Cf介导的ETI和针对稻白叶枯病菌的非寄主抗性的分子调控机理
发布时间：2018-10-31 16:58
【摘要】：根据所涉植物是否为所涉病原物的寄主,植物的抗病性可分为寄主抗性和非寄主抗性(nonhost resistance)。两类抗病性的产生机理既有许多共同点也有一些明显的区别。寄主抗性有病原相关分子模式触发的免疫(PAMP triggered immunity, PTI)和病原物效应蛋白触发的免疫(Effector triggered immunity, ETI)两个层次。番茄与叶霉菌(Cladosporium fulvum)的互作为典型的基因对基因(gene-for-gene)寄主抗性,番茄抗叶霉菌基因Cf介导产生对携带互补Avr基因的叶霉菌的ETI抗性。水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)寄主范围窄,在非寄主本氏烟(Nicotiana benthamiana)中诱导产生强烈的过敏性反应(hypersensitive response, HR)和非寄主抗性。本论文主要针对两个问题开展研究,首先,传统观念认为,介导产生ETI的植物抗病(resistance, R)基因不存在转录和转录后水平的基因表达调控。本论文通过对Cf介导的ETI研究发现,番茄Cf基因存在转录和转录后水平的调控；另外,本氏烟中Xoo诱导的HR和非寄主抗性产生机理尚不明确。本研究采用多种策略筛选鉴定该非寄主抗性调控基因,并明确了农杆菌对该非寄主抗性的抑制作用及机制。本研究获得了以下主要结果： 1.初步明确了番茄抗叶霉病基因Cf-4和Cf-9基因表达调控机理。分别从转录水平—DNA甲基化以及转录后水平—miRNA两个层次,对Cf-4和Cf-9基因表达调控机理进行了分析。在转录水平上,高温抑制Cf基因的表达,Avr强烈诱导Cf基因的表达,Cf-4的表达随着番茄植株株龄增加而升高,而Cf-9不受显著影响。启动子DNA甲基化参与高温抑制的以及常温下Avr9诱导的Cf-9基因的表达调控,另外在由高温到常温的降温过程中Avr4和Avr9对Cf-4和Cf-9基因表达的诱导作用可能受到甲基化的调控。采用VIGS技术,分析了番茄甲基化酶以及去甲基化酶基因对Cf基因表达的影响,发现番茄个别甲基化酶和全部4个去甲基化酶基因单独沉默显著改变Cf-4和Cf-9基因的表达,说明这些番茄甲基化酶和去甲基化酶基因可能参与对Cf表达的调控。在转录后水平,我们预测到了一条能够同时靶标Cf-4和Cf-9基因的长度为21nt的miRNA。通过该miRNA的靶标序列与前体序列共表达等分析,证实该miRNA能有效降解Cf-4和Cf-9mRNA。 2.筛选获得一批Cf-4/Avr4介导的HR的调控基因。通过差异表达蛋白对应基因的功能分析及本氏烟cDNA文库VIGS筛选,我们从500多个基因沉默结构中,筛选得到了28个参与调控Cf-4/Avr4介导的HR的基因,其中核糖核蛋白RNP、一个DNA结合蛋白和一个Dof锌指蛋白等正调控Cf-4/Avr4介导的HR,而一个环指蛋白、个含BTB/POZ和TAZ结构域蛋白、CP结合蛋白、SUMO结合酶SCE1、红色叶绿素代谢产物还原酶RCCR等22个蛋白负调控Cf-4/Avr4介导的HR。 3.建立和优化了本氏烟—Xoo非寄主抗性互作体系并研究明确H2O2积累是Xoo诱导的HR和非寄主抗性产生所必需。研究结果表明,用1×108cfu/ml的Xoo接种本氏烟完全展开的叶片,28℃下培养将产生最迅速最强烈的非寄主抗性。Xoo接种先在浸润区域内诱导活性氧大量积累,而后导致产生强烈的HR,并强烈诱导PR和HR标志基因的表达,最终导致Xoo的菌量急剧下降。采用过氧化氢酶清除H2O2的药理学实验结果表明,H2O2在Xoo诱导的HR和非寄主抗性中至关重要。 4.筛选获得一批Xoo诱导的本氏烟非寄主抗性调控基因。通过差异表达蛋白对应基因功能分析及本氏烟cDNA文库VIGS筛选,我们从500多个基因沉默结构中筛选获得了8个调控Xoo诱导的本氏烟HR的基因,其中碳酸酐酶、硫氧还蛋白h1以及核糖核蛋白是该HR的负调控因子,而丝氨酸/苏氨酸蛋白激酶、尿嘧啶特异的内切酶DnaJ同源亚家族基因成员、GTP结合蛋白以及三生烟中的硫氧还蛋白h这五个基因则为正调控因子。本氏烟核糖核蛋白基因同时参与Xoo诱导的HR和Cf-4/Avr4介导的HR的调控,但作用相反。 5.阐明了农杆菌对Xoo的抑菌作用以及对Xoo诱导的HR的抑制作用及其机制。结果显示,四种农杆菌菌株对Xoo诱导的HR的抑制作用有显著差异,菌株GV3101,EHA105和LBA4404能强烈抑制,但菌株C58C1无此作用。三种农杆菌对Xoo诱导的HR的抑制作用机制不同,菌株GV3101通过直接抑制Xoo的生长来抑制HR,该抑制作用需要Xoo中三型分泌系统基因hrcU和农杆菌Ti质粒pTiC58的参与。菌株LBA4404和EHA105则无直接抑菌作用,而是通过抑制本氏烟上活性氧的积累来抑制HR的产生。 综上所述,本研究首次揭示了Cf-4和Cf-9基因表达在转录及转录后水平的调控作用及其机理,鉴定了首个调节Cf基因表达的miRNA。此外,利用cDNA文库VIGS筛选技术,从500多个基因沉默结构中筛选获得一批参与Xoo诱导的本氏烟HR和Cf-4/Avr4介导的HR的新调控基因。另外,首次揭示农杆菌对Xoo本身及其诱导的HR的抑制作用及其机理。这些研究结果增进对植物寄主ETI抗性和非寄主抗性产生机理的理解。
[Abstract]:The disease resistance of plants can be divided into host resistance and non-host resistance according to whether the plant involved is the host of the pathogen involved. The mechanism of the two types of disease resistance has a lot of common ground and some obvious differences. Host resistance has two levels of immune (PI) triggered by pathogen-related molecular patterns and immune (ETI) triggered by pathogen-effector proteins. The interaction of tomato and leaf mold (Cladosporium fulvum) is a typical gene-to-gene host resistance, and the resistance to ETI of the leaf mold carrying the complementary Avr gene is mediated by the anti-leaf mold gene of tomato. Bacterial leaf blight of rice (Xanthoryzae pv.) oryzae, xoo), which is narrow in range, induces a strong allergic response (hr) and non-host resistance in non-host tobacco (nictiana bentahams). The paper mainly studies the two problems. Firstly, the traditional concept holds that the gene expression and regulation of the plant disease resistance (R) gene which mediated the ETI does not exist after transcription and post-transcriptional level. In addition, the mechanism of Xoo-induced HR and non-host resistance in the tobacco was unclear. In this study, a variety of strategies were used to identify the non-host resistant regulatory genes and to identify the inhibitory effects and mechanisms of Agrobacterium on the non-host resistance. The following main results were obtained in this study: 1. Preliminary study on the regulation and regulation of the gene expression of the anti-leaf genes of tomato and the expression of p27-9 gene Mechanism. The mechanism of gene expression and regulation of p27-4 and p27-9 was carried out from the two levels of transcription level, DNA methylation, and post-transcriptional gene expression. At the transcriptional level, the expression of survivin gene was inhibited by high temperature, and Avr strongly induced the expression of survivin gene. The expression of caspase-4 increased with the increase of plant age of tomato, while the expression of caspase-9 was not significant. The promoter DNA methylation is involved in the high temperature inhibition and the expression and regulation of the caspase-9 gene induced by Avr9 at normal temperature. In addition, the induction of Avr4 and Avr9 on the expression of p27-4 and p27-9 genes may be methylated during the cooling process from high temperature to normal temperature. The effects of methyl parathion and demethylase gene on the expression of parathion gene in tomato were analyzed by VGS technique. It is shown that these tomato parathion and demethylase genes may be involved in the expression of survivin. At the post-transcriptional level, we predicted that a miR gene with a length of 21nt at the same time, both for both the target caspase-4 and the caspase-9 gene NA. Through the analysis of the target sequence of the miRNA and the co-expression of the precursor sequence and the like, it is confirmed that the miRNA can effectively degrade the IL-4 and the IL-9mR. NA. 2. Screening of a batch of EHR-4/ Avr4-mediated HR According to the functional analysis of the differentially expressed protein corresponding gene and the VIGS screening of the cigarette cDNA library, 28 genes involved in the regulation of the R-4/ Avr4-mediated HR are screened from over 500 gene silencing structures, wherein the ribose core Protein RNP, a DNA binding protein and a Dof p27 protein are regulating the HR, while a ring finger protein, a BTB/ POZ and TAZ domain protein, a CP binding protein, a SUMO binding enzyme SCE1, a red chlorophyll metabolism product reductase RCCR, and the like, and has 22 protein negative regulatory proteins 4/ Avr4. The interaction system of Xoo's non-host resistance was established and optimized. The results showed that H2O2 accumulation was Xoo-induced HR and non-mail. The results of the study showed that the most rapid growth of the leaves at 28 鈩，
本文编号：2302958
[Abstract]:The disease resistance of plants can be divided into host resistance and non-host resistance according to whether the plant involved is the host of the pathogen involved. The mechanism of the two types of disease resistance has a lot of common ground and some obvious differences. Host resistance has two levels of immune (PI) triggered by pathogen-related molecular patterns and immune (ETI) triggered by pathogen-effector proteins. The interaction of tomato and leaf mold (Cladosporium fulvum) is a typical gene-to-gene host resistance, and the resistance to ETI of the leaf mold carrying the complementary Avr gene is mediated by the anti-leaf mold gene of tomato. Bacterial leaf blight of rice (Xanthoryzae pv.) oryzae, xoo), which is narrow in range, induces a strong allergic response (hr) and non-host resistance in non-host tobacco (nictiana bentahams). The paper mainly studies the two problems. Firstly, the traditional concept holds that the gene expression and regulation of the plant disease resistance (R) gene which mediated the ETI does not exist after transcription and post-transcriptional level. In addition, the mechanism of Xoo-induced HR and non-host resistance in the tobacco was unclear. In this study, a variety of strategies were used to identify the non-host resistant regulatory genes and to identify the inhibitory effects and mechanisms of Agrobacterium on the non-host resistance. The following main results were obtained in this study: 1. Preliminary study on the regulation and regulation of the gene expression of the anti-leaf genes of tomato and the expression of p27-9 gene Mechanism. The mechanism of gene expression and regulation of p27-4 and p27-9 was carried out from the two levels of transcription level, DNA methylation, and post-transcriptional gene expression. At the transcriptional level, the expression of survivin gene was inhibited by high temperature, and Avr strongly induced the expression of survivin gene. The expression of caspase-4 increased with the increase of plant age of tomato, while the expression of caspase-9 was not significant. The promoter DNA methylation is involved in the high temperature inhibition and the expression and regulation of the caspase-9 gene induced by Avr9 at normal temperature. In addition, the induction of Avr4 and Avr9 on the expression of p27-4 and p27-9 genes may be methylated during the cooling process from high temperature to normal temperature. The effects of methyl parathion and demethylase gene on the expression of parathion gene in tomato were analyzed by VGS technique. It is shown that these tomato parathion and demethylase genes may be involved in the expression of survivin. At the post-transcriptional level, we predicted that a miR gene with a length of 21nt at the same time, both for both the target caspase-4 and the caspase-9 gene NA. Through the analysis of the target sequence of the miRNA and the co-expression of the precursor sequence and the like, it is confirmed that the miRNA can effectively degrade the IL-4 and the IL-9mR. NA. 2. Screening of a batch of EHR-4/ Avr4-mediated HR According to the functional analysis of the differentially expressed protein corresponding gene and the VIGS screening of the cigarette cDNA library, 28 genes involved in the regulation of the R-4/ Avr4-mediated HR are screened from over 500 gene silencing structures, wherein the ribose core Protein RNP, a DNA binding protein and a Dof p27 protein are regulating the HR, while a ring finger protein, a BTB/ POZ and TAZ domain protein, a CP binding protein, a SUMO binding enzyme SCE1, a red chlorophyll metabolism product reductase RCCR, and the like, and has 22 protein negative regulatory proteins 4/ Avr4. The interaction system of Xoo's non-host resistance was established and optimized. The results showed that H2O2 accumulation was Xoo-induced HR and non-mail. The results of the study showed that the most rapid growth of the leaves at 28 鈩，
本文编号：2302958
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2302958.html
