微丝骨架Actin介导的拟南芥免疫反应及其抗小麦条锈病机制研究

发布时间：2018-01-17 21:15

  本文关键词：微丝骨架Actin介导的拟南芥免疫反应及其抗小麦条锈病机制研究　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 小麦 拟南芥 Actin RIN4 CAP1

【摘要】：微丝骨架作为真核细胞中动态变化的三维网状结构,在植物感知和抵抗病原菌侵染中发挥重要作用。微丝骨架参与植物的免疫反应,但其如何参与免疫信号传导过程,目前所知甚少。小麦条锈病是由条形柄锈菌(Puccinia striiformis f.sp.tritici)引起的小麦生产上危害严重的一种真菌病害,但是小麦抗条锈病机制研究尚不完全清楚。因此,从模式植物拟南芥以及小麦重要病害条锈病入手,研究微丝骨架actin(肌动蛋白)介导的植物免疫机制,对全面了解微丝骨架参与拟南芥免疫机制,以及进一步阐明小麦与条锈菌互作的分子机理,具有重要意义。本研究以拟南芥和小麦作为研究对象,主要通过微丝骨架荧光标记、酵母双杂交、免疫共沉淀、蛋白磷酸化、病毒诱导基因沉默、LC/MS(liquid chromatography/mass spectrometry,液相色谱/质谱)技术和MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)等实验技术和方法,筛选与微丝骨架肌动蛋白ACT7互作蛋白,对拟南芥微丝骨架肌动蛋白ACT7、肌动蛋白结合蛋白CAP1和小麦肌动蛋白解聚因子ADF4的功能进行了深入研究。主要研究结果如下:1.对拟南芥cDNA文库进行筛选,选出24个与ACT7互作的蛋白,其中包括actin结合蛋白ADF1、ADF4和ARPC3等,与植物免疫相关的RIN4、ROC4、LOS2蛋白,以及CAP1等多个目前在植物免疫中功能尚不清楚的蛋白。利用酵母双杂交和免疫共沉淀技术,进一步验证了ACT7与RIN4、CAP1的蛋白相互作用。2.利用拟南芥-丁香假单胞菌模式互作系统,发现ACT7和CAP1参与DC3000和DC3000AvrRpm1引起的拟南芥免疫反应,ACT7负调控拟南芥抗病性,CAP1正调控拟南芥抗病性。与野生型Col-0相比,act7-2突变体抑制DC3000繁殖,相反cap1突变体促进DC3000大量繁殖。cap1突变体产生严重坏死斑,act7-2突变体几乎无坏死斑产生。与野生型Col-0相比,act7-2突变体抑制DC3000AvrRpm1生长,坏死面积降低,表现更为抗病;cap1突变体促进DC3000AvrRpm1生长,抑制HR反应,坏死面积增加,表现更为感病。3.利用flg22诱导的MAPK实验,证明突变CAP1显著抑制flg22诱导的MAPK3/MAPK6的积累,而突变ACT7促进flg22诱导的MAPK3/MAPK6的积累。与对照相比,flg22处理后10 min,cap1突变体中MAPK3/MAPK6积累量显著降低;act7-2中MAPK3/MAPK6积累量升高。qRT-PCR检测发现,PTI信号途径marker基因FRK1在act7-2突变体中显著上调表达;在cap1突变体中显著抑制表达。表明ACT7和CAP1参与flg22诱导的MAPK信号级联反应PTI途径。4.采用Phos-tag技术对DC3000AvrRpm1诱导RIN4在act7-2和cap1突变体中磷酸化情况进行分析,发现与野生型相比,ACT7突变后,RIN4磷酸化被促进并增强,而RIN4磷酸化在CAP1突变后受到抑制。接种病原菌DC3000AvrRpm1后,act7-2中RIN4积累量显著降低,而cap1中RIN4的积累却未降低。qRT-PCR分析显示,ETI免疫相关基因RPM1、PR1以及WRKY70在ACT7突变后表达量升高,在CAP1突变后表达量显著降低。表明ACT7和CAP1参与RIN4介导的拟南芥ETI免疫信号途径。5.从小麦cDNA文库中,克隆了6个小麦ADF基因,分别命名为TaADF3、TaADF4、TaADF5、TaADF6、TaADF8和TaADF11。酵母双杂交及免疫共沉淀结果,证明TaADF4与TaACT1、AtADF4与AtACT7直接互作,同时TaADF4与AtACT7、AtADF4与TaACT1交叉互作。蛋白三维结构建模,TaADF4与AtADF4蛋白模型几乎完全重合,表明TaADF4和AtADF4结构功能相似。小麦ADFs与TaACT1特异性互作分析显示,TaADF8不与TaACT1互作,且TaADF8蛋白结构模型独立于另外5个小麦ADF蛋白结构模型,表明不同的小麦ADFs可能存在功能特异性。激光共聚焦显微观察,发现TaADF4-RFP与TaACT1-GFP荧光标记蛋白信号共定位于微丝骨架。蛋白磷酸化实验,证明AtADF4和TaADF4活性被CPK3磷酸化调节。6.qRT-PCR分析显示,TaADF4参与小麦生物与非生物应答,TaADF4被高温、茉莉酸甲酯和脱落酸诱导上调表达,盐和低温显著抑制其表达。TaADF4在小麦与条锈菌CYR23非亲和互作中表达量显著高于小麦与条锈菌CYR31亲和互作。利用VIGS技术沉默TaADF4后,小麦对条锈菌CYR23的抗性降低。LatB处理微丝骨架诱导TaADF4上调表达,抑制条锈菌菌丝生长,促进活性氧的积累和过敏性坏死。利用LC/MS技术,对植物激素JA(jasmonic acid茉莉酸)和SA(salicylic acid水杨酸)的积累进行检测,结果显示TaADF4沉默后,JA的积累量较对照组明显降低。以上结果证明,TaADF4通过参与小麦JA信号途径转导,积极调节小麦对条锈菌小种CYR23的抗性作用。综上所述,本研究进一步解析了微丝骨架在植物免疫过程中的重要作用,首次发现并证明了微丝骨架肌动蛋白ACT7和环化酶相关蛋白CAP1,参与flg22诱导的MAPK拟南PTI信号途径和RIN4介导的ETI信号传导途径,最后探索了小麦肌动蛋白解聚因子TaADF4在小麦抵抗条锈病侵染过程中的作用,为全面了解植物的病害分子机制提供了理论依据。
[Abstract]:As a three-dimensional network structure of actin cytoskeleton dynamics in eukaryotic cells, play an important role in plant resistance to pathogen infection and perception. The immune response of actin cytoskeleton in plant, but how to participate in the process of immune signaling. Little is known about the wheat stripe rust by Puccinia striiformis (Puccinia striiformis f.sp.tritici) caused by the wheat production of a fungal disease of serious harm, but the study of wheat stripe rust resistance mechanism is still not clear. Therefore, starting from the model plant Arabidopsis and wheat stripe rust disease, actin of microfilaments (actin) plant immune mediated mechanism, to fully understand the actin cytoskeleton in Arabidopsis thaliana and further molecular immune mechanism. To clarify the interaction between wheat and stripe rust mechanism, which is of great significance. In this study, Arabidopsis and wheat as the research object, mainly through micro Wire skeleton fluorescent labeling, yeast two hybrid, CO immunoprecipitation, protein phosphorylation, virus induced gene silencing, LC/MS (liquid chromatography/mass spectrometry, liquid chromatography / mass spectrometry (mitogen-activated) technology and MAPK protein kinase, mitogen activated protein kinase) and other experimental techniques and methods of screening and actin cytoskeleton actin ACT7 interacting protein, the Arabidopsis ACT7 actin microfilaments, actin binding protein CAP1 and wheat actin depolymerizing factor ADF4 function are studied. The main results are as follows: 1. of the Arabidopsis cDNA library was screened and selected 24 proteins interacting with ACT7, including the actin binding protein ADF1, ADF4 and ARPC3, related to plant immune RIN4 ROC4, CAP1, LOS2 protein, and other functions in plant immunity is unclear. The protein using yeast two hybrid and co immunoprecipitation Technology, further validation of the ACT7 and RIN4, CAP1 protein.2. interaction using Arabidopsis Pseudomonas syringae interaction system model, found that Arabidopsis immune response of ACT7 and CAP1 in DC3000 and DC3000AvrRpm1 by ACT7, the negative regulation of disease resistance in Arabidopsis, CAP1 positively regulates Arabidopsis resistance. Compared with the wild type Col-0, act7-2 inhibition of DC3000 mutant breeding, instead of cap1 mutant promote DC3000 multiply.Cap1 mutants have serious necrosis, act7-2 mutant almost no necrosis spot. Compared with wild-type Col-0, mutant act7-2 inhibit the growth of DC3000AvrRpm1, the necrotic area decreased, especially for disease resistance; cap1 mutant promote DC3000AvrRpm1 growth inhibition, HR reaction, necrosis area increased more for the experiment of MAPK susceptible.3. induced by flg22, demonstrated that the mutant CAP1 significantly inhibited flg22 induced MAPK3/MAPK6 accumulation and mutation of ACT7 promoted flg2 2 induced the accumulation of MAPK3/MAPK6. Compared with the control group, after treatment with flg22 10 min MAPK3/MAPK6 cap1 mutant accumulation was significantly decreased; MAPK3/MAPK6 accumulation increased.QRT-PCR detected in act7-2, up regulate the expression of PTI signaling pathway of marker gene FRK1 in act7-2 mutant; in mutant cap1 inhibit the expression of ACT7 and CAP1 in the.4. show. The MAPK signaling cascade PTI pathway induced by flg22 using Phos-tag technology on DC3000AvrRpm1 induced phosphorylation of RIN4 in act7-2 and cap1 mutants in the analysis of the situation, found that compared with the wild type, ACT7 mutant, RIN4 phosphorylation was promoted and enhanced, and the inhibition of RIN4 phosphorylation in the CAP1 mutant after inoculation. DC3000AvrRpm1, act7-2 in the accumulation of RIN4 was obviously decreased, while cap1 did not reduce the accumulation of RIN4 in.QRT-PCR analysis showed that ETI gene related to immune RPM1, PR1 and WRKY70 in ACT7 mutant After the expression increased, significantly reduced the expression of CAP1 in mutant. The results indicated that ACT7 and CAP1 participate in the.5. of Arabidopsis ETI immune signaling pathway mediated by RIN4 from wheat cDNA library, cloned 6 ADF genes of wheat, which were named TaADF3, TaADF4, TaADF5, TaADF6, TaADF8 and TaADF11. yeast two hybrid and co the results showed that TaADF4 and TaACT1 precipitation, AtADF4, direct interaction with AtACT7, and TaADF4 and AtACT7, AtADF4 and TaACT1 cross interaction. Protein three-dimensional structure modeling, TaADF4 model and AtADF4 protein almost completely overlap, indicating that TaADF4 and AtADF4 structure. ADFs and TaACT1 function similar to wheat specific interaction analysis showed that the TaADF8 interaction with TaACT1, TaADF8 and protein structure model is independent of the other 5 wheat ADF protein structure model shows that different wheat ADFs may have specific function. Laser confocal microscopic observation, TaADF4-RFP and TaACT1-GFP. Label protein signals were localized in the actin cytoskeleton. Protein phosphorylation experiments show that AtADF4 and TaADF4 activity by CPK3 phosphorylation of.6.qRT-PCR analysis showed that TaADF4 is involved in wheat biological and non biological response, TaADF4 temperature, methyl jasmonate and abscisic acid induced expression, salt and low temperature significantly inhibited the expression of.TaADF4 in wheat and stripe rust CYR23 incompatible expression was significantly higher than that of wheat stripe rust and CYR31 affinity interaction. Using VIGS technology to silence TaADF4, resistance to stripe rust of wheat CYR23.LatB can reduce the expression of actin cytoskeleton induced by TaADF4, the growth inhibition of stripe rust hypha, promote the accumulation of active oxygen and hypersensitive. Using LC/MS technology. JA (jasmonic acid on plant hormone jasmonic acid) and SA (salicylic acid salicylic acid) accumulation were detected, the results showed that TaADF4 gene silencing, JA accumulation was lower than the control group Low. The above results proved that the TaADF4 of wheat JA by participating in signal transduction, actively regulate the resistance of wheat to stripe rust race CYR23. In conclusion, this study further analyzes the important role of actin cytoskeleton in plant immune process, first discovered and proved the microfilaments and actin ACT7 cyclase associated protein CAP1, participate in MAPK flg22 induced quasi ETI signal transduction pathway South PTI pathway and RIN4 mediated, and finally explore the actin depolymerizing factor TaADF4 of wheat resistance to stripe rust infection function in the process of wheat, and provides a theoretical basis for the comprehensive understanding of the molecular mechanism of plant disease.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.121.42

【相似文献】

相关期刊论文 前10条

1 雷宇华,闫芝芬,严玉平,魏建昆;微丝骨架与信号转导研究进展[J];华北农学报;2000年01期

2 刘俊梅,李岩,张红;银杏花粉的萌发及其内部微丝骨架骨的研究[J];农业生物技术学报;2001年04期

3 朱桦,陈阅增;金黄滴虫细胞核微丝系统的初步观察[J];动物学报;1986年01期

4 胡适宜;李春贵;朱o，

本文编号：1438004