【作者】 陈桃；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2012， 博士

【摘要】 百脉根共生受体激酶基因SymRK是第一个被鉴定出来参与根瘤菌和菌根真菌信号转导的基因,是根瘤菌结瘤因子及菌根真菌菌根因子共生信号的交汇点,在共生信号传递过程中扮演重要的角色。本研究以SymRK为诱饵,从百脉根酵母双杂交的cDNA文库中分离到与之相互作用蛋白SIP2(SymRK-interacting protein2),SIP2是一个典型的有丝分裂原激活蛋白激酶激酶(MAPKK)。MAPK级联反应由MAPKKK、MAPKK和MAPK组成,该级联反应广泛存在于酵母、动物和植物等真核生物中,并参与各种生物或非生物的胁迫反应、激素反应、细胞分裂和发育过程的信号转导,MAPKK处于此级联的中间环节,通过磷酸化接收MAPKKK传来的信号,又通过磷酸化的方式将信号向下传递,这个级联将细胞外信号逐级放大并传导至细胞核,导致转录组的变化。本论文深入研究了MAPK级联反应在豆科植物共生信号传递过程中的作用机制和功能,其主要研究结果如下：1、共生受体激酶SymRK特异性地与MAPKK相互作用：利用酵母双杂交技术证实了百脉根MAPKK (SIP2)能与SymRK蛋白激酶结构域相互作用,不与其它的受体激酶NFR1及NFR5的蛋白激酶结构域相互作用,而百脉根MAPKK2和MAPKK10也不能与SymRK相互作用；同时,发现另一豆科植物苜蓿NORK (SymRK同系物)和SIMKK(SIP2同系物)也存在相互作用,并有交叉反应；体外pull-down进一步证实了SIP2不同区段与SymRK-PK间的互作；双分子荧光互补(BiFC)也验证了两个全长蛋白在烟草表皮细胞中的相互作用。这些结果表明SymRK与MAPKK特异性的相互作用在豆科植物中很保守,这两个蛋白在豆科植物中可能有着共同的作用机制。2、SIP2是一个有功能的蛋白激酶：SIP2编码一个丝/苏氨酸的蛋白激酶,它是否具有磷酸化和自磷酸化活性?在大肠杆菌中表达His-SIP2和GST-SIP2融合蛋白,体外磷酸化结果表明SIP2具有自磷酸化和底物水平磷酸化活性,表明SIP2是一个有功能的蛋白激酶,并且MPK6是其磷酸化底物,表明SIP2是一个典型的MAPKK;为了考察SymRK是否与SIP2互为磷酸化底物,首先将SymRK-PK和SIP2结合ATP位点进行点突变,得到SymRK-PK-KR和SIP2-KR两个磷酸化活性缺失的突变体,磷酸化结果表明SIP2不能磷酸化SymRK-PK,也不能被SymRK所磷酸化,即SymRK和SIP2不能互为磷酸化底物。3.SymRK抑制SIP2底物水平磷酸化的活性：为了进一步了解SymRK与SIP2间相互作用机制,我们在测定MPK6是SIP2磷酸化底物时,加入SymRK-PK或SymRK-PK-KR蛋白,考察SymRK对SIP2激酶活性的影响,结果表明随着SymRK-PK或SymRK-PK-KR浓度的增加,SIP2底物磷酸化活性受到抑制,并有很明显的剂量效应,在这个体系中加入NFR1-PK或BSA对SIP2磷酸化活性没有影响,表明SymRK特异性地抑制SIP2磷酸化MPK6的活性。4、SIP2在百脉根中的表达特征及亚细胞定位：鉴于SymRK在共生早期呈组成型表达,SIP2的表达是否呈现与SymRK一样的表达模式?分别收集接种和未接种根瘤菌的百脉根幼根、茎、叶、瘤等材料,利用实时荧光定量PCR检测了SIP2基因在百脉根不同组织中的表达特征,研究结果表明,在接种和未接种根瘤菌的百脉根幼根、茎、叶、瘤中,SIP2表达水平差别不大,呈组成型表达；通过发根农杆菌介导的毛根转化技术,将SIP2：：GFP融合子导入百脉根根中,在激光共聚焦显微镜下观察到SIP2：：GFP融合蛋白定位在细胞质和细胞膜,而单独的GFP蛋白在细胞质、膜与核中都有荧光信号；利用基因枪技术在洋葱表皮细胞中定位,通过质-壁分离处理后,SIP2：：GFP融合蛋白的荧光信号主要集中在细胞膜和细胞质,而GFP对照的荧光遍布整个胞质,充分证明SIP2定位在细胞膜和细胞质。5、百脉根SIP2调控根瘤菌侵染及根瘤原基的形成：为了鉴定SIP2在共生过程中的生物学功能,我们构建了两个基因沉默载体,通过毛根转化导入百脉根根中,在根瘤菌存在条件下进行盆栽试验,4周后通过荧光定量PCR测定SIP2基因的表达水平并观察植株结瘤表型,结果显示,抑制SIP2基因表达导致植株结瘤能力显著下降,侵入线和根瘤原基形成的效率显著降低；进一步分析表明SIP2-RNAi毛根中与早期根瘤菌侵染相关的3个标记基因的表达都受到不同程度的抑制,而其它两种MAPKKs的表达不受影响,这表明豆科植物SIP2参与根瘤菌早期侵染和根瘤原基的形成,在豆科植物与根瘤菌共生体形成过程中起调控作用。6、SymRK-SIP2的相互作用不影响菌根真菌与植物的共生：利用SIP2-RNAi毛根检测AM真菌的感染,结果显示RNAi-1和RNAi-2植株的毛根与转化空载体毛根对照一样都能被AM真菌感染并在细胞内形成丛枝,表明SIP2基因与菌根共生体形成无关,而是特异地调控根瘤共生体的形成。更多还原

【Abstract】 SymRK (symbiosis receptor-like kinase) is first cloned to recognition of both arbuscular mycorrhizal fungal and nitrogen-fixing rhizobial bacteria, likely active near the junction of fungal and rhizobial signaling cascades. SymRK is required for an early signal transduction pathway in the rhizobium-legume symbiosis. In our study, we used SymRK kinase domain as a bait to screen a yeast two-hybrid cDNA library prepared from early rhizobium-inoculated Lotus roots, one cDNA encoding a novel protein was designated as SIP2(SymRK-interacting protein2), which is a typical MAP kinase kinase (MAPKK). MAPK cascades are minimally composed of three kinase modules, which are involed in the regulation of development, growth, differentiation, programmed cell death, hormonal and stress response. Signaling through MAPK cascades is a fundamental and conserved process in animals, plants and yeast. MAPKK is in the middle part of MAPK cascades, needed to be activatied by upstream receptors MAPKKKs through phosphorylation, and phosphorylate MAPKs as downstream targets. Extracellular signal progressively amplified and transmitted to the cell nucleus through MAPK signaling cascades, led to changes in the transcriptone. This work studied the role and mechanism of MAPK signaling cascade during the rhizobium-legume symbiosis signaling pathway in Lotus japonicus. The main results are as follows:1. SymRK is specific interact with MAPKK. Using the yeast two-hybrid system, MAPKK (SIP2) interacted with the protein kinase domain of SymRK, but didn’t interact with protein kinase domain of NFR1or NFR5. Other MAPKKs, such as MAPKK2and MAPKK10from L. japonicus didn’t interact with SymRK neither. In addition, the interaction is conserved in medicago. NORK from alfalfa is the ortholog of SymRK interact with SIMKK, which is the ortholog of SIP2from alfalfa, the cross interactions among them. We further verified the interactions between SIP2constructs and SymRK-PK using an in vitro protein-protein pull-down assay. The technique of BiFC (bimolecular fluorescence complementation) confirms this interaction in Nicotiana benthamiana epidermis leaves. These results indicate that the interaction between SymRK and MAPKK is specific and conserved in the legume, the two proteins may have a common mechanism in the leguminous plants. 2. SIP2is a functional protein kinase. SIP2is a MAPKK, did it has autophosphorylate or transphosphorylate a protein substrate activity? Purified GST-SIP2and His-SIP2proteins were subjected to in vitro kinase assays using casein as a substrate, after autoradiography, we detected that SIP2was able to autophosphorylate itself and phosphorylate casein. SIP2can use MPK6as a substrate for phosphorylation, so SIP2is a typical MAPKK. To test if SymRK could be phosphorylated by SIP2or SIP2could be phosphorylated by SymRK. The kinase activity of SymRK and SIP2had to be abolished, we created kinase negative forms by replacing the absolutely conserved K residue with R in the ATP-binding site. The results showed that SIP2could not phosphorylate SymRK-PK-KR and SymRK failed to phosphorylate the SIP2-KR, these suggesting that SymRK is neither a potential phosphorylation target nor a kinase source of S1P2.3. SymRK is an inhibitor of SIP2kinase. To learn more about the interaction mechanism between SymRK and SIP2, we added an increasing amount of either kinase-active (SymRK-PK) or kinase-negative SymRK (SymRK-PK-KR) as an effector to the kinase-active SIP2assays in the presence of MPK6-KR as a substrate. The results showed that the kinase activity of SIP2decreased as the increase of SymRK in a dose-dependent manner. When added an increasing amount of NFR1-PK or bovine serum albumin (BSA) to the SIP2kinase assays, we did not observe any inhibitory effect on the SIP2kinase activity, suggesting that SymRK is a specific inhibitory effect on the kinase activity of SIP2towards the MPK6substrate.4. Expression of SIP2gene and subcellular localization of SIP2protein. Lotus SymRK is constitutively expressed in early symbiosis signaling pathway, we asked if the SIP2expression patterns similar with SymRK. In this study, young roots, stem, leaves and nodules were harvested at different time points after inoculation with M. loti MAFF303099, the expression levels of SIP2were measured using real-time PCR. Our results showed that SIP2was expressed in all tissues tested, including roots, stem, leaves and nodules, did not observe significant changes in SIP2mRNA level, SIP2is constitutively expressed. Through Agrobacterium rhizogenes LBA1334-mediated transformation, GFP:SIP2expressed in Lotus hairy roots, the fluorescence was observed under a confocal laser scanning microscope, GFP::SIP2localized to the plasma membrane and cytoplasm, GFP alone was used as a control, which was distributed in the cytoplasm and nucleus. The plasmid GFP::SIP2was delivered to the onion epidermal cells via particle bombardment. After plasmolysis, the fluorescence signal was observed in the cytoplasm and plasma membrane, while the GFP control fluorescence throughout the cytoplasm. We conclude that the SIP2protein is localized to the plasma membrane and cytoplasm.5. Lotus SIP2regulate of Rhizobium infection and nodule primordia formation. To identify the SIP2biological functions in the symbiotic process, two RNAi constructs were used to generate L. japonicus hairy roots via A. rhizogenes LBA1334infection, the hairy roots were inoculated with M. loti MAFF303099to induce nodule formation and their nodulation phenotypes were analyzed4weeks post inoculation. The suppression of SIP2expression was confirmed using real-time PCR amplification of a region in the SIP2mRNA. The average nodule number per root of SIP2-RNAi were significantly lower than that of the control hairy roots, IT formation and nodule initiation appeared to be impaired by the knockdown expression of SIP2in RNAi hairy roots. The expression levels of SIP2and three marker genes for infection thread and nodule primordial formation were down-regulated drastically, while the expression of two other MAPKKs genes were not altered. These observations demonstrate an essential role of SIP2in the early symbiosis signaling and nodule organogenesis.6. SymRK-SIP2interaction is not required for arbuscular mycorrhizal colonization. We infected the SIP2-RNAi hairy roots with Glomus intraradices, a common arbuscular mycorrhizal fungus. There was no observable difference in the efficiency of hyphal and arbuscular colonization between SIP2-RNAi hairy roots and the control hairy roots expressing the empty vector. Therefore, we conclude that SymRK-SIP2interaction is specific for the root response to Rhizobium infection and is not required for arbuscular mycorrhizal colonization.更多还原