【作者】 刘若雪；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2010， 博士

【摘要】 自然界中的植物经常遭受病原菌或病原激发子的侵袭,而植物利用自身的先天免疫系统形成一套复杂而有效的防御机制来抵御这种侵害,这些机制包括过敏性细胞死亡(hypersensitive cell death, HCD)和系统获得性抗性(systemic acquired resistance, SAR)。HCD又叫过敏反应(hypersensitive response, HR),是植物抗病反应的一种典型症状,可在24 h内观察到。其特征是病原菌所侵染部位的细胞呈现快速枯死,病原菌得不到营养而致其不能生长和扩散。HR的产生与两种类型的病原菌与植物产生互作有关：一种是病原菌与寄主之间的不亲和互作(avr-R);另外一种是由病原菌与非寄主植物的互作所引起。已有研究表明,病原真菌中引起非寄主过敏性反应的物质主要是由疫霉属病原菌所产生的激发子elicitinS。ParA1是由寄生疫霉Phytophthra parasitica var nicotianae分泌产生的一种elicitin。ParA1处理烟草叶片的表皮毛,可以引起表皮毛细胞发生一系列的HCD相关反应。这表明,表皮毛中存在着一套接受外源激发子ParA1信号的分子机制。那么,ParA1是如何被植物体识别的?表皮毛发育蛋白NtTTG1是如何参与到ParA1引发的HCD过程之中的?转录因子在植物应对生长发育过程中遭受的各种生物和非生物胁迫的过程中起重要的作用。转录因子通过与其调控的下游基因启动子区的顺式作用元件结合而直接调控靶基因的表达,或形成同源、异源二聚体,或与其它蛋白互作成为某种活化形式而参与乙烯、水杨酸(SA)、茉莉酸(JA)与脱落酸(ABA)等信号传导途径,形成复杂的基因表达调控网络,提高植物对环境胁迫的适应能力。HrpNEa是由梨火疫病原细菌Erwinia amylovora产生的一种蛋白激发子。外施HrpNEa能够诱导拟南芥中多种转录基因的表达,也可以通过激发不同信号通路,诱导植物产生抗病、抗虫、抗旱以及促生长等多种有利表型。EIN2 (ethylene insensitive2)是乙烯信号通路的关键调控因子,那么,转录因子和EIN2之间会有什么样的联系?转录因子在乙烯信号传导通路中起到什么作用?本研究着重剖析NtTTG1在ParA1引发的烟草过敏细胞死亡中的作用机制以及AtMYB44参与乙烯信号传导通路的作用机制。1、ParA1与功能缺失蛋白C51S的获得及其功能验证根据以往的研究得知,蛋白激发子ParA1注射烟草叶片,可以引发过敏性细胞死亡；喷施烟草叶片时,可以引发肉眼不可见的微敏反应(microscopic hypersenseitve response, micro-HR),并伴随有活性氧爆发(ROS burst)和染色质凝集现象(Chromatin condensation, Cc)的发生。ParA1中含有6个保守的半胱氨酸,且两两结合形成三对二硫键维持着对于ParA1的功能行使所必需的特异的空间构象。本研究主要通过点突变(site directed mutagenesis)的方法将ParA1中的第51位半胱氨酸替换为丝氨酸,得到了基因C51S,经原核表达得到了点突变蛋白C51S。通过与对照ParA1的功能验证发现,C51S完全丧失了在烟草叶片上诱导HR的能力。而且DAB、trypan blue和DAPI染色结果发现,C51S处理表皮毛12h后,仍检测不到氧爆发、微敏反应和染色质凝集现象的发生。这些结果都表明,C51S已经完全丧失其生物功能。2、ParA1与NtTTG1在烟草表皮毛细胞的细胞膜附近的相互作用根据拟南芥中参与叶片表皮毛发育的几个关键基因,在烟草中筛选可以受ParA1诱导表达的同源物。发现只有AtTTG1的同源物受诱导表达,并依此克隆到NtTTG1基因。NtTTG1在表皮毛中被ParA1诱导表达的程度明显强于叶肉细胞。并通过酵母双杂交和pull-down assay证明了ParA1与NtTTG1可以发生互作,而C51 S与NtTTG1却不能相互作用。蛋白结构分析表明,51位的半胱氨酸位于ParA1蛋白的边缘,这个位置很可能在与其它蛋白的互作中起作用。ParA1与NtTTG1之间的互作经两个蛋白在植物表皮毛中的亚细胞定位而得到进一步验证。将融合有红色荧光蛋白RFP的NtTTG1在烟草中瞬时表达,这个融合基因瞬时表达12h后在烟草表皮毛中有大量的转录。荧光观察表明,NtTTG1-RFP明显的定位在烟草表皮毛细胞的细胞膜上。而且荧光叠加结果表明,ParA1-eGFP可以与NtTTG1-RFP相结合,而C51S-eGFP不能与之结合。双分子荧光互补实验也进一步验证了荧光叠加的结果,而且,这种互作发生在表皮毛细胞的细胞膜附近。3、烟草叶片表皮毛在ParA1引发过敏性细胞死亡过程中的导航作用表皮毛作为烟草最外部的第一道天然屏障,势必最早接触蛋白激发子ParA1,并参与其诱导的过敏性细胞死亡(hypersensitive cell death, HCD)过程。我们以HCD发生过程中的重要信号分子H202和细胞内的染色质凝集为切入点,确定了表皮毛对于ParA1激发子诱导HCD的响应(见本博士论文研究报告第一章)。DCFH-DA染色及荧光观察结果表明,ParA1处理后1h,表皮毛内发生氧爆发,这比叶肉细胞早2h,其产生的活性氧还有向下部叶肉细胞传导的趋势。同时,药物抑制实验证明了活性氧的主要成份是H2O2。DAPI荧光分析表明,表皮毛内染色质凝集的发生要比叶肉细胞早5h左右。Trypan blue染色,并配合质壁分离实验,结果证明,在ParA1处理后1h到12h中,细胞死亡从表皮毛向底部细胞逐渐扩散,表皮毛细胞死亡发生比叶肉细胞早大约5h,且表皮毛在每个时间段内的细胞死亡数都高于叶肉细胞。RT-PCR方法检测了HCD反应中的标志基因PAL、hin1和hsr203的表达,三个基因在ParA1处理12h后的表皮毛中都有明显表达,而在叶肉细胞内表达稍晚一些。这些结果表明,HCD信号是从表皮毛向叶肉细胞中传导的。4、介导HrpNEa诱导拟南芥抗绿桃蚜作用的37个转录因子的初步筛选HrpNEa是由病原细菌产生的一类harpin蛋白质,它可以通过激发不同信号通路,诱导植物产生抗病、抗虫、抗旱以及促生长等多种有利表型。HrpNEa处理拟南芥可以通过激活乙烯信号通路来诱导植物对绿桃蚜的抗性反应。EIN2是乙烯信号通路中的关键调节因子,在诱导抗虫反应中起重要作用。为了阐明乙烯信号在诱导抗虫反应中的调控机制,我们对在拟南芥上由乙烯诱导的37个转录因子进行了研究。HrpNEa处理野生型Col-0后,与对照相比,发现有22个基因被上调表达,4个被下调,还有9个没有变化,其中AtMYB44的上调表达度最强。通过研究37个转录因子突变体中蚜虫趋避以及蚜虫繁殖情况时发现,有24种突变体和野生型差不多,有4种抗性增强,还有9种抗性减弱。atmyb44对蚜虫的趋避性减弱程度最为明显,而且削弱了HrpNEa诱导的抗虫反应。我们又分别检测了乙烯信号通路的标志基因PDF1.2的表达情况,发现其在野生型中表达,但在atmyb44和另外的4种突变体里不表达,这说明了乙烯信号通路已被阻断。本研究证明了拟南芥突变体atmyb44能消除HrpNEa诱导的EIN2基因的表达,从而说明了AtMYB44与EIN2有着很紧密的联系。5、转录因子AtMYB44与EIN2的互作研究AtMYB44是拟南芥MYB家族的一种转录因子,可以对乙烯信号发生感应,从而介入植物防卫反应。植物防卫反应经常受乙烯信号传导支配,而乙烯信号的中心调控因子是EIN2。目前,对AtMYB44调控的蛋白靶标及EIN2的作用机理的研究,都还不够深入。通过遗传学、药理学及微生物学等方法,我们发现AtMYB44、EIN2和乙烯在对HrpNEa诱导拟南芥对蚜虫产生抗性的过程中都是必需的因子。本研究通过geⅠmobility shift assay (GMSA)和chromatin immunoprecipitation assay (ChIP)两种方式证明了AtMYB44与EIN2能在植物体外体内产生互作。6、转基因双突变体ein2-1 atpp2-a1和ein2-1 abi2-1的产生与鉴定拟南芥AtPP2类韧皮部蛋白是一种独特的结合几丁质的凝集素,其氨基酸序列高度保守且均有韧皮部组织特异性,对刺吸式昆虫的侵袭有着特殊的防卫功能,在植物韧皮部相关防卫(plant phloem-related defense, PRD)反应过程中起重要作用。逆境激素脱落酸(ABA)能调控植物营养生长和生殖生长过程中的气孔关闭、抵抗逆境、种子后熟和休眠等许多重要事件。乙烯是植物生长发育重要的内源信号分子,其介导的植物抗病防卫基本信号通路对于植物的基本防卫十分重要。而EIN2是乙烯信号通路中的关键调节因子。本研究通过RNAi介导的基因沉默的转化方法,构建了AtPP2-A1与ABl2基因的沉默发夹结构,分别转化拟南芥乙烯不敏感突变体ein2-1,将AtPP2-A1与ABl2基因沉默,成功获得了双突变体品系ein2-1 atpp2-a1和ein2-1 abi2-1。为深入研究植物乙烯与PRD、ABA信号通路在植物防卫信号传导过程中的关系提供了材料与依据。更多还原

【Abstract】 In the nature, plant suffers from the pathogen or the pathogenic exciton attack frequently, but they can use their own innate immune system to form a set complex and effective defense mechanism for resisting these kinds of violation, including hypersensitive cell death (HCD) and system acquired resistance (SAR). HCD is also called hypersensitive response (HR), is one of the typical symptoms for the plant disease-resistant response. The HR production are related with two types of pathogen interacted with the plant. One kind is the interaction between the pathogen and the host like the avr-R. Another kind is the interaction between the pathogen and the non-host plant. Previous researches indicate that elicitins are the main fungi which can cause HR to the non-host plant. ParAl is one kind of elicitin produced by Phytophthra parasitica var nicotianae. The tobacco leaf trichomes treated by ParA1 may cause a series of HCD correlation response. This indicated that there are a set of molecular mechanism for receiving the ParA1 signal in the tobacco leaf trichomes. However, how does the plant recognize the ParA1? How does the trichome development protein NtTTG1 participate in the HCD process induced by the ParA1?Transcription factor plays a vital role in plant growth and development process to answering much kinds of biological and non-biological intimidate. According to binding the cis-element of downstream gene promoter, or forming homologous or heterogenous dimmers, or interacting with other proteins, transcription factor can regulate the expression of target genes, or participate in the ethylene, salicylic acid (SA), jasmine acid (JA) and abscisic acid (ABA) signal transductions, so they can form the complex gene expression regulation network and enhance the ability for the plant to adapt to the environment. HrpNEa is one kind of protein produced by Erwinia amylovora, which can stimulate different signal pathways to cause many kinds of advantageous phenotypes. EIN2 (ethylene insensitive2) is the key regulation factor in the ethylene signal pathway, however, how the relationship between the transcription factor and EIN2? What does the transcription factor play in the ethylene signal transduction pathway?This study analyzes the tobacco trichome protein NtTTGl and the Arabidopsis transcription factor AtMYB44 function to regulate transduction of two distinct defensive signals (ParA1 and HrpNEa) via the membrane and in the plant cell.1、Production of the loss of function protein C51S and its functional analysis compared with ParAlAccording to the previous studies about ParA1, it is a kind of proteinous elicitor with low molecular weight derived from Phytophthora parasitica var. nicotianae. Infiltrated in the tobacco leaves, ParA1 can induce hypersensitive cell death (HCD). Sprayed on the tobacco leaves, it may induce the microscopic hypersensitive response (micro-HR), companied with ROS burst and chromatin condensation (Cc) in the corresponding plant cells. The leaves were excised at 12h post treatment (hpt) and stained with green fluorescence dye 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) and ROS could be detected, but not the C51S treatment. Cc was studied by 4’,6’-diamino-2-phenylindole (DAPI) fluoluminescence assay. There are 6 conserved cysteines that form three disulfides to maintain the specific structure in ParA1, and this structure is required for the function of ParAl. This study focused on the site direct mutant of ParA1. We replaced the cysteine at the site of 51 by serine, and obtained the mutagenesis protein C51S. Compared with ParA1, C51S lost the function of inducing HR in tobacco leaves. And we could not detect ROS burst、micro-HR and chromatin condensation in trichome cells by further experiments. Our results suggest that C51S has been lost of biological functions.2、Analysis of the interaction between ParA1 and NtTTG1 in tobacco trichome cell membraneAccording to cloning the homologues genes AtGL1, AtGL3, AtTTG1, and AtTTG2, which are essentially involved in the development of Arabidopsis thaliana leaf trichomes, we found only AtTTG1 homologue responded to ParA1 and NtTTG1 was cloned. Also amino acid sequence of NtTTG1 is highly identical with that of AtTTG1. The expression of NtTTG1 was strongly induced by ParA1 in tobacco trichomes rather than leaves. ParA1, but not C51S, could interact with NtTTG1 by yeast two-hybrid assay and pull-down assay. Analysis of ParA1 structure model,51’ cysteine is positioned on the surface of the outer edge of the protein, which is implicated in interacting with other proteins. These also suggested that the importance of 51/95 disulfide bone with 3/71 in ParA1. ParA1-NtTTG1 interaction conformed to subcellular localization of both proteins in tobacco trichomes that transiently expressed NtTTG1 fused to a red-fluorescence protein (RFP) gene. Fluorescence microscopy showed different distribution of these proteins in trichomes. NtTTG1-RFP showed conspicuous localization at the trichome cell membrane. At equivalent locations, ParAl-eGFP was affluent but C51S was little when applied externally and treated with a washing solution. This observation suggested that ParAl was bound tightly but C51S was not. The following experiment about mergence of red and green fluorescence indicated that ParA1-eGFP, instead of C51S-eGFP, bound NtTTG1-RFP, and that the interacting sign clearly localized at the cell membrane.3、Tobacco leaf trichomes contribute to ParAl-induced signalling and cell deathAs the first and the most external barriers of the plant, trichomes always touch the ParAl elicitor at the earliest time, and also contribute to the ParA1-induced hypersensitive cell death (HCD). We have showed that trichomes could respond to ParA1, with ROS burst and chromatin condensation being detected. After localized treatment of trichomes, the leaves were excised at 6h post treatment (hpt) and stained with green fluorescence dye 2’, 7’-dichlorofluorescein diacetate (DCFH-DA), apparently, ROS had spread from the trichome to leaf epidermal cells and the mesophyll in 6 hpt. Simultaneity, we proved that H2O2 occupies a prominent part of the ROS production that is induced by ParA1 by applying with diphenyleneiodonium (DPI) or catalase (Cat). Typical Cc was detected in trichomes and mesophylls following the use of ParA1 versus EVP. Nuclei in cells of control trichomes and in cells of ParAl-treated trichomes within 1 hpt had a clear central nucleolus surrounding by a uniform stained chromatin; whereas, in cells of ParA1-treated trichomes over 1 hpt, chromatin had a swollen aspect and nuclei were lobated. In one-way F tests (P< 0.01), trichomes significantly exceeded mesophylls in levels of cell death at each time point since 1 hpt. HCD marker genes PAL, hin1, and hsr203 were expressed evidently in trichomes 12 hpt with ParA1. Mesophylls were inferior to trichomes in time and extents of the gene expression. All these results suggested that HCD signal transduct from tobacco trichomes to mesophylls.4、Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in ArabidopsisThe harpin protein HrpNEa induces Arabidopsis resistance to the green peach aphid by activating the ethylene signaling pathway and by recruiting EIN2, an essential regulator of ethylene signaling, into defense response in the plant. Here we investigated 37 ethylene-inducible Arabidopsis transcription factor genes for effects on the activation of ethylene signaling and insect defense. In response to HrpNEa,22 genes increased in transcription levels with AtMYB44 being the most inducible,6 genes had decreased transcript levels, and 9 remained unchanged. When Arabidopsis mutants defected at the 37 genes were surveyed,24 mutants were similar to the wild-type (WT) plant while 4 mutants were more resistant and 9 mutants were more susceptible than WT to aphid infestation. The atmyb44 mutant with a defect in the AtMYB44 gene was most susceptible to aphid infestation and most compromised in HrpNEa-induced resistance. Resistance accompanied the expression of PDF 1.2, a an ethylene signaling marker gene that requires EIN2 for transcription, in WT but not in atmyb44 and other 4 aphid-susceptible mutants, suggesting a disruption of ethylene signaling in the five mutants. However, atmyb44 was an mutant with an abrogation in HrpNEa-induced EIN2 expression, suggesting a close relationship between AtMYB44 and EIN2.5、Analysis of the interaction between the transcription factor AtMYB44 and ethylene signaling regulator EIN2AtMYB44 is a transcription factor responsive to ethylene and implicated in Arabidopsis thaliana defense response. Plant defense is often subject to ethylene signaling that recruits EIN2 as a central regulator. Previously we have shown that EIN2 plays a critical role during the development of insect defense in Arabidopsis treated with HrpNEa, a harpin protein produced by bacterial plant pathogen. Regulatory targets of AtMYB44 and regulation of EIN2 activation have not been characterized. Through genetic, molecular, and pharmacological studies, we have shown that AtMYB44, EIN2, and ethylene are required for HrpNEa to induce Arabidopsis resistance against the green peach aphid. In this study, we testified that AtMYB44 could combine the specific consensus MYB recognition sequence 5’-TAACTG-3’ in the promoter of EIN2 by gel mobility shift assay (GMSA) and chromatin immunoprecipitation assay (ChIP).6、Production and identification of the two transgenic double mutant ein2-1 atpp2-a1 and ein2-1 abi2-1AtPP2 class phloem protein belongs to a unique tissue-specific agglutinin combinating with chitin in Arabidopsis thaliana, and its amino acid sequence is highly conserved. It has a special defense capability on the invasion of sucking insects and plays an important role during the plant phloem-related defense (PRD) processes. Abscisic acid (ABA) as the stress hormone can modulate many important events in plant growth and development including stoma closing, resistance to stresses, promoting seeds maturity and dormancy. Ethylene is the most important endogenic signal in plant growth and development and mediate the basal plant defense and disease resistant signal pathways. EIN2 is a central regulator in ethylene signal pathway. The interaction between PRD and ethylene or ABA and ethylene in plant growth and development is not very clear at present. We constructed AtPP2-A1-silencing and ABI2-silencing AtPP2-A1vector, which could be able to silence AtPP2-A1 and ABI2 in ein2-1, and obtained the two transgenic double mutant ein2-1 atpp2-a1 and ein2-1 abi2-1. This study will provide further materials and evidences for research of relationship between PRD and ethylene or ABA and ethylene pathways in the plant development and signal transduction.更多还原