CaROP1与HPR互作在辣椒高温高湿下抗青枯病中作用及其机制分析

Pepper(Capsicum annuum) is an important vegetable and typical Solanaceae with various soil-borne diseases, and these disease tend to be more serious under high temperature and high humidity.The potential application of dissection of molecular mechanism of pepper disease resistance under high temperature and high humidity is the genetic improvement of pepper disease rsistance. Our provious studies suggested that the overexpression of CaROP1 and DN-CaROP1 enhanced significantly disease resistance under high temperature and high humidity,suggesting its role as a key molecular swatch in the immunity signaling and by yeast two hybird system analysis, DN-CaROP1 was found to interact with HPR, a important protein involves in photo respiration and also possibly play a role in plant immunity by previous studies. In the present study, the possible interaction between CA-CaROP1,DN-CaROP1 and CaROP1 with HPR will be confirmed by CoIP and BiFC, and the effect of this interaction on enzyme activity of HPR, the subcellular localization, the transcriptional expression of HPR agasinst high temperature and high humidity as well as Ralstonia solanacearum inoculation will also be analysized. By overexpression in transgenic Nicotiana benthamiana or transient overexpression and VIGS in pepper plants, the function of HPR and its interaction with CaROP1 in pepper diseases resistance under high temperature and high humidity will also be studied. The results of this project can not only indentify the the role of HPR in disease resistance under high temperature and high humidity mediated by CaROP1, but also put forward new molecular mechanism of pepper disease resistance.

辣椒是重要蔬菜和典型茄科植物，土传病害多，高温高湿下发病严重导致减产。高温高湿下抗病机制剖析是开展辣椒抗病遗传改良的重要基础。我们前期发现CaROP1是辣椒高温高湿下抗病重要分子开关，其DN突变体超表达可显著提高烟草高温高湿下的抗病水平，经酵母双杂交发现并经CoIP验证DN-CaROP1可与羟基丙酮酸还原酶（HPR）互作，后者据前人报道与植物耐高温和抗病相关。本项目拟进一步通过BiFC分析HPR与CaROP1互作在细胞中发生部位及对HPR酶活性的影响，通过qPCR分析 HPR在高温高湿、青枯菌侵染以及其它逆境作用下的转录表达，通过在烟草中超表达（或辣椒植株上瞬间超表达）和VIGS介导的基因沉默分析HPR及其与CaROP1互作在CaROP1介导的辣椒高温高湿下抗病中的作用。研究结果有利于明确HPR在CaROP1介导的辣椒高温高湿下抗病中的作用及机制，为辣椒高温高湿下抗病遗传改良提供依据。

摘要：辣椒是重要蔬菜和典型茄科植物，土传病害多，高温高湿下发病严重导致减产，高温高湿下抗病机制剖析是开展辣椒抗病遗传改良的重要基础。我们前期研究发现CaHPR1与CaROP1功能关联,可能在辣椒耐高温和抗病中起重要的作用。本项目通过转基因、病毒诱导基因沉默等功能基因组学手段进一步研究了CaHPR1的功能及其表达调控机制，结果表明：（1） CaHPR1分布于整个细胞，其表达受到高温处理处理和青枯菌接种侵染的诱导；与CaROP1的功能相似的是， CaHPR1超表达可显著提高转基因烟草的耐高温水平以及耐高温相关标记基因的表达，而其基因沉默则导致辣椒植株耐高温水平降低，（2）CaHPR1不仅可与DN-CaROP1互作，还可与CA-CaROP1互作，但上述互作在辣椒耐高温高湿和抗病中的作用还有待进一步研究予以证实；（3）常温下感病的辣椒植株中，CaHPR1主要受CaWRKY40的直接调节，而在高温高湿和感染病原菌的作用下，CaWRKY40在CaVAMP72等作用下转而直接调节CaEBS2,而由CaEBS2直接调节CaHPR1的转录表达。这些结果表明，CaHPR1在辣椒耐高温高湿和抗病及辣椒高温高湿下抗病中可能都起到重要的作用。鉴于CaEBS2含有BAH和PHD两个表观遗传学调节相关的结合域，暗示CaHPR1转录表达涉及到CaEBS2介导的表观遗传学机制，为进一步深入剖析辣椒高温高湿下的抗病机制提供了有价值的研究线索。