【作者】 陈蕾；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 革兰氏阴性植物病原细菌产生的harpin蛋白质在植物上可以引起多种效应,包括抗病、抗虫、抗旱,促进植物生长等。作为一种多功能激发子,它非常有利于研究植物在生长发育、抗病防卫、抗虫和抗逆等过程中的相关信号传导机制。于是,harpin如何被植物识别,如何引发各种效应,并且这些反应中有哪些信号通路参与,以及harpin如何实现这些功能等多个问题成为了研究的重点。本研究着重剖析harpin促进植物生长、诱导抗病和抗虫的信号传导机制以及转录调控机制。一、水稻细菌性条斑病菌harpin蛋白HpaGXooc诱导植物抗病和促进植物生长的功能片段鉴定Harpin是由植物病原细菌产生的一类蛋白质激发子,可以激发非寄主植物产生过敏性细胞死亡(hypersensitive cell death, HCD),诱导植物产生防卫反应,促进植物生长。其中,后两种效应对植物有益,并且可能是通过蛋白质不同的功能域实现的。本研究分离鉴定和检测了来自于水稻条斑病细菌(Xanthomonas oryzae pv. oryzicola)的一个137个氨基酸的harpin蛋白HpaGXooc的九个功能片段,产生的蛋白质分别施用于烟草(Nicotiana tabaccum)和水稻(Oryza sativa)能产生不同的效应。相比较HpaGXooc全长蛋白,HpaG10-42诱导水稻对白叶枯病菌(Xanthomonas oryzae pv. oryzae)和稻瘟病菌(Magnaporthe grisea)产生抗性能力最强；HpaG1-94促进水稻生长能力最强；而HpaG62-137诱导过敏性细胞死亡(hypersensitive cell death,HCD)能力最强。同时,HpaG62-137诱导HCD标志基因效果强于HpaGXooc全长蛋白质,HpaG10-42对水稻防卫反应基因和生长相关基因的诱导活性明显强于HpaGXooc。由此可以解释我们所观察到的蛋白质有益效应。二、HpaGXooc小分子功能片段HpaG10-42诱导水稻抗病性和提高稻米产量在第一章的研究中,产生的功能片段HpaG10-42,相比较HpaGXooc全长蛋白,在烟草上不诱导明显的细胞死亡,能促进水稻更快的生长,同时能诱导水稻对白叶枯病菌(X. oryzae pv. oryzae)和稻瘟病菌(Magnaporthe grisea)产生较理想的抗性。本研究将HpaG10-42应用在水稻田间,研究HpaG10-42对水稻稻瘟病和水稻白叶枯病的控制效果以及对产量的影响。结果显示：在水稻苗期、返青期、分蘖后期、始穗期连续使用四次,同对照处理相比,6,6,6,6μg/ml的HpaG10-42使水稻白叶枯病(X.oryzae pv. oryzae)和穗颈瘟(Magnaporthe grisea)的病情指数均显著降低,并使产量明显提高,而且效果好于高浓度的HpaGXooc;在水稻四个生长时期仅用6μg/ml HpaG10-42处理一次,也可诱导水稻该生长时期的抗病性,但不如连续多次使用效果好；经过比较,HpaG10-42与化学农药的使用效果相当；在9个测试水稻品种上均能诱导抗病性和增加产量,但在不同水稻品种之间存在差异；在田间大面积示范实验中HpaG10-42效果稳定。三、HrpNEa诱导蚜虫趋避和抑制取食需要EIN2介导的韧皮部防卫反应HrpNEa是由梨火疫病菌(Erwinia amylovora)产生的harpin激发子。本实验室发现HrpNEa诱导拟南芥抑制蚜虫的繁殖需要乙烯信号通路重要因子EIN2,但是没有研究蚜虫趋避情况,同时也没有明确蚜虫取食与蚜虫趋避的关系。本研究结果表明,HrpNEa诱导蚜虫趋避。用罗丹明B标记、EPG(Electrical Penetration Graph)技术监测蚜虫的取食情况,结果表明HrpNEa抑制蚜虫的取食活动,同趋避效应有关。HrpNEa处理后,植物乙烯含量升高,并且乙烯生物合成抑制剂部分抑制HrpNEa的作用。在乙烯信号通路突变体ein5-1中,HrpNEa能够象在Col-0中一样诱导趋避效应,但在ein2-1中不能。表明HrpNEa诱导趋避效应需要EIN2。原位杂交结果表明,HrpNEa处理后可以诱导韧皮部相关蛋白基因AtPP2在韧皮部的表达,与韧皮部防卫反应(plant phloem-related defense, PRD)的作用模式一致,而PRD在防卫刺吸式昆虫取食中起作用。RT-PCR结果显示,HrpNEa可以诱导AtPP2在野生型拟南芥Col-O中的表达,但是在ein2-1突变体中不能。韧皮部相关蛋白在韧皮部的表达通常伴随有胼胝质的沉积,化学发光实验结果证明,HrpNEa同样可以诱导胼胝质在Col-0中的沉积,但是在ein2-1突变体中不能。因此,本研究结果表明HrpNEa诱导抗虫趋避效应、激发PRD反应,且ET信号传导因子EIN2参与了该过程。四、参与HrpNEa诱导拟南芥抗虫作用转录因子的筛选通过RT-PCR方法对拟南芥Col-O中受HrpNEa诱导的转录因子进行筛选。在研究的37个转录因子中,初步筛选到21个受HrpNEa诱导上调的转录因子,随机选取13个基因用Northern方法验证,其中有12个基因被诱导表达上调。Realtime RT-PCR进一步筛选的结果显示,AtMYB51,AtMYB38,AtMYB108, AtMYB30, AtMYB44, AtERF11, RAP2.12, F15H21.12和AtHB-7被不同程度的诱导表达,AtMYB44被诱导的效果最为明显。同时对37个突变体中蚜虫趋避以及蚜虫繁殖情况进行了研究。结果表明,在atmyb44突变体中,HrpNEa诱导后趋避率最小,对蚜虫繁殖情况没有明显影响。因此,HrpNEa诱导抗虫的效果在atmyb44突变体中被严重阻断。通过转录因子基因被HrpNEa诱导的情况,结合处理后突变体中HrpNEa诱导抗虫效果两方面的实验结果,筛选到一个感兴趣的转录因子AtMYB44,参与调控HrpNEa诱导的拟南芥抗虫性。五、AtMYB44通过作用于乙烯信号传导因子EIN2调控拟南芥对桃蚜的诱导抗性AtMYB44是拟南芥的一种转录因子,可以对乙烯发生感应,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,而乙烯信号需要EIN2作为中心调控因子才能完成传导。我们以前研究表明EIN2在HrpNEa诱导拟南芥对蚜虫产生防卫反应的过程中起关键作用,但AtMYB44调控的靶标及EIN2的转录调控机理都还不清楚。在HrpNEa处理Col-0后,植物乙烯含量升高,蚜虫趋避且蚜虫繁殖受到抑制。但是在atmyb44突变体中,HrpNEa虽然能够诱导乙烯含量升高,但是不能诱导蚜虫趋避效应和抑制蚜虫繁殖。外源乙烯作用与HrpNEa作用效果相同。遗传学及药理学实验研究表明,乙烯和AtMYB44对于HrpNEa诱导拟南芥抗虫互为必需。在Col-0中,EIN2能够被诱导表达,但是在atmyb44突变体中不能,在回补实验材料中EIN2的表达情况与在Col-0中类似。因此,EIN2诱导表达需要AtMYB44。将AtMYB44原核表达并部分纯化用于GMSA实验,结果表明AtMYB44能与EIN2的启动子序列体外特异性结合。用atmyb44得到遗传互补的转基因植物Mgh/m提取核蛋白用于GMSA实验,结果表明AtMYB44与EIN2启动子发生特异结合。用HrpNEa处理Col-0和Mgh/m植株,既促进了AtMYB44蛋白的产生,也提高了AtMYB44与EIN2互作的能力,还增强了EIN2的表达水平。Col-0和Mgh/m植株不受HrpNEa诱导的条件下,人工定殖的蚜虫可以诱导这两类植物表达AtMYB44和EIN2基因,同时诱导这两类植物对第二次定殖的蚜虫产生抗蚜性反应,而基因表达与抗虫反应在atmyb44体内均不能被蚜虫所诱导。这些试验结果表明,AtMYB44通过启动EIN2的表达来控制拟南芥对蚜虫的抗性。这一调控过程无论对诱导抗性还是免疫反应均显示功能,代表了植物抵抗植食性昆虫的一种重要防卫机制。六、创新点1.实验阐述了水稻细条斑病菌产生的harpin蛋白HpaGXooc不同结构域与功能的关系。挑选出来的三个片段HpaG1-94、HpaG10-42、HpaG62-137分别在促进植物生长、诱导抗病和诱导过敏性细胞死亡方面表现最为突出。其中,HpaG10-42促进植物生长、诱导植物抗病性,但是不诱导肉眼可见的过敏性细胞死亡,为高效重组表达HpaGXooc蛋白提供理论基础。将HpaG10-42应用在水稻田间,效果好于全长片段HpaGXooc,且与化学农药效果相当,能够诱导抗病性,并最终使稻米产量增加。2.研究明确EIN2介导植物韧皮部防卫反应,并在HrpNEa诱导拟南芥对桃蚜的抗性发生过程中起重要作用。3.对HrpNEa诱导的转录因子进行筛选鉴定,结果证明AtMYB44在HrpNEa诱导拟南芥抵抗桃蚜的过程中起重要作用,进一步实验表明,AtMYB44通过启动EIN2的表达来控制拟南芥对蚜虫的抗性。更多还原

【Abstract】 Harpin proteins that produced by Gram-negative plant pathogenic bacteria can induce multiple benificial responses such as disease resistance, insect resistance, enhanced plant growth and enhanced tolerance to environment adversity like drought. As a multiple eilicitor, harpin is perspective in studying signalling of plant development and plant defense.Hence, how harpin is recognized by plant? How to induce different effects, which signalling pathway takes part in the effects of harpin? And how these effects come true? These questions are important to be answered. Our study aimed at the signalling of harpin in enhanced plant growth、disease resistance and insect resistance as well as its transcription regulation.1. Identification of specific fragments of HpaGXooc, a harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in riceHarpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defence, and they can enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. HpaGXooc, produced by Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial streak of rice, is a 137-amino acid harpin as a member of Harpin group of proteins. Here we report the identification and testing of nine functional fragments of HpaGXooc using PCR-based mutagenesis. These specific proteins caused different responses following their application to Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Noticeably, HpaG10-42 was over 90% less active than other proteins in inducing HCD, but its effet in inducing the resistance to Xanthomonas oryzae pv. oryzae and Magnaporthe grisea was greatest. HpaG1-94 was most active in growth enhancement. Cell death level was 2-fold greater when induced by HpaG62-137 compared to HpaGXooc. Fragments HpaG62-137 and HpaG10-42 induced more intense HCD and did not cause evident cell death in tobacco, respectively, but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGxooc.2. Studies on effects of HpaG10-42, a specific fragment of HpaGxooc from Xanthomonas oryzae pv. oryzicola, in inducing disease resistance and enhancing growth in riceIn the previous study, we selected a fragment of HpaGXooc called HpaG10-42, which did not cause evident cell death in tobacco but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGXooc.Now we studying the effects of HpaG10-42 in the field, in controlling rice blast, sheath blight and leaf blight, as compared to HpaGXooc.The results showed that plants were sprayed once in nursery and three times in the field at vegetative growth V6 and V11 stages (collar formation on leaves 6 and 11 on main stem, respectively) and the reproductive stage R2 (collar formation on flag leaf) with 6,6,6,6μg/ml HpaG10-42, can deduce the severrity index of leaf blight and blast, as a result, the production was also enhanced obviously, as compaired to HpaGxooc·In the case of single ues of 6μg/ml HpaG10-42 in the four stages, its effect is better than using 12μg/ml HpaGxooc, but is worse than the 4-times appliction; HpaG10-42 was compared to chemicals in control of plant diseases; the effects of HpaG10-42 on nine different rice varieties was observed but were different; and HpaG10-42 effects was confirmed by large-scale experimentation.3. HrpNEa induces repellency and the inhibition of aphids feeding requiring EIN2-mediated plant phloem-related defense (PRD) in ArabidopsisHrpNEa is a multiple functional elicitor produced by Erwinia amylovora. In the previous study, we found that HrpNEa induces insect resistance though EIN2, but the repellency and the inhibition of feeding were not studied.In this study, we found HrpNEa can induce repellency. We used Rodanmin B as a marker and EPG to study the aphids feeding activities in Arabidopsis, and we found it identical to repellency. HrpNEa can promote the level of ethylene, and the inhibitor of the ethylene biosythesis can inhibit the effect of HrpNEa. HrpNEa can induce repellency in ein5-1 as in Col-0 rather than in ein2-1, so the effect of HrpNEa to induce repellency required EIN2. The in situ hybridization result showed the the plant phloem-related protein AtPP2 genes can be induced to express in the phloem, and its modle was the same as that of PRD, while PRD functions against attacks by sap-sucking insects. HrpNEa can induce the expression of AtPP2 in Col-0 but not in ein2-1. The expression of AtPP2 is always companied by callose deposition. Our results show that callose deposition is also induced by HrpNEa in Col-0 rather than in ein2-1. In all, our studyt shows that HrpNEa can induce repellency and PRD, and EIN2 mediated this progress.4. Screening of hrpNEa-responsive transcription factors in Arabidopsis defenses against the green peach aphidWe screen transcription factor genes that are HrpNEa-responsive by RT-PCR. Selected 37 TF genes were investigated for expression in WT Arabidopsis following treatment with HrpNEa, which was used in contrast to EVP. Twenty-one genes increased in plants 12 h post treatment (hpt) with HrpNEa, compared with EVP. As confirmed by Northern blot analysis,12 of 13 selected genes accumulated great amounts of transcripts in 12 hpt with HrpNEa-To determine transcript levels, the 9 genes were subjected to real time RT-PCR protocols. In 12 hpt, transcripts of AtMYB44 in HrpNEa-treated plants were greater than those in EVP-treated plants. To correlate the expression of TF genes with Arabidopsis resistance to the green peach aphid, the plant mutants disrupted at the 37 TF genes were tested for variations in supporting the insect to colonize plant leaves and multiply on leaves. In the atmyb44 mutant, the repellency was least, and aphids multiplication were not repressed in atmyb44 responding to HrpNEa, indicating that the effect of HrpNEa was abolished, indicating AtMYB44 was a possible regulator of insect repellency. Finally, we get a selected transcription factor AtMYB44, which may paticipate the induced insect resistance in Arabidopsis.5. AtMYB44 Targets EIN2 to Regulate Arabidopsis Resistance against the Green Peach AphidAtMYB44 is a transcription factor responsive to ethylene and implicated in Arabidopsis 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. HrpNEa can promote the ethylene content in Col-0, and induce repellency and repress production. In atmyb44, can also promote the ethylene content, but the effect of inducing repellency and repressing production were lost. Through genetic, molecular, and pharmacological studies, we show that HrpNEa induce Arabidopsis resistance against the green peach aphid required AtMYB44. This effect is arrested in atmyb44. EIN2 can be induced by HrpNEa in Col-0 but not in AtMYB44 indicating the expression of EIN2 required AtMYB44. In wild-type and atmyb44-complemented plants, extrinsic ethylene duplicates the function of intrinsic ethylene to induce resistance and the expression of AtMYB44 and EIN2. In atmyb44-complemented plants, moreover, AtMYB44 binds EIN2 promoter. HrpNEa treatment promotes AtMYB44 production and the binding activity as well as EIN2 expression. When atmyb44-complemented and wild-type plants are subjected to aphid infestation, AtMYB44 and EIN2 expression is induced and plants become resistant to secondary infestation. Therefore, AtMYB44 controls Arabidopsis resistance to the insect by activating the expression of EIN2. This regulatory pathway functions as an important mechanism that protects plants from insect herbivores.6. Concluding remarks1. We identify and test nine functional fragments of HpaGxooc, and these proteins have different effects on Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Among them, HpaG62-137 induced more intense HCD, HpaG10-42 did not cause evident cell death in tobacco, but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGXooc.We used a selected fragment of HpaG10-42 in the field, studying the effects of HpaG10-42 in controlling rice blast、sheath blight and leaf blight, as compared to HpaGxooc.The results show that HpaG10-42 can induce disease resistance and finally increase the production as chemicals.2. EIN2 mediated HrpNEa-induced plant phloem-related defense which functions against attacks by sap-sucking insects.3. We selected transcription factors that are responsive to HrpNEa, and AtMYB44 is a candidate, which may also take part in the effect of HrpNEa in regulating insect resistance in Arabidopsis. Our results showed that AtMYB44 controls Arabidopsis resistance to the insect by activating the expression of EIN2, providing a mechanism of EIN2 transcription regulation.更多还原