【作者】 包菲；

【导师】 杨淑华；

【作者基本信息】 中国农业大学 ， 植物学， 2014， 博士

【摘要】 病害和极端温度是降低农作物产量和品质的两大因素。植物的抗病防卫机制在长期与病原体的较量过程中进化得复杂而严密。温度作为重要的环境因素之一,影响着植物生长发育的同时,也调节着植物的抗病防卫反应。R蛋白是介导植物识别并抵抗病原菌的一类重要抗病蛋白,一些R蛋白可以受低温诱导激活,比如,拟南芥中的SNCl和烟草中的N蛋白,它们在NB-ARC结构域特殊位点的突变造成R蛋白在22℃激活,植株叶片表现细胞死亡等抗病防卫反应激活的表型。而拟南芥的CHS2/RPP4(Recognition of Hyaloperonospora Parasitica4)也是这一类蛋白。突变体chs2(rpp4-1d)在R蛋白RPP4的NB-ARC结构域处的功能获得性突变,导致在低温条件下抗病防卫反应组成型激活,植株表现卷缩、矮小,叶片细胞死亡,严重至整株植物死亡的冷敏感表型。为了进一步研究rpp4介导的冷响应和抗病防卫反应之间的关系,我们筛选了rpp4-1d的抑制子。在rpp4-1d的EMS诱变库中筛选到了抑制子rarl、sgt1b、hsp90.2和hsp90.3。突变体hsp90基本抑制了rpp4-1d的低温诱导防卫反应激活和相关的冷敏感表型。突变的hsp90蛋白对正常HSP90的功能有干扰作用,并且这一干扰作用有剂量依赖效应。突变体hsp90在由RPM1(Resistance to Pseudomonas Maculicola1)、RPS4(Resistant to P. Syringae4)和RPP4介导的病原菌抗病反应中表现出比野生型较易感病的表型。但是,这些R蛋白介导的抗病反应对HSP90的依赖程度是不一样的。已有研究报道,伴侣蛋白复合体RAR1-SGT1B-HSP90可以与一些R蛋白相互作用,是维持R蛋白正常结构和生理功能所必需的(Hubert et al.,2003; Takahashi et al.,2003; Zhang et al.,2004)。本研究也证明野生型的RPP4和突变型的rpp4蛋白都能够与HSP90蛋白互相作用形成蛋白复合体,并且低温不影响rpp4与HSP90之间复合体的形成。在sp90突变体中,rpp4蛋白的稳定性也没有受到影响。另外,RPP4和rpp4蛋白在正常温度条件下在细胞质和细胞核中都有积累,但是与RPP4不同,低温会降低rpp4突变蛋白在细胞核中的积累。在hsp90突变体中,rpp4蛋白低温细胞核积累降低的现象也没有改变。对rpp4-1d的基因内抑制子进行遗传分析,结果表明TIR结构域可以部分激活抗病反应,而NB-ARC和LRR结构域对RPP4介导的温度依赖的防卫反应信号转导有重要的作用。另外,rpp4-1d的冷敏感表型基本不依赖WRKY70和MOS (modifier of snc1)基因。综合以上结果,本研究证明RPP4介导的冷响应和抗病信号都依赖HSP90蛋白,HSP90与rpp4蛋白在体内以复合体形式存在,hsp90功能的缺失不影响rpp4蛋白的稳定性和亚细胞定位。rpp4的作用机制与同源蛋白sncl不同。rpp4介导的冷敏感细胞死亡类似于抗病过程中的超敏反应。突变体hsp90编码的突变蛋白对正常HSP90的功能有干扰作用。它不仅抑制了rpp4介导的低温信号响应,而且影响了多个R蛋白介导的抗病防卫反应。本研究对探索温度对植物抗病的影响提供了实验证据,给研究多种形式的R蛋白介导的抗病机理提供了新的线索。更多还原

【Abstract】 Disease and extreme temperature are negative factors that affect the yield and quality of crops. During the long time competing with pathogens, plants have developed complicated mechanisms to defend themselves against disease. And this plant defense response can be regulated by temperature, an important factor of environment that affects plant growth and development. In the course of identifying pathogen, R proteins play an important role. Some R proteins can be activated under low temperature, such as SNC1in Arabidopsis and N protein in tobacco. A specific site mutations in NB-ARC domain of SNC1or N protein cause R proteins to be activated and the leaves to perform cell death at22℃. The CHS2/RPP4(Recogition of Hyaloperonospora Parasitica4) in Arabidopsis is also this kind of R protein. We found that a gain-of-function mutation in NB-ARC domain of RPP4leads to constitutive activation of the defense response under low temperature. And this rpp4-1d mutant exhibits chilling sensitive phenotypes, including curling, dwarfism, cell death, or even the death of whole plant.To further study the relationship between rpp4-mediated disease resistance and cold response, the suppressors of rpp4-1d was screened from an EMS mutagenesis library of rpp4-1d mutant. Suppressors rar1, sgt1b, hsp90.2and hsp90.3have been identified. The hsp90mutants largely suppress the activation of defense response and chilling sensitive phenotypes of rpp4-1d. The mutated hsp90could interfere with the wild-type HSP90in a dose-dependent manner. The hsp90mutants exhibited compromised RPM1(Resisance to Pseudomonas Maculcola1)-, RPS4(Resistant of Pseudomonas Syringae4)-and RPP4-mediated pathogen resistance, compared to the wild-type. However, the requirement degrees of HSP90in the pathogen resitance mediated by these R proteins are different.It has been reported that chaperonin complex RAR1-SGT1B-HSP90could interact with some R proteins to maintain the normal structures and physiological functions of proteins. Our study also proved the both wild-type RPP4and the mutated form rpp4can interact with HSP90to form protein complexes. And the formation of this complex could not be affected by low temperature. Further more, the stability of rpp4protein in hsp90mutant had not changed as well. RPP4and rpp4proteins were localized in both cytoplasm and nucleus under normal temperature, Nevertheless, not like RPP4, low temperature reduced the accumulation of rpp4protein in nucleus, which was not affected in hsp90mutants. Genetic analysis of the intra-genic suppressors of rpp4-1d revealed the auto-activation of TIR domain and the important functions of the NB-ARC and LRR domains of RPP4in temperature-dependent defense signaling. In addition, the chilling sensitivity of rpp4-1d was largely independent of the WRKY70or MOS (Modifier of snc1) genes.In summary, this study suggests that RPP4-mediated disease resistance and cold response both depend on HSP90proteins. HSP90and rpp4proteins form a complex in vivo. The mutated hsp90does not affect the stability and subcellular localization of rpp4protein. The mechanisms of rpp4and sncl-mediated responses are different. The chilling-sensitive cell death mediated by rpp4is similar to the hypersensitivity in disease resistance. The mutated hsp90protein has interfering effect on the wild-type HSP90, which not only inhibits rpp4-mediated chilling sensitive phenotypes, but also reduces R protein-mediated defense resistance. Our research has provided experimental evidence to the influence of temperature on disease resistance and a new clue to study various mechanisms of R protein-mediated disease resistance.更多还原