Ca~(2+)信号通路对本氏烟叶位介导的核盘菌抗性的影响
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摘要
组合采用药理学、分子生物学和反向遗传学等技术,分析叶位对本氏烟抗核盘菌(Sclerotinia sclerotiorum)的影响及其机制.结果表明,叶位显著影响本氏烟对核盘菌的抗性,随着叶位自上而下,该抗性逐渐增强.药理学分析结果显示,Ca2+通道抑制剂LaCl3和NaVO3处理消除了本氏烟叶片对核盘菌抗性的叶位间差异.定量反转录聚合酶链反应检测结果显示,3个Ca2+信号通路基因NbCNGC20、NbCAMTA3和NbCML1在本氏烟不同叶位叶片中的表达存在显著差异,随着叶位自上而下逐渐增加.病毒诱导的基因沉默(virus-induced gene silencing,VIGS)分析结果表明,钙调素类似蛋白基因NbCML1的沉默导致叶位介导的本氏烟对核盘菌抗病性的丧失.这些结果说明叶位对本氏烟抗核盘菌具有显著影响,揭示了包括NbCML1基因在内的Ca2+信号通路对叶位介导的核盘菌抗性的重要调控作用.
Leaf position significantly affects plant disease resistance.The majority of known examples demonstrate that plants are generally more susceptible to disease in lower leaves than upper leaves.Among them there are the resistances of cabbage to Hyaloperonospora parasitica,tomato to Phytophthora infestans and adlay to Bipolaris coicis.The exception is grapevine-Uncinula necator pathosystem where the lower leaves show a higher resistance to powdery mildew pathogen U.necatorthan the upper leaves.To date,the molecular mechanisms controlling leaf position-associated resistance remain unclear.Sclerotinia sclerotiorum(Lib.)de Bary is one of the most destructive plant pathogenic fungi in the world.The white mould/stem rot disease caused by S.sclerotiorumis a serious world-wide problem,resulting in a huge yield loss every year.On the other hand,the role of Ca2+ signaling pathway in plant disease resistance has been revealed.Nevertheless,whether it affects the leaf position-associated resistance is still unclear.The aim of this study was to investigate the effect of leaf position on resistance of Nicotiana benthamianato S.sclerotiorum and to further reveal the role of Ca2+ signaling pathway in this leaf position-associated resistance and thus to improve the understanding of the molecular mechanisms underlying this resistance.The effect of leaf position on the resistance of N.benthamianato S.sclerotiorum was analyzed by comparison among the resistance of leaves at various positions in the same plants,which was evaluated through inoculation experiments.Contribution of Ca2+ signaling pathway to this leaf position-associated resistance was demonstrated through three layers of assays,pharmacological assay to make clear effect of Ca2+ channel inhibitors LaCl3 and NaVO3 on leaf position-associated resistance,quantitative reverse transcriptase-polymerase chain reaction(qRTPCR)assay to probe the expression of three Ca2+signaling-related genes NbCNGC20,NbCAMTA3 and NbCML1 in leaves at different positions and virus-induced gene silencing(VIGS)assay to explore the effect of the Ca2+ signaling-related gene NbCML1 on leaf position-associated resistance to S.sclerotiorumin N.benthamiana.The results of inoculation experiments showed that the leaf position significantly influenced the resistance of N.benthamianato S.sclerotiorum.The upper,middle and lower leaves of 12-leaf-stage plants formed lesions of 18.0mm,13.7mm and 11.9mm at diameter,respectively.This demonstrates that the resistance increases in leaves of positions from upper to lower,which is in contrast to most of the reported pathosystems.When pre-infiltrated with1mmol/L LaCl3 and 50μmol/L NaVO3,leaves at different positions exhibited lesions of larger size in comparison with those of the untreated control plants,indicating that the two inhibitors of Ca2+ signaling eliminate the leaf position-associated resistance to S.sclerotiorum in N.benthamiana.Additionally,the expression of genes NbCNGC20,NbCAMTA3 and NbCML1 varied obviously in leaves at different positions,and all of them were increased from upper to lower leaves.Moreover,in NbCML1-silenced plants,all leaves of different positions displayed lesions of larger size,when compared with those of the non-silenced control plants,revealing that the silencing of NbCML1 in N.benthamianaerases the leaf position-associated resistance to S.sclerotiorum.In summary,the data of this study reveal that leaf position significantly affects the resistance of N.benthamianato S.sclerotiorum.In contrast to most of the reported pathosystems,this resistance is much stronger in lower leaves than in upper ones.Our finding demonstrates that the magnitude trend of leaf position-associated resistance in leaves of various positions is pathosystem-dependent.Furthermore,this study unveils that Ca2+ signaling pathway,including NbCML1,makes great contribution to the leaf position-associated resistance to S.sclerotiorumin N.benthamiana.This finding provides new insights into molecular mechanisms underlying the leaf position-associated resistance.
Leaf position significantly affects plant disease resistance.The majority of known examples demonstrate that plants are generally more susceptible to disease in lower leaves than upper leaves.Among them there are the resistances of cabbage to Hyaloperonospora parasitica,tomato to Phytophthora infestans and adlay to Bipolaris coicis.The exception is grapevine-Uncinula necator pathosystem where the lower leaves show a higher resistance to powdery mildew pathogen U.necatorthan the upper leaves.To date,the molecular mechanisms controlling leaf position-associated resistance remain unclear.Sclerotinia sclerotiorum(Lib.)de Bary is one of the most destructive plant pathogenic fungi in the world.The white mould/stem rot disease caused by S.sclerotiorumis a serious world-wide problem,resulting in a huge yield loss every year.On the other hand,the role of Ca2+ signaling pathway in plant disease resistance has been revealed.Nevertheless,whether it affects the leaf position-associated resistance is still unclear.The aim of this study was to investigate the effect of leaf position on resistance of Nicotiana benthamianato S.sclerotiorum and to further reveal the role of Ca2+ signaling pathway in this leaf position-associated resistance and thus to improve the understanding of the molecular mechanisms underlying this resistance.The effect of leaf position on the resistance of N.benthamianato S.sclerotiorum was analyzed by comparison among the resistance of leaves at various positions in the same plants,which was evaluated through inoculation experiments.Contribution of Ca2+ signaling pathway to this leaf position-associated resistance was demonstrated through three layers of assays,pharmacological assay to make clear effect of Ca2+ channel inhibitors LaCl3 and NaVO3 on leaf position-associated resistance,quantitative reverse transcriptase-polymerase chain reaction(qRTPCR)assay to probe the expression of three Ca2+signaling-related genes NbCNGC20,NbCAMTA3 and NbCML1 in leaves at different positions and virus-induced gene silencing(VIGS)assay to explore the effect of the Ca2+ signaling-related gene NbCML1 on leaf position-associated resistance to S.sclerotiorumin N.benthamiana.The results of inoculation experiments showed that the leaf position significantly influenced the resistance of N.benthamianato S.sclerotiorum.The upper,middle and lower leaves of 12-leaf-stage plants formed lesions of 18.0mm,13.7mm and 11.9mm at diameter,respectively.This demonstrates that the resistance increases in leaves of positions from upper to lower,which is in contrast to most of the reported pathosystems.When pre-infiltrated with1mmol/L LaCl3 and 50μmol/L NaVO3,leaves at different positions exhibited lesions of larger size in comparison with those of the untreated control plants,indicating that the two inhibitors of Ca2+ signaling eliminate the leaf position-associated resistance to S.sclerotiorum in N.benthamiana.Additionally,the expression of genes NbCNGC20,NbCAMTA3 and NbCML1 varied obviously in leaves at different positions,and all of them were increased from upper to lower leaves.Moreover,in NbCML1-silenced plants,all leaves of different positions displayed lesions of larger size,when compared with those of the non-silenced control plants,revealing that the silencing of NbCML1 in N.benthamianaerases the leaf position-associated resistance to S.sclerotiorum.In summary,the data of this study reveal that leaf position significantly affects the resistance of N.benthamianato S.sclerotiorum.In contrast to most of the reported pathosystems,this resistance is much stronger in lower leaves than in upper ones.Our finding demonstrates that the magnitude trend of leaf position-associated resistance in leaves of various positions is pathosystem-dependent.Furthermore,this study unveils that Ca2+ signaling pathway,including NbCML1,makes great contribution to the leaf position-associated resistance to S.sclerotiorumin N.benthamiana.This finding provides new insights into molecular mechanisms underlying the leaf position-associated resistance.
引文
[1]Poethig R S.Phase change and the regulation of developmental timing in plants.Science,2003,301(5631):334-336.
[2]Develey-Rivière M P,Galiana E.Resistance to pathogens and host developmental stage:A multifaceted relationship within the plant kingdom.New Phytologist,2007,175(3):405-416.
[3]Lazarovits G,Stossel P,Ward E W B.Age-related changes in specificity and glyceollin production in the hypocotyl reaction of soybeans to Phytophthora megasperma var.sojae.Phytopathology,1981,71:94-97.
[4]Kus J V,Zaton K,Sarkar R,et al.Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae.The Plant Cell,2002,14(2):479-490.
[5]Coelho P S,Valério L,Monteiro A A.Leaf position,leaf age and plant age affect the expression of downy mildew resistance in Brassica oleracea.European Journal of Plant Pathology,2009,125(2):179-188.
[6]Chang S W.Effects of plant age,leaf position,inoculum density,and wetness period on Bipolaris coicisinfection in adlays of differing resistance.Plant Disease,2003,87(7):821-826.
[7]Nelson H E.Bioassay to detect small differences in resistance of tomato to late blight according to leaf age,leaf and leaflet position,and plant age.Australasian Plant Pathology,2006,35(3):297-301.
[8]Doster M A,Schnathorst W C.Effects of leaf maturity and cultivar resistance on development of the powdery mildew fungus on grapevines.Phytopathology,1985,75(3):318-321.
[9]Hugot K,Riviere M P,Moreilhon C,et al.Coordinated regulation of genes for secretion in tobacco at late developmental stages:Association with resistance against oomycetes.Plant Physiology,2004,134(2):858-870.
[10]Boland G J,Hall R.Index of plant hosts of Sclerotinia sclerotiorum.Canadian Journal of Plant Pathology,1994,16(2):93-108.
[11]Reddy A S N,Ali G S,Celesnik H,et al.Coping with stresses:Roles of calcium-and calcium/calmodulin-regulated gene expression.The Plant Cell,2011,23(6):2010-2032.
[12]Li W,Xu Y P,Zhang Z X,et al.Identification of genes required for nonhost resistance to Xanthomonasoryzaepv.oryzae reveals novel signaling components.PLoS ONE,2012,7(8):e42796.
[13]Cai X Z,Wang C C,Xu Y P,et al.Efficient gene silencing induction in tomato by a viral satellite DNA vector.Virus Research,2007,125:169-175.
[14]Wang C C,Cai X Z,Wang X M,et al.Optimisation of Tobacco rattle virus-induced gene silencing in Arabidopsis.Functional Plant Biology,2006,33(4):347-355.
[15]程维舜,徐秋芳,黎飞,等.适于烟草脆裂病毒诱导的本氏烟基因沉默分析的对照载体构建.浙江大学学报:农业与生命科学版,2012,38(1):10-20.Cheng W S,Xu Q F,Li F,et al.Establishment of a suitable control vector for Tobacco rattle virus-induced gene silencing analysis in Nicotiana benthamiana.Journal of Zhejiang University:Agriculture and Life Sciences,2012,38(1):10-20.(in Chinese with English abstract)
[2]Develey-Rivière M P,Galiana E.Resistance to pathogens and host developmental stage:A multifaceted relationship within the plant kingdom.New Phytologist,2007,175(3):405-416.
[3]Lazarovits G,Stossel P,Ward E W B.Age-related changes in specificity and glyceollin production in the hypocotyl reaction of soybeans to Phytophthora megasperma var.sojae.Phytopathology,1981,71:94-97.
[4]Kus J V,Zaton K,Sarkar R,et al.Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae.The Plant Cell,2002,14(2):479-490.
[5]Coelho P S,Valério L,Monteiro A A.Leaf position,leaf age and plant age affect the expression of downy mildew resistance in Brassica oleracea.European Journal of Plant Pathology,2009,125(2):179-188.
[6]Chang S W.Effects of plant age,leaf position,inoculum density,and wetness period on Bipolaris coicisinfection in adlays of differing resistance.Plant Disease,2003,87(7):821-826.
[7]Nelson H E.Bioassay to detect small differences in resistance of tomato to late blight according to leaf age,leaf and leaflet position,and plant age.Australasian Plant Pathology,2006,35(3):297-301.
[8]Doster M A,Schnathorst W C.Effects of leaf maturity and cultivar resistance on development of the powdery mildew fungus on grapevines.Phytopathology,1985,75(3):318-321.
[9]Hugot K,Riviere M P,Moreilhon C,et al.Coordinated regulation of genes for secretion in tobacco at late developmental stages:Association with resistance against oomycetes.Plant Physiology,2004,134(2):858-870.
[10]Boland G J,Hall R.Index of plant hosts of Sclerotinia sclerotiorum.Canadian Journal of Plant Pathology,1994,16(2):93-108.
[11]Reddy A S N,Ali G S,Celesnik H,et al.Coping with stresses:Roles of calcium-and calcium/calmodulin-regulated gene expression.The Plant Cell,2011,23(6):2010-2032.
[12]Li W,Xu Y P,Zhang Z X,et al.Identification of genes required for nonhost resistance to Xanthomonasoryzaepv.oryzae reveals novel signaling components.PLoS ONE,2012,7(8):e42796.
[13]Cai X Z,Wang C C,Xu Y P,et al.Efficient gene silencing induction in tomato by a viral satellite DNA vector.Virus Research,2007,125:169-175.
[14]Wang C C,Cai X Z,Wang X M,et al.Optimisation of Tobacco rattle virus-induced gene silencing in Arabidopsis.Functional Plant Biology,2006,33(4):347-355.
[15]程维舜,徐秋芳,黎飞,等.适于烟草脆裂病毒诱导的本氏烟基因沉默分析的对照载体构建.浙江大学学报:农业与生命科学版,2012,38(1):10-20.Cheng W S,Xu Q F,Li F,et al.Establishment of a suitable control vector for Tobacco rattle virus-induced gene silencing analysis in Nicotiana benthamiana.Journal of Zhejiang University:Agriculture and Life Sciences,2012,38(1):10-20.(in Chinese with English abstract)