Ca^(2+)信号通路对本氏烟叶位介导的核盘菌抗性的影响

Effect of Ca(2+) signaling pathway on leaf position-associated resistance to Sclerotinia sclerotiorumin Nicotiana benthamiana.

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.

Summary 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 . necator than 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 . sclerotiorum is a serious world‐wide problem , resulting in a huge yield loss every year . On the other hand , the role of Ca 2 ＋ 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 benthamiana to S . 〈br〉 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 . 〈br〉 The effect of leaf position on the resistance of N . benthamiana to 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 Ca 2 ＋ channel inhibitors LaCl3 and NaVO3 on leaf position‐associated resistance , quantitative reverse transcriptase‐polymerase chain reaction （ qRT‐PCR） assay to probe the expression of three Ca2 ＋ signaling‐related genes NbCNGC20 , NbCA M TA3 and NbCML1 in leaves at different positions and virus‐induced gene silencing （ VIGS ） assay to explore the effect of the Ca 2 ＋signaling‐related gene NbCML1 on leaf position‐associated resistance to S . sclerotiorum in N . benthamiana .The results of inoculation experiments showed that the leaf position significantly influenced the resistance of N . benthamiana to S . sclerotiorum . The upper , middle and lower leaves of 12‐leaf‐stage plants formed lesions of 18 .0 mm , 13 .7 mm and 11 .9 mm 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 with 1 mmol/L LaCl3 and 50 μmol/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 Ca 2 ＋ signaling eliminate the leaf position‐associated resistance to S . sclerotiorum in N . benthamiana . Additionally , the expression of genes NbCNGC20 , NbCA M TA3 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 . benthamiana erases 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 .benthamiana to 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 Ca 2 ＋ signaling pathway , including NbCML1 , makes great contribution to the leaf position‐associated resistance to S . sclerotiorum in N . benthamiana . This finding provides new insights into molecular mechanisms underlying the leaf position‐associated resistance .