摘要
植物使用不同的信号通路控制对不同类型病原物的抗性。由水杨酸(salicylic acid,
SA)、乙烯(ethylene)、茉莉酸(jasmonic acid,JA)介导的信号传导被称为植物抗病
防卫基本信号通路。它们之间及与其它信号通路之间通过某些通调因子的作用进行交
叉对话,形成复杂的信号传导网络,可以使植物应对不同刺激、快速调动防卫反应。
这些因子如何对不同的外源信号作出反应,通过何种机制形成信号网络并发挥作用,
是抗病防卫研究中的重要内容。
Harpins 是革兰氏阴性(G-)植物病原细菌产生的一类蛋白质激发子,可以在产生
菌的非寄主植物上激发过敏反应即过敏性细胞死亡(hypersensitive cell death, HCD)。该
类蛋白拥有共同生化特征:水溶性酸性蛋白、富含甘氨酸、不含半胱氨酸、热稳定、
对蛋白酶敏感。已从绝大多数 G-植物病原细菌中克隆到编码 harpin 的基因,并在原核
系统中表达出了有生物学活性的harpin蛋白。 HarpinXoo 是水稻白叶枯病菌 Xanthomonas
oryzae pv. oryzae 的 hrfAXoo 基因编码的蛋白,它显示一些独特性质,如它比其它 harpins
都小,富含甘氨酸,并含有其它 harpins 不具备的半胱氨酸。HarpinXoo还具有其它 harpin
所共有的生物学活性,包括诱发过敏反应、诱导抗病、抗虫和促进作物生长等,但对
其作用机制还不知道。
本研究的目的是研究 harpinXoo是否启动抗病和 HCD 信号传导通路,解剖二者的关
系。采用了两种方法,一种是解析外源施用 harpinXoo引发的反应;二是将编码 harpinXoo
的基因 hrfAXoo转化番茄,研究转基因在番茄体内表达激发的有关信号传导的关键环节。
结果表明,外源施用 harpinXoo 在烟草上可同时激发过敏反应和对 TMV 的抗性,而在
番茄上则启动了 Pti 激酶通路和对细菌 Pseudomonas syringae pv. tomato 的抗性;转
hrfAXoo 基因的番茄也获得了对该细菌的抗性。
1.HarpinXoo 启动烟草 HCD 通路,诱发具有程序化细胞死亡特征的反应
病原物等因子诱导的 HCD 与程序化的细胞死亡(programmed cell death, PCD)发
生机制相似,可引发细胞核形态的变化、基因组 DNA 的断裂、信号分子 H2O2的积累
等典型反应。为明确 harpinXoo诱导的烟草 HCD 是否具有 PCD 的特征,我们从以下几
方面进行了研究。
1.1 用 harpinXoo 注射烟草最早在诱导后 20h 开始出现肉眼可见的 macro-HR,为确
1
刘爱新:HarpinXoo诱发植物过敏反应和抗病性信号传导解析
定在 macro-HR 发生前是否有微过敏反应(micro-HR),我们用死细胞特异结合染料 Evans
blue 对 harpinXoo 诱导的烟草叶片染色然后测定光吸收,检测细胞死亡动态。结果是,诱
导后 4-8h 开始有少量光吸收,到 16-20h 光吸收达最大值,表明在 macro-HR 发生前烟
草细胞已陆续发生 micro-HR。
1.2 hin1 和 hsr203J 是 HCD 的分子标志,为检测 harpinXoo 诱导的 HCD 是否伴有
标志基因的表达,用 RT-PCR 法对 hin1 和 hsr203J 的诱导表达进行了研究,结果发现诱
导后 3h,hin1 和 hsr203J 已开始表达,诱导后 5-7h 表达相对较强,至 10h 表达开始减
弱。
1.3 用 DNA 特异结合染料(DAPI)对 harpinXoo 诱导处理的烟草叶片进行染色、在
荧光显微镜下观察发现,细胞核染色质发生了浓缩和片断化;基因组 DNA 琼脂糖凝胶
电泳表明,烟草叶片在诱导后 12h 开始出现 DNA 片断化,到 24-48h,断裂的 DNA 小
片段大量增加。这些现象说明,harpinXoo诱导的烟草 HCD 具有类似细胞凋亡的性质。
1.4 H2O2 是 HCD 通路的重要信号分子,它在植物组织中的积累可以用专化性染料
DAB 染色来显示。为检测 harpinXoo 诱导的烟草 HCD 是否伴有信号分子的积累,我们对
harpinXoo 诱导的烟草叶片做了 DAB 染色观察。结果发现,在 harpinXoo 诱导的烟草叶片
中有 H2O2的明显积累;为了明确 H2O2积累的分子基础,我们对 H2O2代谢相关基因的
诱导表达进行了研究。RT-PCR 分析显示,harpinXoo诱导后 1h 编码 NADPH 氧化酶的基
因 rboh 即开始表达,3-7h 表达较强,10h 开始减弱,其表达趋势与 H2O2积累趋势一致,
编码交替氧化酶的基因 aox1 表达较迟,sodA 基因则在诱导后表达减弱。这些基因的不
同表达模式说明,它们在 harpinXoo诱导的 H2O2代谢过程中可能具有不同的作用。
1.5 我们还对 harpinXoo 诱导的烟草叶片进行了组织化学研究 诱导叶片经乳酚油组
o
织透明、在荧光显微镜下观察,发现死亡细胞发出较强的荧光,表明由 harpinXoo诱导的
烟草 HCD 过程可能涉及了苯丙烷代谢途径。
以上结果说明,harpinXoo 诱导的烟草 HCD 不仅具有程序化细胞死亡的典型特征,
还伴有信号分子 H2O2的积累和组织化学的变化。
2.HarpinXoo诱导烟草对 TMV 的抗性
用 harpinXoo喷雾处理烟草可诱导对 TMV 的抗性,抗病效率平均约 47.97%。对诱导
的烟草叶片经 RT-PCR 检测发现,harpinXoo处理可诱导防卫反应基因 PR-1a、PR-1b 的
表达,这表明 harpinXoo可能通过激活系统获得抗性(SAR)通路诱导了对 TMV 的抗性。
2
山东农业大学博士学位论文
3
Plants defend themselves against different pathogens by activating distinct signal
transduction pathways. Basal defense pathways leading to non-specific resistance to
different categories of pathogens are mediated by plant hormones salicylic acid (SA),
jasmonates (JA) and ethylene (ET). These pathways often cross-talk one to another and with
pathways for insect resistance, plant growth and development in response to stimulations by
different exogenous signals. The cross-talk requires modulation by components that can
regulate different pathways.
Harpins are a group of proteinous elicitors produced by Gram-negative (G-) plant
pathogenic bacteria and are required for induction of the hypersensitive response (HR) in
non-host of the bacteria. They share some common characteristics such as rich in Glycine,
containing no Cysteine, heat-stable and sensitive to protease. Genes encoding harpins have
been cloned from most G- bacteria and harpins with biological functions are expressed in E.
coli. HarpinXoo in this study has been identified recently from a Japanese strain of
Xanthomonas oryzae pv. oryzae and expressed in E. coli BL21. It showed some special
properties such as having small molecular weight, containing Cysteine ect. Application of
harpinXoo to many plants can enhance plant growth, induce resistance to pathogens and
insects. How harpinXoo performes those diverse functions have been unclear.
Studies in this Ph. D project was aimed to dissect the signaling pathway leading to
pathogen defense and hypersensitive cell death (HCD) induced by harpinXoo. Two
approaches were used to determine signaling pathway in two plant species. The first
approach is the assays of plants treated with harpinXoo for signaling events. The second
approach is to test disease resistance and signaling of transgenic tomato plants expressing
hrfAXoo. Tobacco and tomato plants sprayed with harpinXoo can enhance disease resistance to
TMV and Pseudomonas syringae pv. tomato respectively. Tobacco plants infiltrated with
harpinXoo have showed properties of PCD (programmed cell death) but it has not in tomato.
4
山东农业大学博士学位论文
Transgenic tomato expressing harpinXoo showed increased resistance to P. syringae pv.
tomato also.
HCD in tobacco induced by harpinXoo showing properties of PCD
HR/HCD occurred after infiltration of tobacco leaves with harpinXoo. Histochemical and
molecular changes can be tested during the process of HCD including condensation of nucleic
chromatin, cleavage and fragmentation of nucleic DNA, accumulation of H2O2 and
expression of defense genes. In order to determine whether these responses occur in tobacco
induced by harpinXoo or not, we tested the histochemical and molecular changes in tobacco
leaves after infiltration with harpinXoo. The results are read as follows.
1. Death kinetics study showed that micro-HR began to occur gradually before the
occurrence of macro-HR. HR observed by our naked eyes are results of micro-HR. Results of
RT-PCR showed that the expression of hin1and hsr203 started before the occurrence of
macro-HR. The expression module of those two genes showed that they involved in tobacco
HCD.
2. To understand the cellular and molecular response in the process of HCD, tobacco leaves
infiltrated with harpinXoo are stained with DAPI and examined under up-right fluorescence
microscope using UV filters sets. Tobacco cells treated with harpinXoo showed condensation
and fragmentation of nucleic chromatin accompanying HCD. DNA were extracted at different
times after infiltration and separated by 2% agarose gel electrophoresis. DNA fragmentation
began to appear 12 hours after induction; Most DNA was degraded to small fragment 24 to 48
hours after induction. These results showed that tobacco cells induced by harpinXoo had the
properties of programmed cell death.
3. The results of DAB staining showed that H2O2 accumulated in tobacco leaves during
the process of HCD, which prior to the condensa
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