【作者】 李鑫；

【导师】 廖红东；

【作者基本信息】 湖南大学 ， 生物学， 2021， 硕士

【摘要】 自然环境下生长的植物受到多种微生物的影响,而内生菌作为一种有益菌,在为植物提供营养元素的同时也能从植物获取营养促进自身生长,并且不会引起植物明显的病害症状。内生菌在农业上的作用广泛,可以促进作物的生长发育。在定植的过程中,内生菌分泌的效应蛋白影响植物免疫响应以达到共生建立的目的。效应蛋白从靶向结合植物表面类受体激酶（receptor-like kinase,RLK）、调控转录表达等方面抑制植物免疫促进菌丝共生的建立。鉴定新的内生菌效应蛋白有利于深入研究共生关系建立机制。在多种微生物中鉴定到的快速碱化因子（rapid alkalization factor,RALF）类似物为我们研究内生菌效应蛋白奠定了理论基础。在本研究中,我们通过生物信息学鉴定到内生炭疽菌RALF-like蛋白,并对其进行了分析:（1）使用植物RALF典型的序列基序如YISY基序和RGC（5N）C基序在炭疽菌属中搜索到一条同源蛋白序列,并命名为Ct RALF,克隆并体外表达Ct RALF蛋白。外源添加Ct RALF可以抑制拟南芥（Arabidopsis thaliana）野生型Col-0的根长、促进胞外碱化,且抑制根长这一功能依赖于植物RALF受体FERONIA（FER）。这表明内生炭疽菌会分泌RALF-like效应蛋白,并具有植物RALF蛋白的典型生物活性特征。（2）构建FER胞外结构域（extracellular domain,ECD）的原核表达载体,通过蛋白质免疫共沉淀（Co-immunoprecipitation,CO-IP）、GST pull-down实验、竞争性结合实验等技术验证了Ct RALF与FER胞外结构域的相互作用关系。结果显示,与植物RALF一样,Ct RALF可以直接与FER相互作用。（3）通过实时定量实验分析,菌丝在侵染植物过程中可以表达Ct RALF,结合免疫印迹（Western Blot）技术分析Ct RALF对植物免疫信号的影响。结果显示,外源添加Ct RALF可以抑制植物活性氧（reactive oxygen species,ROS）积累、诱导短时间内的钙信号(Ca²⁺)迸发,但其影响程度比植物RALF轻;Ct RALF可以通过FER磷酸化丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）并降低茉莉酸（jasmonic acid,JA）信号途径中调节蛋白MYC2的稳定性。（3）以拟南芥野生型Col-0/C24和FER缺失突变体fer-4/srn为植物材料,通过菌丝侵染实验验证Ct RALF对菌丝定植的影响。不添加Ct RALF时,Col-0/C24根部附着的菌丝比fer-4/srn根部附着的菌丝多;外源添加Ct RALF后,Col-0根部附着的菌丝增多,fer-4根部附着的菌丝定植率没有明显变化。这说明Ct RALF通过FER通路促进菌丝的定植。综上所述,本研究发现内生炭疽菌可以分泌RALF-like蛋白Ct RALF,Ct RALF具有植物RALF典型活性,并通过RALF-FER信号通路影响植物免疫信号促进内生菌菌丝定植。更多还原

【Abstract】 Plants growing in the natural environment are affected by a variety of microorganisms.As a kind of beneficial bacteria,endophyte can obtain nutrients from plants to promote their own growth while providing nutrients for plants,and will not cause obvious disease symptoms of plants.Endophytes have a wide range of functions in agriculture and can promote the growth and development of crops.In the process of hypha colonization,the effector secreted by endophytes affect the immune response of plants to achieve the purpose of symbiosis establishment.The effectors can inhibit plant immunity and promote the establishment of hypha symbiosis from binding to plant receptor-like kinase(RLK)and regulating the transcription expression.The identification of new endophytic effectors is conducive to in-depth study of the mechanism of symbiosis establishment.The rapid alkalization factor(RALF)analogs identified in a variety of microorganisms have laid a theoretical foundation for us to study endophytic effectors.In this study,we identified and analyzed the endophytic Colletotrichum tofieldiae RALF-like protein through bioinformatics:(1)We used the typical motifs of plant RALF such as YISY motif and RGC(5N)C motif to search for a homologous protein sequence named Ct RALF.Ct RALF was cloned and expressed in vitro.Exogenous addition of Ct RALF can inhibit the root length of Arabidopsis wild-type Col-0 by the plant receptor FERONIA(FER)and promote extracellular alkalization.This indicated that the endophytic Colletotrichum tofieldiae secrete RALF-like effector which has the typical biological activity of plant RALF.(2)We constructed a prokaryotic expression vector of the extracellular domain of FER,and verified the interaction between Ct RALF and FER through Coimmunoprecipitation(CO-IP),GST pull-down,and competitive binding experiments.The results showed that Ct RALF can directly interact with FER like plant RALF.(3)Ct can express Ct RALF during the process of infecting plants through realtime quantitative PCR.We analyzed the effect of Ct RALF on plant immune signals combined with Western Blot.The results showed that exogenous addition of Ct RALF can inhibit the accumulation of reactive oxygen species(ROS)in plants and induce the burst of calcium signals within a short period of time,but its impact is less severe than that of plant RALF.Ct RALF can promote mitogen-activated protein kinase(MAPK)phosphorylation through FER and reduce the stability of the regulatory protein MYC2 in the jasmonic acid(JA)signaling pathway.(3)We used Arabidopsis wild-type Col-0/C24 and FERONIA function deficiency mutant fer-4/srn as plant materials to verify the effect of Ct RALF on hypha colonization through hypha infection experiments.Col-0 and C24 had more hyphae attached to the roots than fer-4 and srn roots without Ct RALF.After exogenous addition of Ct RALF,the hyphae attached to the roots of Col-0 increased,and the colonization rate of hyphae attached to the roots of fer-4 did not change significantly.This showed that Ct RALF can promote hyphae colonization through FER pathway.In summary,this study found that the endophytic Colletotrichum tofieldiae can secrete the RALF-like protein-Ct RALF.Ct RALF has the typical activity of plant RALF and affects plant immune signals through the RALF-FER signaling pathway to promote endophytic hypha colonization.更多还原