【作者】 冯勇；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2020， 博士

【摘要】 豆科植物（legume）能够与根瘤菌（rhizobia）产生特异性的相互作用,进而在根部形成高度特化的器官--根瘤（nodule）,这个过程也被称作根瘤共生（Root Nodule Symbiosis,RNS）。在根瘤中,宿主植物为根瘤菌提供碳源以及稳定的生存环境,根瘤菌则通过固氮酶的固氮作用将大气中游离的氮气（N₂）还原成氨（ammonia）以促进植物生长,二者互利互惠和谐共存。近20年以来的研究主要以模式豆科植物百脉根（Lotus japonicus）和蒺藜苜蓿（Medicago truncatula）为材料,通过正向遗传学和反向遗传学等方法鉴定到了约200个参与RNS的基因,极大的丰富了我们对共生信号转导机制的认识。其中共生受体激酶Sym RK是根瘤菌与豆科植物之间早期信号识别的一个关键基因,但是其调控机制尚不完善。本研究以百脉根为材料,利用蛋白相互作用、CRISPR基因敲除、植物遗传转化等技术探讨了Sym RK及其互作蛋白在根瘤共生早期的功能,主要结果如下:1. 利用百脉根根瘤菌MAFF303099、人工合成的细菌鞭毛蛋白N端22个保守氨基酸（flg22）分别处理百脉根植株,都能够激发百脉根产生相似的免疫防御反应。但是先用根瘤菌预处理植株后,再用flg22处理,植株会丧失产生免疫防御反应的功能。说明了百脉根与根瘤菌共生关系的建立能够抑制flg22诱发植物的免疫反应。2. 为了揭示根瘤菌与植物共生过程中,哪个功能基因具有抑制植物免疫防御反应的功能。在百脉根结瘤因子受体激酶NFR1、NFR5功能缺失突变体（nfr1、nfr5）和共生受体激酶Sym RK功能缺失突变体（ems61、symrk-409）中,利用根瘤菌预处理突变体植株后,再用flg22处理。研究表明,在nfr1和nfr5中,根瘤菌也抑制了flg22激发的植物免疫反应;而在2个symrk突变体（ems61,symrk-409）中,根瘤菌则不能抑制flg22激发的植物免疫反应。这一结果暗示根瘤菌的免疫抑制功能可能依赖于Sym RK而非结瘤因子的两个受体NFRs。3. 为了研究Sym RK参与根瘤菌介导抑制免疫反应的机制,我们以Sym RK胞内激酶结构域（Sym RK-CD）作为诱饵,用酵母双杂交（yeast two hybrid）的方法鉴定到一个能够与Sym RK互作的蛋白。该蛋白与拟南芥中的At BAK1（BRI1-Associated Kinase）蛋白质序列具有高度相似,氨基酸同源比对显示二者序列相似度高达82%。因此,将其命名为Lj BAK1。为了验证Lj BAK1的生物学功能,在拟南芥bri1-5背景下超表达Lj BAK1能够一定程度上促进下胚轴的生长,说明Lj BAK1可能与At BAK1都参与了BR信号通路。在Atbak1-4突变体超表达Lj BAK1也能部分回补其发育缺陷。Atbak1-4突变体对flg22处理不敏感,超表达Lj BAK1后flg22激发的MPK磷酸化能恢复到野生型的水平。进一步说明了Lj BAK1与At BAK1一样参与了flg22介导的免疫反应。4. 利用体内、体外蛋白质相互作用的技术手段,我们进一步证实了Lj BAK1的胞内激酶结构域与Sym RK间的相互作用,而其不能与Lj NFR1或Lj NFR5相互作用。说明Lj BAK1与Sym RK间的相互作用具有特异性,且这种相互作用在豆科和非豆科中都具有保守性。5. 在大肠杆菌中分别表达并纯化了Lj BAK1（His-Lj BAK1-CD）和Sym RK（MBP-Sym RK-CD）蛋白,并开展了一系列体外磷酸化实验,验证二者的激酶活性。结果显示二者的激酶结构域都具有自磷酸化和底物水平磷酸化活性,并且能相互磷酸化。同时发现Sym RK能够抑制Lj BAK1的自磷酸化和底物水平磷酸化活性,并且这种抑制作用依赖于Sym RK的激酶活性。为了进一步探索验证Lj BAK1激酶活性与其生物学功能之间的相关性,将Lj BAK1构建到带有（G₄S）₂-FLAG的载体上,导入野生型百脉根并得到稳定的转化植株。转基因植株的根段用flg22处理后,通过免疫沉淀分离Lj BAK1蛋白,分别用FLAG抗体和丝氨酸/苏氨酸/酪氨酸磷酸化的抗体检测。结果表明,flg22可以诱导Lj BAK1发生磷酸化;而用根瘤菌MAFF303099预处理后,磷酸化活性受到了抑制。说明根瘤菌能够抑制flg22介导的Lj BAK1的磷酸化。6. 利用CRISPR-Cas9基因编辑系统,我们得到百脉根bak1两个突变体植株（Ljbak1-1,ljbak1-2）,二者都会导致Lj BAK1蛋白的翻译提前终止。百脉根bak1突变体表型分析结果表明,bak1发育受阻并且对flg22的处理不敏感,在接种根瘤菌后突变体植株的侵染线增多、侵染密度增大。说明Lj BAK1在豆科植物百脉根与根瘤菌共生过程中具有负调控作用。同时,也揭示了豆科共生受体激酶Sym RK调控植物免疫防御反应的分子机制。7. 利用酵母双杂交和Bi FC的方法,我们发现Sym RK的两个互作蛋白SIP1（一种ARID型DNA结合蛋白）和SIE3（一种含有RING[Really Interesting New Gene]结构域的E3连接酶）能够相互作用,同时SIE3能够与自身结合并形成同源二聚体。8. 进一步的研究发现,SIE3的CRA结构域上的第266位的半胱氨酸残基（Cys266）对于其二聚体的形成十分重要,将Cys266突变掉后SIE3不能形成二聚体并且也丧失了其生物学功能,说明SIE3可能通过Cys266形成二硫键之后以二聚体的形式行使功能。更多还原

【Abstract】 Specific interactions between legume and rhizobia result in the formation of a highly specialized plant organ-the root nodule,and this process is also called Root Nodule Symbiosis（RNS）.In the nodules,the host cells provide organic carbon com-pounds to the rhizobia and guarantee a stable living environment,in return,rhizobia reduce the free nitrogen（N₂）in the atmosphere to ammonia by nitrogen fixation to promote plant growth,thus,both the plant host and bacterial symbiont appear to derive benefit.In the past 20 years,nearly 200 genes were identified involved in RNS through forward genetics and reverse genetics in the model legumes Lotus japonicus and Medi-cago truncatula.The symbiosis receptor-like kinase（Sym RK）is a key regulator for early signal recognition between rhizobia and legumes,but the regulatory mechanism is unclear.In this study,the functions of Sym RK and its interacting proteins in the early stage of symbiosis of root nodules were explored using protein interaction,CRISPR knockout,plant genetic transformation and other techniques in Lotus japonicus,and the results are list below:1. Both M.loti MAFF30309 and flg22 trigger immune responses in the roots of wild-type L.japonicus,but pretreatment with M.loti significantly suppresses host de-fense responses triggered by flg22;2. To assess genes involved in suppression of flg22-triggered host defense responses,loss-of-function mutants of Nod Factor Receptor-like Kinase 1 and 5（nfr1,nfr5）and Symbiosis Receptor-like Kinase（ems61,symrk-409）were pretreated with M.loti,followed by treatment with flg22.Suppression of flg22-triggered defense re-sponses were observed in nfr1 and nfr5 but not symrk mutant plants which indicates Sym RK,but not NFR1 or NFR5,is required for suppression of host defense responses during symbiosis.3. To reveal the mechanism of Sym RK involved suppression of flg22-triggered defense responses by pretreatment with M.loti.The kinase domain of Lj Sym RK was used as a bait to screen a yeast two-hybrid library for Lj Sym RK-interacting proteins.Among those candidates identified was a protein that shared high homology with the Arabidopsis leucine-rich repeat receptor kinase At BAK1.We cloned the full-length gene from L.japonicas referred to it as Lj BAK1.Ectopic expression of Lj BAK1 could partially rescue the defective phenotypes of bri1-5,and bak1-4,indicates that Lj BAK1is an ortholog of Arabidopsis BAK1 with similar biological functions regarding induc-tion of innate immunity and plant development.4. To confirm the interaction between Lj Sym RK and Lj BAK1,yeast two-hybrid and co-immunoprecipitation were carried out.Lj BAK1 specific interacts with Sym RK but not NFR1 or NFR5 in yeast cells,and this interaction were conserved among leg-umes and nonlegumes.5. To study the kinase activity of Sym RK and Lj BAK1,we expressed and purified recombinant proteins of Lj BAK1（His-Lj BAK1-CD）and Sym RK（MBP-Sym RK-CD）in E.coli.An in vitro kinase assay showed that both Lj Sym RK and Lj BAK1 had auto-phosphorylation and transphosphorylation activity and can phosphorylated each other.At the same time,Sym RK inhibits the kinase activity of Lj BAK1,and the kinase activ-ity of Sym RK is required for inhibitory effects on Lj BAK1 in vitro.To dissect the cor-relation between the kinase activity of Lj BAK1,two stable transgenic lines expressing Lj BAK1 with（G₄S）₂-FLAG tag were carried out in wild-type Lotus japonicus.Trans-genic plants were treated with flg22 followed by immunoprecipitation with FLAG an-tibody,the output was analysis by western-blot using anti-FLAG and anti-p Ser/Thr/Tyr antibodies.The results showed that flg22 induced phosphorylation of Lj BAK1 was sup-pressed by pretreatment with M.loti.6. We generated two independent Ljbak1 mutant lines using CRISPR-Cas9 editing with two guide RNAs targeting Lj BAK1,both Ljbak1 lines make truncated proteins with a frameshift mutation within the Lj BAK1 gene.Compared with wild-type MG20,Ljbak1 mutant plants showed a semi-dwarfed phenotype and insensitive to flg22,and the densities of Ips and Its in Ljbak1 mutant plants significantly increased.These results indicate Lj BAK1 negative regulates symbiosis process between legumes and rhizobia,and provide a partial mechanism of suppression of defense response for the action of Sym RK during the symbiosis.7. Using Y2H and Bi Fc protein interaction methods,we found Sym RK interacting E3 ligase（SIE3,a RING[Really Interesting New Gene]-containing E3 ligase）and Sym RK interacting protein 1（SIP1,an ARID-type DNA-binding protein）interact with each other.Moreover,SIE3 associated with itself to form a homodimer.8. The cysteine 266 residue（Cys266）in the CRA domain was found to be essential for SIE3 dimerization,and substitution Cys266 with Ser（C266S）abolished the di-meration and biological function of SIE3,these results suggested SIE3 may functions as a homodimer.更多还原