【作者】 李萍；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2020， 博士

【摘要】 水稻白叶枯病是水稻种植中的一类重要细菌病害。稻黄单胞菌分泌的效应蛋白对于病害的发生极为重要,本文从两个方面介绍了水稻白叶枯病菌效应子PthXo1转运的机制,从水稻水通道蛋白和水稻核输入蛋白入手,阐明了效应蛋白PthXo1从细胞质转运到水稻细胞核的过程中水稻水通道蛋白和核输入蛋白对于PthXo1转运所发挥的调控作用。革兰氏阴性植物病原细菌主要是通过Ⅲ型分泌系统将有毒性或无毒性的效应蛋白转运到寄主细胞内从而引发一系列的抗病或感病性互作反应。许多植物致病菌依赖于向植物细胞中分泌毒性的类似转录激活效应(TALE)蛋白来激活寄主植物的感病基因的表达而导致植物发病。这一过程需要依赖于TALE与植物细胞核中寄主靶标基因启动子上的特定元件的结合。稻黄单胞菌(Xanthomonas oryzae pv.oryzae,Xoo)分泌的效应蛋白TALEs可以定位在寄主植物的细胞核内,结合靶标基因的启动子区域进而发挥其转录调控的作用。水通道蛋白(aquaporins,AQPs)是可以促进水和中性小分子物质跨大多数生物的生物膜运输的膜通道蛋白质。在稻黄单胞菌侵染水稻的过程中,水稻中的水通道蛋白质在侵染过程中发挥着重要的作用,本研究发现,OsPIP1家族的蛋白质OsPIP1;3可以影响白叶枯病菌在侵染水稻时在水稻叶片上的定殖以及促进TALE效应蛋白的转运。经前期筛选,发现了 harpin蛋白与水稻的水通道蛋白之间存在着相互作用。1水稻水通道蛋白调控效应子PthXo1转运机制水通道蛋白可以促进水和中性小分子物质跨生物膜运输的膜通道蛋白质。在本课题组前期的研究基础上,已证实水稻水通道蛋白I家族中的OsPIP1;3和白叶枯病原菌的harpins蛋白hpal互作。但Hpa1Xoo-OsPIP1;3二者特异性互作的生物学功能仍然未曾有报道。在本章节研究中,我们在已构建好的沉默株系、过表达株系以及TALEN敲除突变体上分别测定了 PthXo1的转运作用。通过水稻叶片含菌量测定以及荧光定量法进一步测定了 PthXol的靶标基因Os8N3表达检测等手段探究不同OsPIP1;3转基因株系对水稻白叶枯病菌存在抗感性差异的调控机理。结果表明,通过接种白叶枯病菌PXO99A菌株在野生型水稻品种日本晴以及OsPIP1;3过表达、OsPIP1;3敲除或沉默株系上引起的水渍状病斑发病程度来测定其致病性。与野生型菌株PXO99A相比较,hpa1基因缺失突变体菌株在不同OsPIP1;3转基因株系上引起的水渍状病斑发病症状较对照明显的减弱。我们应用传统的腺苷酸环化酶(adenylate cyclase,Cya)报告系统进一步对PXO99A的主要毒力TAL效应蛋白PthXol的转运过程进行了检测。进一步明确了 PthXo1-cya融合蛋白从细菌细胞到水稻细胞质中的转位过程。本研究除了测定二者的生物学功能之外,还进一步的分析了其关键的互作功能域,通过利用水稻东格鲁杆状病毒Rice tungro bacilliform virus(RTBV)介导的过表达系统,分别构建了 OsPIP1;3和OsPIP1;1的LE功能域互换的水稻株系。在不同的功能域替换株系上测定了 PthXol的转运,阐明了 Hpa1Xoo-OsPIP1;3二者起关键互作的作用位点在OsPIP1;3的LE功能域。综上,Hpa1Xoo-OsPIP1;3二者的互作影响TAL效应蛋白PthXo1从白叶枯病菌向水稻胞内的转运过程,进而调控水稻水通道蛋白与白叶枯病菌的互作,影响水稻白叶枯菌的致病性。2水稻白叶枯病菌TAL效应子PthXo1在水稻中跨核运输机制在稻黄单胞菌分泌的效应蛋白中,有一类被称为TAL效应子(Transcription Activator-Like Effectors,简称TALEs),可定位到寄主细胞核内,通过结合靶标基因的启动子区域进行转录调控,从而引起寄主的感病或抗病反应。在此过程中,TALEs的核质转运是其能否发挥调控作用的关键步骤。通过酵母双杂交和双分子荧光互补试验,筛选与TAL效应子PthXo1互作的水稻核输入蛋白质(16个水稻核输入相关蛋白),筛选得知,效应子PthXo1可以与水稻核输入蛋白OsIMPα1a和OsIMPα1b互作。为了进一步的研究二者互作的生物学意义,运用水稻东格鲁杆状病毒系统构建了OsIMPα1a和OsIMPα1b的过表达突变体,对其致病力进行了测定,结果表明,过表达OsImpα1a和OsImpα1b可以促进TAL效应子PthXo1在水稻上的毒力作用。应用烟草脆裂病毒体系在烟草上沉默了OsImpα的同源基因NbImpα,研究发现NbImpα影响烟草的生长,并且使得黄单胞菌在非寄主烟草上引起的过敏反应加重。进一步说明PthXol的跨核转运过程中水稻核输入蛋白质OsIMPαla和OsIMPαlb发挥着重要作用。这一转运过程对于水稻黄单胞菌诱发的靶向感病基因的表达是至关重要。3 OsImpα1a和OsImpα1b转基因水稻的构建与鉴定在水稻的核输入蛋白质家族中,已经证实了水稻IMPα家族的两个蛋白质可以和黄单胞菌的TAL效应子PthXol互作。本文利用CRISPR-Cas9技术,成功构建了CRISPR-Cas9基因编辑骨架载体,对筛选的两个互作蛋白进行了基因编辑,成功获得了 OsImpα1a和OsImpα1b的突变体。经Sanger法鉴定获得了 T1代的纯合体植株,进一步地对其基因编辑效率进行了评估。生殖生长期,与野生型水稻日本晴相比,突变体地植株明显较矮小,水稻核输入相关的蛋白OsIMPα1a和OsIMPα1b会影响水稻地生长,尤其是株高。致病性测定结果表明,缺失OsImps后,影响黄单胞菌对水稻的毒力,以及效应子PthXo1的靶标基因OsSWEET11的表达。通过测定缺失突变体上效应子PthXo1的转运,进一步地说明了水稻核输入相关的OsIMPα1a和OsIMPα1b影响TAL效应子PthXo1的转运。效应子PthXo1从细胞质转运到水稻细胞核的过程中水稻核输入蛋白对于转运是极为重要的。4 OsIMPα与PthXo1互作的结构学基础探究OsIMPα是核输入蛋白家族中重要的成员之一,作为核转运受体,在蛋白质的入核过程中充当着十分重要的角色。核质转运是真核生物的一个重要过程。在前期研究的基础上,我们已经证实了 OsIMPα1a和OsIMPα1b与PthXo1互作,但是二者互作的结构学机理尚未清楚。本文对OsIMPα1与PthXo1的各个片段进行了原核表达,并通过体外的Pull down试验对其进行了筛选,研究结果表明,OsIMPα家族的OsIMPα1a的全长及N72、ΔIBB均可以与PthXo1 C220互作,OsIMPα1b的全长及OsIMPα1b C76可以与PthXo1 C220互作。运用阴离子交换和凝胶过滤层析纯化技术对筛得的互作蛋白质进行共纯化,进一步得到了高纯度的复合蛋白,对复合蛋白进行晶体生长条件的摸索,发现条件Crystal Screen I可以长出晶体,以期探究适合复合蛋白生长出高质量的晶体条件。本文围绕稻黄单胞的效应子PthXo1的转运问题,分别对水稻水通道蛋白和核输入蛋白质进行了互作筛选,阐明了 Hpa1xoo-OsPIP 1;3二者的互作对于PthXol转运的调控机制,以及证实了效应子PthXol从细胞质转运到水稻细胞核的过程中水稻核输入蛋白对于PthXo1转运过程中发挥着重要的作用。更多还原

【Abstract】 Bacterial blight is an important bacterial disease in rice cultivation.The effector protein secreted by Xanthomonas oryzae is very important for the occurrence of the disease.This paper introduces the mechanism of PthXo1 transport from two aspects,starting with rice aquaporin and rice nuclear importin protein,and clarifies the regulatory role of rice aquaporin and nuclear importin protein on PthXo1 transport in the process of rice PthXol transport from cytoplasm to rice nucleus.Gram-negative plant pathogenic bacteria mainly transfer toxic or non-toxic effector proteins to host cells through type Ⅲ secretory system,which leads to a series of resistance or susceptibility interactions.Many plant pathogens rely on secreting toxic TALE proteins into plant cells to activate the expression of host plant susceptible genes and cause plant disease.This process depends on the binding of TALE to specific elements on the promoter of the host target gene in the plant nucleus.The effector protein TALEs can be located in the nucleus of the host plant and bind to the promoter region of the target gene to play its role in transcriptional regulation.Aquaporin is a membrane channel protein that can promote the transport of water and neutral small molecules across the biofilms of most biomass.Water channel proteins in rice play an important role in the process of rice infection by Xoo.In this study,it was found that OsPIP1;3,a member of the OsPIP1 family,could affect the colonization of Xoo on rice leaves and promote the transport of TALE effector proteins.After pre-screening,it was found that there was an interaction between harpin protein and aquaporin in rice.1 The mechanism of rice aquaporin regulating effector PthXol transportAquaporin is a membrane channel protein that promotes the transport of water and neutral small molecules across biofilms.On the basis of previous studies of our group,it has been confirmed that OsPIP1;3,a member of rice aquaporin family,interacts with harpins protein hpal of Xoo.However,the specific interaction between Hpa1Xoo-OsPIP1;3 and its biological function has not been reported yet.In this paper,we determined the transport of PthXo1,the pathogenic phenotype of hpal and OsPIP1;3 on the constructed silent lines,overexpression lines and TALEN knockout mutants,and further detected the expression of Os8N3,the target gene of PthXo1,in rice leaves by fluorescence quantitative method to explore the regulatory mechanism of different OsPIP1;3 transgenic lines in resistance to Xoo paddy rice bacterial blight.The results showed that the pathogenicity of PXO99A was determined by inoculating wild type Nipponbare and OsPIP1;3 overexpression,OsPIP1;3 knockout or silencing lines to determine the pathogenicity of waterlogged disease spots.Compared with wild-type strain PXO99A,the symptoms of watery spot caused by hpal gene deletion mutants in different OsPIP1:3 transgenic lines were significantly weaker than those in the control.We used the traditional adenylate cyclase reporting system to further detect the transport process of PthXo1,the main virulence TAL effector protein of PXO99A.The translocation process of PthXol-cya fusion protein from bacterial cells to rice cytoplasm was further clarified.In this study,in addition to determining their biological functions.we further analyzed their key interaction domains,and constructed rice lines with interchangeable LE domains of OsPIP1;3 and OsPIP1;1 respectively by using rice tungro bacilliform virus mediated overexpression system.The transport of PthXol was determined on different domain replacement lines,and it was clarified that the key interaction site between Hpa1Xoo-OsPIP1;3 and OsPIP1;3 was the LE domain of OsPIP1;3.In summary,the interaction between Hpa1Xoo-OsPIP1;3 and TAL affects the transport of TAL effector protein PthXol to rice cells,which regulates the interaction between rice aquaporins and Xanthomonas oryzae,and affects the pathogenicity of Xoo.2 Mechanism of transnuclear transport of TAL effector PthXolAmong the effector proteins secreted by Xoo,a class of effectors called TAL effectors can be located in the host nucleus and transcribed by binding to the promoter region of the target gene,thus causing host disease susceptibility or resistance.In this process.the nucleocytoplasmic transport of TALEs is a key step in whether it can play a regulatory role.The rice nuclear importin proteins(16 rice nuclear importin related proteins)interacting with TAL effector PthXol were screened by yeast two-hybrid and bipartite fluorescence complementarity test.The results showed that effector PthXol could interact with rice nuclear importin proteins OsIMPala and OsIMPalb.In order to further study the biological significance of their interaction,the overexpression mutants of Oslmpa1a and OsImpa1b were constructed by using rice tungro bacilliform virus mediated overexpression system,and their pathogenicity was determined.The results showed that overexpression of OsImpa1a and OsImpa1b could promote the virulence of TAL effector PthXo1 in rice.Tobacco fragility virus system was used to silence the homologous gene NbImpa of OsImpa in tobacco.It was found that NbImpa affected the growth of tobacco and aggravated the allergic reaction caused by Xoo in non-host tobacco.It is further suggested that OsIMPα1a and OsIMPα1b play an important role in the transnuclear transport process of PthXo1.This transport process is very important for the expression of targeted susceptible genes induced by Xoo.3 Construction and Identification of Oslmpa1a and Oslmpa1b transgenic RiceIn the nuclear importin protein family of rice,it has been confirmed that two proteins of rice IMPa family can interact with TAL effector PthXo1 of Xoo.In this paper,the CRISPR-Cas9 gene editing skeleton vector was successfully constructed by CRISPR-Cas9 technology,and the mutants of Oslmpala and OsImpalb were successfully obtained.The homozygous plants of T1 generation were identified by Sanger,and the gene editing efficiency was further evaluated.During the reproductive period,compared with the wild-type rice Nipponbare,the plant of the mutant was obviously shorter,and OsIMPala and OsIMPalb of rice would affect the growth of rice,especially the plant height.The results of pathogenicity test showed that the deletion of OsImps affected the virulence of Xanthomonas to rice and the expression of OsSWEET11 the target gene of effector PthXo1.By measuring the transport of effector PthXol on the deletion mutant,it was further explained that the transport of TAL effector PthXo1 was affected by rice nuclear importin related OsIMPα1a and OsIMPalb.Rice nuclear importin protein is very important for the transport of effector PthXo1 from cytoplasm to rice nucleus.4 A study on the structural basis of the interaction between OsIMPa and PthXo1OsIMPα is an important member of the nuclear importin protein family.As a nuclear transport receptor,it plays a very important role in the process of protein entry into the nucleus.Nucleoplasmic transport is an important process in eukaryotes.On the basis of previous studies,we have confirmed the interaction between OsIMPα1a and OsIMPalb and PthXo1,but the structural mechanism of the interaction is not clear.In this paper,the fragments of OsIMPα and PthXo1 were expressed in prokaryotic cells and screened by Pull down test in vitro.The results showed that the full length of OsIMPα1a and N72 and Δ IBB of OsIMPα family could interact with PthXo1 C220,and the full length of OsIMPa lb and OsIMPα 1b C76 could interact with PthXo1 C220.The screened interaction protein was co-purified by anion exchange and gel filtration chromatography,and the high purity composite protein was further obtained.Through the exploration of the crystal growth conditions of the composite protein,it was found that the condition Crystal Screen I could grow crystals,in order to explore the conditions suitable for the growth of high-quality crystals of the composite protein.In this paper,the interaction between rice aquaporin and nuclear importin protein was screened for the transport of effector PthXo1 respectively,and the regulation mechanism of Hpa1Xoo-OsPIP1;3 interaction on PthXo1 transport was clarified,and the mechanism of PthXo1 transport by rice nuclear importin protein in the process of effector PthXo1 transport from cytoplasm to rice nucleus was confirmed.更多还原