.Annual Review of Phytopathology,2015,53:541-563.
[8]Hengge R.Principles of c-di-GMP signalling in bacteria.Nature Reviews Microbiology,2009,7(4):263-273.
[9]Pérez-Mendoza D,Aragón IM,Prada-Ramírez HA,Romero-Jiménez L,Ramos C,Gallegos MT,Sanjuán J.Responses to elevated c-di-GMP levels in mutualistic and pathogenic plant-interacting bacteria.PLoS ONE,2014,9(3):e91645.
[10]Wang YW,Xu J,Chen AM,Wang YZ,Zhu JB,Yu GQ,Xu L,Luo L.GGDEF and EAL proteins play different roles in the control of Sinorhizobium meliloti growth,motility,exopolysaccharide production,and competitive nodulation on host alfalfa.Acta Biochimica et Biophysica Sinica,2010,42(6):410-417.
[11]Hengge R,Grundling A,Jenal U,Ryan R,Yildiz F.Bacterial signal transduction by cyclic di-GMP and other nucleotide second messengers.Journal of Bacteriology,2016,198(1):15-26.
[12]Newell PD,Monds RD,O'Toole GA.LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1.Proceedings of the National Academy of Sciences of the United States of America,2009,106(9):3461-3466.
[13]Amikam D,Galperin MY.PilZ domain is part of the bacterial c-di-GMP binding protein.Bioinformatics,2005,22(1):3-6.
[14]Sudarsan N,Lee ER,Weinberg Z,Moy RH,Kim JN,Link KH,Breaker RR.Riboswitches in eubacteria sense the second messenger cyclic di-GMP.Science,2008,321(5887):411-413.
[15]Fang X,Ahmad I,Blanka A,Schottkowski M,Cimdins A,Galperin MY,Romling U,Gomelsky M.GIL,a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.Molecular Microbiology,2014,93(3):439-452.
[16]Duerig A,Abel S,Folcher M,Nicollier M,Schwede T,Amiot N,Giese B,Jenal U.Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression.Genes&Development,2009,23(1):93-104.
[17]Conner JG,Zamorano-Sánchez D,Park JH,Sondermann H,Yildiz FH.The ins and outs of cyclic di-GMP signaling in Vibrio cholerae.Current Opinion in Microbiology,2017,36:20-29.
[18]Chou SH,Galperin MY.Diversity of cyclic Di-GMP-binding proteins and mechanisms.Journal of Bacteriology,2016,198(1):32-46.
[19]Peng JL,Hao BH,Liu L,Wang SM,Ma BG,Yang Y,Xie FL,Li YG.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells.PLoS One,2014,9(4):e93626.
[20]Abd-El-Karem Y,Elbers T,Reichelt R,Steinbüchel A.Heterologous expression of Anabaena sp.PCC7120cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium(Ensifer)meliloti 1021.Applied Microbiology and Biotechnology,2010,89(4):1177-1192.
[21]Sch?per S,Krol E,Skotnicka D,Kaever V,Hilker R,S?gaard-Andersen L,Becker A.Cyclic di-GMP regulates multiple cellular functions in the symbiotic Alphaproteobacterium Sinorhizobium meliloti.Journal of Bacteriology,2015,198(3):521-535.
[22]Robledo M,Rivera L,Jiménez-Zurdo JI,Rivas R,Dazzo F,Velázquez E,Martínez-Molina,Hirsch AM,Mateos PF.Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces.Microbial Cell Factories,2012,11:125.
[23]Tian CF,Garnerone AM,Mathieu-Demazière C,Masson-Boivin C,Batut J.Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.Proceedings of the National Academy of Sciences of the United States of America,2012,109(17):6751-6756.
[24]Zhang YB,Wei C,Jiang WD,Wang L,Li CR,Wang YY,Dow JM,Sun WX.The HD-GYP domain protein RpfG of Xanthomonas oryzae pv.oryzicola regulates synthesis of extracellular polysaccharides that contribute to biofilm formation and virulence on rice.PLoS ONE,2013,8(3):e59428.
[25]Lovering AL,Capeness MJ,Lambert C,Hobley L,Sockett RE.The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.mBio,2011,2(5):e00163-11.
[26]Zhang Y,Kitajima M,Whittle AJ,Liu WT.Benefits of genomic insights and CRISPR-Cas signatures to monitor potential pathogens across drinking water production and distribution systems.Frontiers in Microbiology,2017,8:2036.
[27]Gao T,Meng Q,Gao HC.Thioesterase YbgC affects motility by modulating c-di-GMP levels in Shewanella oneidensis.Scientific Reports,2017,7:3932.