寄主植物中抑制十字花科黑腐病菌Ⅲ型分泌系统小分子化合物的鉴定
|
摘要
寻找抑制植物病原菌Ⅲ型分泌系统的植物源活性小分子化合物,是研发生物安全农药的重要途径之一。本研究采用水煮提取法从十字花科黑腐病菌寄主植物满身红萝卜中提取分离活性小分子化合物,利用高效液相-质谱联用解析出活性物质的单体结构。然后用荧光素酶基因luxAB构建融合报告系统以及定量PCR检测活性物质对十字花科黑腐病菌Ⅲ型分泌系统的抑制效果,最后采用剪叶接种和压渗接种的方法研究活性小分子物质对十字花科黑腐病菌的生防作用。研究表明,植物鞘氨醇和二氢鞘氨醇对十字花科黑腐病菌Ⅲ型分泌系统基因的转录表达有一定程度的抑制作用,但是对Ⅰ、Ⅱ、Ⅳ型分泌系统基因的表达没有明显的抑制作用。植物鞘氨醇在XCM1上影响菌的生长,而二氢鞘氨醇不影响菌的生长。同时,还发现这两种物质能显著降低Xcc在寄主植株满身红萝卜上的病害症状以及能够使Xcc在非寄主植物辣椒上引起过敏反应的能力丧失。该研究结果为深入研究小分子化合物对十字花科黑腐病菌Ⅲ型分泌系统的作用机制及后续开发植物源抑制剂提供了一定的理论依据。
Screening of host plants derived active small molecule substances inhibiting the type Ⅲ secretion system (T3SS) of phytopathogen is one of the important strategy for development of natural biosafety pesticides.In this study,the small molecule substances were extracted by boiling extraction from Chinese radish,the host plant of Xanthomonas campestris pv. campestris (Xcc). The monomer structure of the active substance was analyzed by high performance liquid chromatography-mass spectrometry. The inhibition effect of active substances on T3SS of Xcc was detected through bacterial luciferase report system and quantitative PCR (qPCR). The biocontrol effect of the active small molecules was studied by leaf-cutting inoculation and infiltration inoculation methods. The results showed that phytosphingosine and sphinganine exhibited stronger inhibition effects on the transcriptional expression of Xcc T3SS genes,while the expression of type Ⅰ,Ⅱ and Ⅳ secretion system genes were not suppressed. Phytosphingosine affects the growth of bacteria on XCM1,but sphinganine does not. In addition,these two substances can significantly reduce the disease symptoms of Xcc on host plant Chinese radish and attenuate HR induction of Xcc on non-host pepper ECW-10 R. The study will provide a theoretical base for further characterization of the interaction mechanism between small molecule compounds and Xcc T3SS and for the subsequent development of plant derived inhibitors.
Screening of host plants derived active small molecule substances inhibiting the type Ⅲ secretion system (T3SS) of phytopathogen is one of the important strategy for development of natural biosafety pesticides.In this study,the small molecule substances were extracted by boiling extraction from Chinese radish,the host plant of Xanthomonas campestris pv. campestris (Xcc). The monomer structure of the active substance was analyzed by high performance liquid chromatography-mass spectrometry. The inhibition effect of active substances on T3SS of Xcc was detected through bacterial luciferase report system and quantitative PCR (qPCR). The biocontrol effect of the active small molecules was studied by leaf-cutting inoculation and infiltration inoculation methods. The results showed that phytosphingosine and sphinganine exhibited stronger inhibition effects on the transcriptional expression of Xcc T3SS genes,while the expression of type Ⅰ,Ⅱ and Ⅳ secretion system genes were not suppressed. Phytosphingosine affects the growth of bacteria on XCM1,but sphinganine does not. In addition,these two substances can significantly reduce the disease symptoms of Xcc on host plant Chinese radish and attenuate HR induction of Xcc on non-host pepper ECW-10 R. The study will provide a theoretical base for further characterization of the interaction mechanism between small molecule compounds and Xcc T3SS and for the subsequent development of plant derived inhibitors.
引文
[1]Ho Y P,Tan C M,Li M Y,et al.The AvrB_AvrCdomain of AvrXccC of Xanthomonas campestris pv.campestris is required to elicit plant defense responses and manipulate ABA homeostasis[J].M olecular Plant-M icrobe Interactions,2013,26(4):419-430.
[2]Vicente J G,Holub E B.Xanthomonas campestris pv.campestris(cause of black rot of crucifers)in the genomic era is still a w orldw ide threat to brassica crops[J].Molecular Plant Pathology,2013,14(1):2-18.
[3]Galán J E,Collmer A.Type III secretion machines:bacterial devices for protein delivery into host cells[J].Science,1999,284:1322-1328.
[4]Hueck C J.Type III protein secretion systems in bacterial pathogens of animals and plants[J].M icrobiology and M olecular Biology Review s,1998,62(2):379-433.
[5]Qian W,Jia Y T,Ren S X,et al.Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv.campestris[J].Genome Research,2005,15(6):757-767.
[6]Kauppi A M,Nordfelth R,Uvell H,et al.Targeting bacterial virulence:inhibitors of type III secretion in Yersinia[J].Chemistry and Biology,2003,10(3):241-249.
[7]Gauthier A,Robertson M L,Lowden M,et al.Transcriptional inhibitor of virulence factors in enteropathogenic Escherichia coli[J].Antimicrobial Agents and Chemotherapy,2005,49(10):4101-4109.
[8]Pan N,Lee C,Goguen J.High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis[J].Advances in Experimental M edicine and Biology,2007,603:367-375.
[9]Pan N J,Brady M J,Leong J M,et al.Targeting type III secretion in Yersinia pestis[J].Antimicrobial Agents and Chemotherapy,2009,53(2):385-392.
[10]Felise H B,Nguyen H V,Pfuetzner R A,et al.An inhibitor of gram-negative bacterial virulence protein secretion[J].Cell Host and M icrobe,2008,4(4):325-336.
[11]Harmon D E,Davis A J,Castillo C,et al.Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis[J].Antimicrobial Agents and Chemotherapy,2010,54(8):3241-3245.
[12]Li Y,Hutchins W,Wu X,et al.Derivative of plant phenolic compound inhibits the type III secretion system of Dickeya dadantii via Hrp X/Hrp Y tw o-component signal transduction and Rsm systems[J].M olecular Plant Pathology,2015,16(2):150-163.
[13]Fan S,Tian F,Li J,et al.Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system[J].M olecular Plant Pathology,2017,18(4):555-568.
[14]Al-Reza S M,Rahman A,Ahmed Y,et al.Inhibition of plant pathogens in vitro and in vivo w ith essential oil and organic extracts of Cestrum nocturnum L.[J].Pesticide Biochemistry and Physiology,2010,96(2):86-92.
[15]Zhang X,Ma Z Q,Feng J T,et al.Review on research and development of botanical pesticides[J].Chinese Journal of Biological Control,2015,5(31):685-698.
[16]Gu L,Zhou S,Zhu L,et al.Small-molecule inhibitors of the type III secretion system[J].M olecules,2015,20(9):17659-17674.
[17]Tang X Y,Xiao Y M,Zhou J M.Regulation of the type III secretion system in phytopathogenic bacteria[J].Molecular Plant-Microbe Interactions,2006,19(11):1159-1166.
[18]Jiang B L,He Y Q,Cen W J,et al.The type IIIsecretion effector XopXccN of Xanthomonas campestris pv.campestris is required for full virulence[J].Research in M icrobiology,2008,159(3):216-220.
[19]Daniels M J,Barber C E,Turner P C,et al.Cloning of genes involved in pathogenicity of Xanthomonas campestris pv.campestris using the broad host range cosmid p LAFRl[J].The European M olecular Biology Organization(EM BO)Journal,1984,3(13):3323-3328.
[20]Jiang G F,Jiang B L,Yang M,et al.Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv.campestris[J].Brazilian Journal of M icrobiology,2014,44(3):945-952.
[21]Balakrishnan L,Hughes C,Koronakis V.Substratetriggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli[J].Journal of M olecular Biology,2001,313(3):501-510.
[22]Korotkov K V,Johnson T L,Jobling M G,et al.Structural and functional studies on the interaction of GspC and GspD in the type II secretion system[J].PLoS Pathogens,2011,7(9):e1002228.
[23]Oliveira L C,Souza D P,Oka G U,et al.VirB7 and VirB9 interactions are required for the assembly and antibacterial activity of a type IV secretion system[J].Structure,2016,24(10):1707-1718.
[24]Desveaux D,Singer A U,Wu A J,et al.Type IIIeffector activation via nucleotide binding,phosphorylation,and host target interaction[J].PLoS Pathogens,2007,3(3):3042-3048.
[25]He S Y,Nomura K,Whittam T S.Type III protein secretion mechanism in mammalian and plant pathogens[J].Biochimica et Biophysica Acta(BBA)-M olecular Cell Research,2004,1694(1-3):181-206.
[26]Baron C.Antivirulence drugs to target bacterial secretion systems[J].Current Opinion in M icrobiology,2010,13(1):100-105.
[27]Kline T,Felise H B,Sanowar S,et al.The type IIIsecretion system as a source of novel antibacterial drug targets[J].Current Drug Targets,2012,13(3):338-351.
[28]Duncan M C,Linington R G,Auerbuch V.Chemical inhibitors of the type three secretion system:disarming bacterial pathogens[J].Antimicrobial Agents and Chemotherapy,2012,56(11):5433-5441.
[29]Keyser P,Elofsson M,Rosell S,et al.Virulence blockers as alternatives to antibiotics:type III secretion inhibitors against Gram-negative bacteria[J].Journal of Internal M edicine,2008,264(1):17-29.
[30]Cheng J,Park T S,Chio L C,et al.Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans[J].M olecular and Cellular Biology,2003,23(1):163-177.
[31]Yin R,Gao H J,Zhao X Y.Biocontrol effect of Chaetomium globosum and sphingosine on Plasmodiaphora brassicae(in Chinese)[J].Journal of Huazhong Agricultural University(华中农业大学学报),2016,35(5):58-62.
[32]Peer M,Stegmann M,Mueller M J,et al.Pseudomonas syringae infection triggers de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana[J].The Federation of European Biochemical Societies(FEBS)Letters,2010,584(18):4053-4056.
[33]Siamer S,Guillas I,Shimobayashi M,et al.Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae dow n-regulates the sphingolipid biosynthetic pathw ay leading to grow th arrest[J].Journal of Biological Chemistry,2014,289(26):18466-18477.
[34]Jessen D L,Bradley D S,Nilles M L.A type IIIsecretion system inhibitor targets YopD w hile revealing differential regulation of secretion in calcium-blind mutants of Yersinia pestis[J].Antimicrobial Agents and Chemotherapy,2014,58(2):839-850.
[35]Wang D,Zetterstr9m C E,Gabrielsen M,et al.Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds[J].Journal of Biological Chemistry,2011,286(34):29922-29931.
[36]Yang F,Korban S S,Pusey P L,et al.Small-molecule inhibitors suppress the expression of both type IIIsecretion and amylovoran biosynthesis genes in Erwinia amylovora[J].M olecular Plant Pathology,2014,15(1):44-57.
[37]Bowlin N O,Williams J D,Knoten C A,et al.Mutations in the Pseudomonas aeruginosa needle protein gene pscF confer resistance to phenoxyacetamide inhibitors of the type III secretion system[J].Antimicrobial Agents and Chemotherapy,2014,58(4):2211-2220.
[38]Garrity-Ryan L K,Kim O K,Balada-Llasat J M,et al.Small molecule inhibitors of LcrF,a Yersinia pseudotuberculosis transcription factor,attenuate virulence and limit infection in a murine pneumonia model[J].Infection and Immunity,2010,78(11):4683-4690.
[2]Vicente J G,Holub E B.Xanthomonas campestris pv.campestris(cause of black rot of crucifers)in the genomic era is still a w orldw ide threat to brassica crops[J].Molecular Plant Pathology,2013,14(1):2-18.
[3]Galán J E,Collmer A.Type III secretion machines:bacterial devices for protein delivery into host cells[J].Science,1999,284:1322-1328.
[4]Hueck C J.Type III protein secretion systems in bacterial pathogens of animals and plants[J].M icrobiology and M olecular Biology Review s,1998,62(2):379-433.
[5]Qian W,Jia Y T,Ren S X,et al.Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv.campestris[J].Genome Research,2005,15(6):757-767.
[6]Kauppi A M,Nordfelth R,Uvell H,et al.Targeting bacterial virulence:inhibitors of type III secretion in Yersinia[J].Chemistry and Biology,2003,10(3):241-249.
[7]Gauthier A,Robertson M L,Lowden M,et al.Transcriptional inhibitor of virulence factors in enteropathogenic Escherichia coli[J].Antimicrobial Agents and Chemotherapy,2005,49(10):4101-4109.
[8]Pan N,Lee C,Goguen J.High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis[J].Advances in Experimental M edicine and Biology,2007,603:367-375.
[9]Pan N J,Brady M J,Leong J M,et al.Targeting type III secretion in Yersinia pestis[J].Antimicrobial Agents and Chemotherapy,2009,53(2):385-392.
[10]Felise H B,Nguyen H V,Pfuetzner R A,et al.An inhibitor of gram-negative bacterial virulence protein secretion[J].Cell Host and M icrobe,2008,4(4):325-336.
[11]Harmon D E,Davis A J,Castillo C,et al.Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis[J].Antimicrobial Agents and Chemotherapy,2010,54(8):3241-3245.
[12]Li Y,Hutchins W,Wu X,et al.Derivative of plant phenolic compound inhibits the type III secretion system of Dickeya dadantii via Hrp X/Hrp Y tw o-component signal transduction and Rsm systems[J].M olecular Plant Pathology,2015,16(2):150-163.
[13]Fan S,Tian F,Li J,et al.Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system[J].M olecular Plant Pathology,2017,18(4):555-568.
[14]Al-Reza S M,Rahman A,Ahmed Y,et al.Inhibition of plant pathogens in vitro and in vivo w ith essential oil and organic extracts of Cestrum nocturnum L.[J].Pesticide Biochemistry and Physiology,2010,96(2):86-92.
[15]Zhang X,Ma Z Q,Feng J T,et al.Review on research and development of botanical pesticides[J].Chinese Journal of Biological Control,2015,5(31):685-698.
[16]Gu L,Zhou S,Zhu L,et al.Small-molecule inhibitors of the type III secretion system[J].M olecules,2015,20(9):17659-17674.
[17]Tang X Y,Xiao Y M,Zhou J M.Regulation of the type III secretion system in phytopathogenic bacteria[J].Molecular Plant-Microbe Interactions,2006,19(11):1159-1166.
[18]Jiang B L,He Y Q,Cen W J,et al.The type IIIsecretion effector XopXccN of Xanthomonas campestris pv.campestris is required for full virulence[J].Research in M icrobiology,2008,159(3):216-220.
[19]Daniels M J,Barber C E,Turner P C,et al.Cloning of genes involved in pathogenicity of Xanthomonas campestris pv.campestris using the broad host range cosmid p LAFRl[J].The European M olecular Biology Organization(EM BO)Journal,1984,3(13):3323-3328.
[20]Jiang G F,Jiang B L,Yang M,et al.Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv.campestris[J].Brazilian Journal of M icrobiology,2014,44(3):945-952.
[21]Balakrishnan L,Hughes C,Koronakis V.Substratetriggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli[J].Journal of M olecular Biology,2001,313(3):501-510.
[22]Korotkov K V,Johnson T L,Jobling M G,et al.Structural and functional studies on the interaction of GspC and GspD in the type II secretion system[J].PLoS Pathogens,2011,7(9):e1002228.
[23]Oliveira L C,Souza D P,Oka G U,et al.VirB7 and VirB9 interactions are required for the assembly and antibacterial activity of a type IV secretion system[J].Structure,2016,24(10):1707-1718.
[24]Desveaux D,Singer A U,Wu A J,et al.Type IIIeffector activation via nucleotide binding,phosphorylation,and host target interaction[J].PLoS Pathogens,2007,3(3):3042-3048.
[25]He S Y,Nomura K,Whittam T S.Type III protein secretion mechanism in mammalian and plant pathogens[J].Biochimica et Biophysica Acta(BBA)-M olecular Cell Research,2004,1694(1-3):181-206.
[26]Baron C.Antivirulence drugs to target bacterial secretion systems[J].Current Opinion in M icrobiology,2010,13(1):100-105.
[27]Kline T,Felise H B,Sanowar S,et al.The type IIIsecretion system as a source of novel antibacterial drug targets[J].Current Drug Targets,2012,13(3):338-351.
[28]Duncan M C,Linington R G,Auerbuch V.Chemical inhibitors of the type three secretion system:disarming bacterial pathogens[J].Antimicrobial Agents and Chemotherapy,2012,56(11):5433-5441.
[29]Keyser P,Elofsson M,Rosell S,et al.Virulence blockers as alternatives to antibiotics:type III secretion inhibitors against Gram-negative bacteria[J].Journal of Internal M edicine,2008,264(1):17-29.
[30]Cheng J,Park T S,Chio L C,et al.Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans[J].M olecular and Cellular Biology,2003,23(1):163-177.
[31]Yin R,Gao H J,Zhao X Y.Biocontrol effect of Chaetomium globosum and sphingosine on Plasmodiaphora brassicae(in Chinese)[J].Journal of Huazhong Agricultural University(华中农业大学学报),2016,35(5):58-62.
[32]Peer M,Stegmann M,Mueller M J,et al.Pseudomonas syringae infection triggers de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana[J].The Federation of European Biochemical Societies(FEBS)Letters,2010,584(18):4053-4056.
[33]Siamer S,Guillas I,Shimobayashi M,et al.Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae dow n-regulates the sphingolipid biosynthetic pathw ay leading to grow th arrest[J].Journal of Biological Chemistry,2014,289(26):18466-18477.
[34]Jessen D L,Bradley D S,Nilles M L.A type IIIsecretion system inhibitor targets YopD w hile revealing differential regulation of secretion in calcium-blind mutants of Yersinia pestis[J].Antimicrobial Agents and Chemotherapy,2014,58(2):839-850.
[35]Wang D,Zetterstr9m C E,Gabrielsen M,et al.Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds[J].Journal of Biological Chemistry,2011,286(34):29922-29931.
[36]Yang F,Korban S S,Pusey P L,et al.Small-molecule inhibitors suppress the expression of both type IIIsecretion and amylovoran biosynthesis genes in Erwinia amylovora[J].M olecular Plant Pathology,2014,15(1):44-57.
[37]Bowlin N O,Williams J D,Knoten C A,et al.Mutations in the Pseudomonas aeruginosa needle protein gene pscF confer resistance to phenoxyacetamide inhibitors of the type III secretion system[J].Antimicrobial Agents and Chemotherapy,2014,58(4):2211-2220.
[38]Garrity-Ryan L K,Kim O K,Balada-Llasat J M,et al.Small molecule inhibitors of LcrF,a Yersinia pseudotuberculosis transcription factor,attenuate virulence and limit infection in a murine pneumonia model[J].Infection and Immunity,2010,78(11):4683-4690.