Harpins蛋白诱导植物防卫和生长及四种激素信号的交叉调控作用
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摘要
植物防卫反应的激活是通过植物识别激发子开始的，激发子是由病原物编码的分子，如：微生物蛋白、小肽及多糖等。当遭遇外界各种不同的刺激时，植物会迅速启动反应机制，调用多个信号通路中相关的调控因子，重新编排形成高效的信号网络，应对面临的生存危机。植物如何根据不同的外源信号调用相关的因子，并使其“各就各位”发挥作用，是目前植物科学领域研究的重要话题。
革兰氏阴性植物病原细菌产生的蛋白质激发子harpins可诱导植物多种反应，如抗病，抗虫，促生长和抗旱等。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，harpins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，它们相互之间有没有交叉对话，如何交叉对话、以及harpins如何实现这些功能等多个问题成为了研究的重点。
1．不同HpaG_(Xooc)功能片段的分离鉴定及对植物的诱导抗病和促生长作用
Harpins是由革兰氏阴性植物病原细菌产生的一类蛋白质激发子，他们的共同特点是：富含甘氨酸、热稳定、对蛋白酶敏感，注射到产生菌的非寄主植物叶片细胞间隙后，可以诱导过敏性细胞死亡(hypersensitive cell death,HCD)，诱导植物产生防卫反应、促进植物生长。Harpin蛋白的不同效应，有些是对植物有益的，而这些效应的发生可能是依赖于蛋白质不同的功能域实现的。分离、鉴定蛋白质有益和有害的功能区域，更有利于我们将harpin相关蛋白应用于农作物，而不产生诸如细胞坏死之类的负面效应。
水稻细菌性条斑病菌(Xanthomonas oryzae pv．oryzicola)Hpa G_(Xooc)作为Harpin蛋白家族中的一员，包含两个富含甘氨酸的结构域(glycine-rich motif,GRM)，一个半胱氨酸残基(cysteine)。GRM是Harpin类蛋白质共有的特征，而半胱氨酸则是其他Harpin类蛋白质所没有的。本研究根据HpaG_(Xooc)不同功能域和区段，基于PCR的体外诱变技术产生九个HpaG_(Xooc)的蛋白质功能片段，分别为HpaG_(1-105)、HpaG_(1-94)、HpaG_(1-61)、HpaG_(1-47)、HpaG_(7-61)、HpaG_(62-137)、HpaG_(10-42)、HpaG_(95-137)、HpaG_(84-94)，右下角的数字分别代表了蛋白质所包括的HpaG_(Xooc)相应的氨基酸区域．将这九个片段体外表达，产生的蛋白质施用于烟草(Nicotiana tabaccum)和水稻(Oryza sativa)能产生不同的效应。HapG_(1-94)、HpaG_(95-137)、HpaG_(84-94)和HpaG_(Xooc)诱导细胞死亡的能力相近，HpaG_(1-105)、HpaG_(1-61)、HpaG_(1-47)、HpaG_(7-62)及△GRM诱导细胞死亡的能力高于HpaG_(Xooc)63％以上；HpaG_(62-137)诱导细胞死亡的能力是HpaG_(Xooc)的2倍，但是HpaG_(10-42)诱导细胞死亡的能力低于其他蛋白约90％以上。
同HpaG_(Xooc)全长蛋白相比较，HpaG_(62-137)能在烟草上诱导更强的HCD，而HpaG_(10-42)不表现明显的细胞死亡，但都能在水稻上能激发更强的防卫反应和促进植株更快的生长。在检测的9个片段及全长蛋白质中，对水稻白叶枯病菌(Xanthomonasoryzae pv.oryzae)的抗性诱导效应中，HpaG_(10-42)诱导效应最强，HpaG_(1-61)和HpaG_(7-61)次之，HpaG_(62-137)的诱导抗病性效应表现为最低；而对稻瘟病菌(Magnaporthe grisea)的诱导抗性效应中，HpaG_(62-137)处理的叶片上仅能看到侵染点，没有明显的坏死斑；而HpaG_(10-42)处理的叶片上甚至连坏死斑都无法看见。因此，HpaG_(10-42)能同时抑制细菌和真菌病原的致病性。在对水稻的促生长效应中，以HpaG_(Xooc)为对照，HpaG_(1-105)、HpaG_(1-62)、HpaG_(1-47)、HpaG_(62-137)和HpaG_(84-94)的促生长效应较弱；但是△GRM、HpaG_(1-94)、HpaG_(7-61)及HpaG_(10-42)处理后根系分别增长了15％、55％、20％和48％。因此HpaG_(1-94)和HpaG_(10-42)在促进水稻生长的效应最强。
在蛋白粗提液实验中HpaG_(1-94)、HpaG_(10-42)、HpaG_(62-137)和△GRM对植物的作用明显强于HpaG_(Xooc)，因此我们将这几种蛋白质进行镍柱纯化，并进一步检测、验证他们的生物活性以确证上面的实验结果。从结果可以看出不管是粗蛋白提取液还是纯化蛋白质均表现相同的效应。
由于在检测的所有蛋白质中HpaG_(62-137)和HpaG_(10-42)表现为更高水平的HCD诱导活性及所有其他有益效应，因此我们使用HpaG_(62-137)和HpaG_(10-42)同HpaG_(Xooc)相比较，来检测他们在诱导植物HCD和激活生长防卫相关信号传导通路的活性。HpaG_(62-137)处理后HCD标志基因hsr203和hin1基因在处理6小时后就开始积累转录，并随着处理时间的推移叶片中的表达量也逐渐增加；处理48小时后，HpaG_(62-137)处理的叶片中这两个基因的表达量明显强于HpaG_(Xooc)的处理。此外HpaG_(10-42)处理6个小时后防卫反应基因NPR1和PR1开始被诱导表达；特别是PR1基因的表达明显地依赖于诱导，因为在处理0小时时没有明显的组成性表达。从HpaG_(10-42)和HpaG_(Xooc)处理48小时后NPR1和PR1基因表达的结果可以看出，HpaG_(10-42)的诱导表达效应明显强于HpaG_(Xooc)。由于HpaG_(10-42)的诱导活性明显强于HpaG_(Xooc)全长蛋白质的活性，因此可以解释我们所观察到的植物上有益效应的表型。我们同时发现某些特异性片段相比较全长蛋白更能有效地激活某些信号通路。总之，本研究结果阐释了一个HpaG_(Xooc)的特异性片段HpaG_(10-42)能促进水稻生长和诱导抗病性，具有潜在的农业应用价值。
2．HrpN_(Ea)诱导植物生长和膨胀素基因表达及乙烯与赤霉素的调控作用
膨胀素(Expansin)是植物细胞生长期间释放的一种能使细胞壁松弛的蛋白质，是细胞壁的关键调节剂，对细胞生长有重要作用。有研究表明植物激素介导了膨胀素的功能，但是植物激素是特异性作用还是共同起作用及作用机制仍然不是很清楚。本文报道了HrpN_(Ea)促进植物生长和诱导expansin基因表达所启动的不同信号通路。
拟南芥浸种处理15天后在MS平板上可以看见，HrpN_(Ea)可以明显的促进根系生长，根系增长89％，幼苗鲜重增加67％；同样盆播幼苗在HrpN_(Ea)喷雾处理15天和30天后也明显的促进的植株地上部分的生长。HrpN_(Ea)浸种番茄后可以促进种子萌发、根系生长及后期的营养生长，HrpN_(Ea)处理12天后植株高度增加46％，鲜重增加52％。烟草在HrpN_(Ea)处理20天后可以看见促生长作用，到40天促生长作用更明显，高度和鲜重分别增加32％和50％，到处理60天营养生长后期，与对照相比，地上部分生长明显加快，茎节粗壮，叶片肥大，长势旺盛。HrpN_(Ea)处理水稻后极大的促进了种子萌发和根系生长，处理12天后可以明显观察到促生长作用，高度和鲜重分别增加56％和48％，直接促进了幼苗的生长．此外与对照相比，HrpN_(Ea)不仅增加了植株叶面积，而且还增加了叶片数量。HrpN_(Ea)明显增加植物体内全氮含量，其提高幅度分别比对照高28％(拟南芥)，36．4％(番茄)，28．9％(烟草)和25．4％(水稻)。HrpN_(Ea)浸种处理四种植物10天后，叶绿素a和叶绿素b及叶绿素总量均明显增加。此外，HrpN_(Ea)处理后，叶绿素a／b比值也显著高于对照处理。当HrpN_(Ea)喷雾处理10天与20天的植株地上部分后，叶片全氮量和叶绿素含量同样增加。因此，HrpN_(Ea)通过影响基本生理过程来强化植物的生物合成功能和增加植物生物学产量．
我们检测了HrpN_(Ea)处理四种不同植物后对生长相关基因的诱导表达。HrpN_(Ea)处理后拟南芥AtEXP7在叶片和茎中表达不明显，而在根中表达量较高；LeEXP2和LeEXP5在茎中表达而在叶片和根中不表达；另外8个EXPs，包括AtEXP10和AtEXP2在叶片中表达量高，而在根和茎中表达量较低。同时AtEXP16和NtEXP2的诱导表达分别相似于AtEXP10和NtEXP2，在叶片中特异性表达的EXPs如AtEXP10、LeEXP2和NtEXP2，在根和茎中转录水平明显降低。在不同的器官、不同的处理模式及诱导时间的不同，基因表达水平也不相同。AtEXP2、LeEXP18和NtEXP2表达模式相同，在处理72小时和96小时内随着处理时间的推移表达量逐渐增加。相反地，其他的EXPs基因在处理6小时后直到72小时始终保持高水平表达量。
为了阐释HrpN_(Ea)诱导EXPs基因表达和促生长作用同乙烯信号通路的相关性，我们测定了HrpN_(Ea)处理后乙烯信号通路中关键基因的表达情况及同EXPs表达的关系。拟南芥中ETR1的表达受HrpN_(Ea)诱导，并且随着诱导时间表达量逐渐增强；EIN2表达是组成性的，并且在HrpN_(Ea)处理6小时后表达量快速增加达到顶峰，随后不再增加。HrpN_(Ea)处理后乙烯信号通路的标志基因PDF1．2和PR-3b被诱导表达，说明HrpN_(Ea)激活乙烯信号通路。同样地，在番茄和烟草中编码乙烯受体类的基因LeETR1和NtETR1在HrpN_(Ea)处理后也诱导表达，并且在对照和处理0小时时没有明显的组成性表达。HrpN_(Ea)喷雾处理20天拟南芥野生型和突变体etr1-1幼苗后，突变体中的AtEXP10的表达量相比较野生型极大的降低，处理2天后，突变体中基因表达水平仅是野生型的5％。当用HrpN_(Ea)和乙烯抑制剂1-MCP混合处理番茄、烟草和水稻幼苗后，LeEXP2、NtEXP2和OsEXP4基因的表达量比HrpN_(Ea)单独处理时有明显的下降。HrpN_(Ea)和1-MCP共同处理番茄、烟草和水稻后，植株的生长明显受到抑制。因此，在供试的四种植物中，乙烯的接受在HrpN_(Ea)促生长和诱导EXP基因的表达过程中是关键的。
我们比较了HrpN_(Ea)和GA_3对水稻OsEXPs基因的影响。水稻中EXPs基因均能被HrpN_(Ea)和GA_3诱导增强表达。水稻中HrpN_(Ea)诱导OsEXP2、OsEXP4和OsEXP16表达增强，作用类似于GA_3。但HrpN_(Ea)的作用明显滞后于GA_3，并且诱导效应低于GA_3。尤其是OsEXP2和OsEXP16，在GA_3处理6小时后开始持续增强表达，而HrpN_(Ea)处理12小时后表达水平才逐渐增强。OsEXP4基因在处理12小时后表达开始明显增强，然后一直保持在正常表达水平。我们使用了赤霉素合成抑制剂pp333来验证HrpN_(Ea)在诱导EXP基因的表达和促生长作用是否需要赤霉素信号传导。HrpN_(Ea)处理水稻幼苗后OsEXP4被极大地诱导增强表达，HrpN_(Ea)和抑制剂pp333共处理后，OsEXP4的表达却被抑制了。在番茄和烟草中，pp333同样抑制了HrpN_(Ea)对LeEXP2和NtEXP2诱导表达效应。以上结果可知，HrpN_(Ea)诱导EXPs基因的表达和促生长效应需要赤霉素信号通路。为了检测拟南芥中赤霉素是否参与了HrpN_(Ea)促生长和诱导AtEXP基因过程，研究使用了野生型(Col-0)植株和赤霉素合成突变体ga5-1。HrpN_(Ea)诱导AtEXP7基因在根中增强表达，而AtEXP10基因在叶中被诱导增强表达；同野生型相比，突变体ga5-1在HrpN_(Ea)处理后这两个基因均没有明显的表达。浸种试验中，HrpN_(Ea)不能诱导ga5-1的促生长效应。因此，阻断赤霉素合成同样消弱了HrpN_(Ea)的促生长和诱导EXPs基因表达的作用。
本研究结果说明HrpN_(Ea)促生长和诱导EXPs基因表达需要乙烯和赤霉素信号通路。
3．烟草诱导防卫与生长调控相关基因CES1的克隆和功能初步分析
本实验室以前使用病毒TMV N14和CMV SV52分别诱导处理NC89后，通过体细胞无性系繁殖得到两个突变体ces1-1、ces2-1，三者在抗病性和长势上存在明显差异，在幼苗期ces2-1的长势最强，ces1-1的长势最弱；5-6叶期之后NC89的长势最强，而ces2-1的长势减弱，ces1-1的长势最弱。5-6叶期接种烟草赤星病菌(Alternaria alternata)发现，ces1-1、ces2-1的抗病性较NC89明显强，且ces2-1较ces1-1强。
本研究中利用差显片段设计特异性引物，RACE法克隆到全长基因，CES1基因ORF长372 bp碱基，翻译出的蛋白质一级机构包括124个氨基酸，命名为CES1(constitutive expresser of systemic acquired resistancel)。通过功能分析发现，此蛋白质在53-69 bp之间有一个富含脯氨酸/苏氨酸区域(Pro／Thr-rich domain)、含有一个氨基葡聚糖结合位点、两个蛋白激酶C磷酸化位点、两个酪氨酸蛋白激酶Ⅱ磷酸化位点以及三个N端酰基化位点。在线预测该蛋白质发现有2个α-螺旋、3个β-片、6个翻转。由于此蛋白质研究较少，没有预测到此蛋白质的空间结构。二级结构预测没有发现信号肽。通过序列比对发现该基因是在高等植物中是保守的，并且在N末端和C末端高度保守。2,4-D和HrpN_(Ea)处理后均增强了突变体的抗病性，并诱导增强防卫相关基因的表达。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明：CES1定位于细胞核内。通过抗病性分析发现，CES1正向调节抗病性，负向调节生长。
4．乙烯与脱落酸信号共同调控HrpN_(Ea)诱导的韧皮部防卫反应与昆虫驱避
植物韧皮部相关防卫(plant phloem-related defense，PRD)能够抵抗刺吸式昆虫的取食。已知HrpN_(Ea)类生物激发子能够激发抗虫信号传导途径，可能参与了韧皮部防卫反应的诱导，但是抗虫途径如何调节PRD反应仍是未知的。本章初步研究了拟南芥ABA和ET信号传导通路协同作用参与了HrpN_(Ea)介导的PRD反应及抗虫驱避效应，并且初步筛选了13个受HXpN_(Ea)上调并且参与调控ABA和ET信号通路的转录调节因子。原位杂交及化学发光实验结果证明HrpN_(Ea)处理后可以增加在韧皮部产生韧皮部相关蛋白的表达及沉积胼胝质，两者共同构成了PRD反应，并且有利于驱避蚜虫、减少蚜虫韧皮部取食活动；实验结果证明HrpN_(Ea)诱导的蚜虫驱避效应需要EIN2扣ABI2因子的协同作用，同时蚜虫的取食活动同驱避效应有关。
对拟南芥生态型Col-0和Ler-0在HxpN_(Ea)处理后蚜虫趋避效应以及蚜虫刺吸电子信号分析，结果表明蚜虫更喜好取食Col-0型植株；同时对照处理中，蚜虫驱避效应主要发生在20-40分钟和3-12小时两个阶段；而在HrpN_(Ea)处理中，蚜虫驱避主要集中在1-12小时。此外，HrpN_(Ea)处理增加了蚜虫对叶片的试探性的刺吸，而韧皮部取食活动明显降低，说明处理后降低了蚜虫对植株的取食喜好。
为了确定HrpN_(Ea)处理后ET和ABA信号传导途径是否影响蚜虫的取食活动，我们使用了拟南芥ET和ABA信号通路相关的突变体。HrpN_(Ea)处理后，突变体ein5-1和abi1-1在HrpN_(Ea)处理后，同野生型处理一样降低了蚜虫取食的喜好。相反地，突变体ein2-1和abi2-1处理后这种降低效应不明显，蚜虫喜好没有明显降低，对蚜虫的活动没有明显的影响。因此，HrpN_(Ea)诱导植株的蚜虫趋避效应需要EIN2和ABI2的协同作用，主要作用在蚜虫韧皮部取食活动阶段，降低蚜虫对植物的喜好。
遗传学和化学药物抑制双重实验结果说明，ET和ABA信号通路交叉调控HrpN_(Ea)诱导的抑制蚜虫繁殖力的效应，植株ET和ABA的产生及接受能力对于蚜虫趋避是很关键的。化学抑制ABA不敏感突变体abi2-1中ET的接受以及化学抑制乙烯不敏感突变体ein2-1中ABA生物合成均抑制了HrpN_(Ea)的诱导效应。
同时罗丹明B及胼胝质沉积的化学发光实验也证明HrpN_(Ea)处理后降低了蚜虫对植株的取食喜好，增加了韧皮部胼胝质沉积，并且这一过程需要EIN2和ABI2的协同作用。通过分析ET和ABA信号通路中关键基因和标志基因以及韧皮部相关基因的表达谱，表明HrpN_(Ea)处理后，ET和ABA信号途径协同作用，并且EIN2作用于ABI2的上游。通过对花柱顶端横切面的原位杂交结果可以看出，AtPP2-A1和AtPP2-A2基因定位在韧皮部，并且在HrpN_(Ea)处理的植株中杂交信号强于对照植株。
5．总结
通过以上研究结果，我们对harpins诱导防卫和调控生长的作用机理有了更进一步的认识。第一，本研究根据HpaG_(Xooc)不同功能域和区段，分离、鉴定了九个HpaG_(Xooc)的蛋白质功能片段，并对不同的功能片段进行了生物活性测定，结果显示不同的功能片段在植物上的效应也不相同，其中功能片段HpaG_(62-137)能在烟草上诱导更强的HCD，而HpaG_(10-42)不表现明显的细胞死亡，但他们都能在水稻上激发更强的防卫反应和促进植株更快的生长。第二，HrpN_(Ea)促进植物生长过程中，诱导了细胞壁松弛蛋白基因EXPs基因的表达，并且两者需要通过乙烯和赤霉素信号通路。在番茄、烟草和拟南芥中，抑制赤霉素合成和乙烯接受同样也抑制了HrpN_(Ea)诱导EXP基因表达和促生长效应。第三，本论文初步研究了拟南芥脱落酸(ABA)和乙烯(ET)信号传导通路参与了HrpN_(Ea)介导的PRD反应及抗虫趋避效应，结果证明HrpN_(Ea)诱导的蚜虫趋避效应需要EIN2(ETHYLENE INSENSITIVE 2)和ABI2(ABA INSENSITIVE 2)因子的协同作用，原位杂交及化学发光实验结果证明HrpN_(Ea)处理后可以增加在韧皮部产生韧皮部相关蛋白的表达及沉积胼胝质，并且初步筛选了13个受HrpN_(Ea)上调并且参与调控ABA和乙烯信号通路的转录调节因子。
The activation of defense responses in plants is initiated by host recognition of pathogen-encoded molecules called elicitors, e.g., microbial proteins, small peptides, and oligosaccharides, etc. The interaction of pathogen elicitors with host receptors (many of which may be encoded by R genes) likely activates a signal transduction cascade that may involve protein phosphorylation, ion fluxes, reactive oxygen species (ROS), and other signaling events. Subsequent transcriptional and/or posttranslational activation of transcription factors eventually leads to the induction of plant defense genes. In addition to eliciting primary defense responses, pathogen signals may be amplified through the generation of secondary plant signal molecules such as SA. Both primary pathogen elicitors and secondary endogenous signals may activate a diverse array of plant protectant and defense genes. So it is ubiquitous for these pathways to crosstalk with each other, depending on which signaling components are recruited into the pathway in response to different stimuli. Such signaling components are the keys to link different pathways and construct an efficient signaling network for plant to confront challenges encountered.
1. Identification of specific fragments of HpaG_(Xooc), a Harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in rice
Harpin 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. Identification of beneficial and deleterious regions might facilitate the advantageous use of harpin-related proteins on crops without causing negative effects like cell death. HpaG_(Xooc) 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 that contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Here we report the identification and testing of nine functional fragments of HpaGxooc using PCR-based mutagenesis. These specific proteins named hpaG_(1-105), hpaG_(1-94), hpaG_(1-61), hpaG_(1-47), hpaG_(7-61), hpaG_(62-137), hpaG_(10-42), hpaG_(95-137) and hpaG_(84-94) which span the indicated amino acid residues of the HpaG sequence, which caused different responses following their application to Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Cell death levels were close when induced by HapG_(1-94), HpaG_(95-137), HpaG_(84-94), and HpaG_(Xooc), over 63% greater in response to HpaG_(1-105), HpaG_(1-16), HpaG_(1-47), or HpaG_(7-62), and AGRM, but 2-fold greater when induced by HpaG_(62-137) compared to HpaG_(Xooc)- Noticeably, HpaG_(10-12) was over 90% less active than other proteins in the effect. Fragments HpaG_(62-137) and HpaG_(10-42) 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, HpaG_(Xooc).
When the 10 variants of HpaG_(Xooc) were used to induce resistance to Xanthomonas oryzae pv. oryzae, which causes bacterial leaf blight of rice, HpaG_(1-61) and HpaG_(7-61) were much more active than others except HpaG_(10-42), whereas HpaG_(10-42) was most effective, HpaG_(62-137) surprisingly provided the lowest level of disease resistance. Of the nine fragments and full-length HpaG_(Xooc) tested to induce the resistance to Magnaporthe grisea, which causes rice blast, blast symptoms shown as tissue necrosis appeared on several sites of leaves treated with EVP, but were limited on HpaG_(Xooc)-treated leaves. On leaves treated with HpaG_(62-137), infection signs were found but necrosis was not evident, while even infection signs were not evident on leaves treated with HpaG_(10-42) So HpaG_(10-42) was consistent in impeding pathogenicity by the bacterial and fungal pathogens.
We studied rice root growth by soaking seeds separately in solutions of cell-free preparations of the 12 proteins. Compared to control, all the proteins tested supported better growth of roots as observed at 6 dpt. When compared with HpaG_(Xooc), HpaG_(1-105), HpaG_(1-62), HpaG_(1-47), HpaG_(62-137), and HpaG_(84-94) were less active; in contrast,△GRM, HpaG_(1-94), HpaG_(7-61), and HpaG_(10-42) increased root length by 15%, 55%, 20%, and 48%, respectively. Clearly, HpaG_(1-94) and HpaG_(10-42) were robust in promoting rice growth.
Because HpaG_(1-94), HpaG_(10-42), HpaG_(62-137), and AGRM markedly preponderated over HpaG_(Xooc) to affect plants in assays with cell-free protein preparations, purified forms of these proteins were tested for bioactivities to corroborate results shown above. We obtained the same results in testing both cell-free and purified proteins. Because HpaG_(62-137) and HpaG_(10-42) tested versus other proteins provided higher levels of HCD and all the defined beneficial effects, respectively, both variants were compared to HpaG_(Xooc) in activating plant signaling events associated with HCD and associated with plant defense and growth.
In leaves treated with HpaG_(62-137), hsr203 and hin1 both were induced to accumulate transcripts since 6 hpt and expression leaves were increased with time. When compared at 48 hpt, markedly greater transcripts of both genes were detected in leaves treated with HpaG_(62-137) than HpaG_(Xooc). We studied responses of NPR1 and PR1 to HpaG_(10-42) tested in comparison with HpaG_(Xooc), both genes were expressed at levels increased with time after treatment with HpaG_(10-42), as monitored at intervals in 48 hpt. The low levels of expression in 6 hpt suggested that both genes were induced for transcription. Especially, the expression of PR1 seemed dependent of induction because little transcripts were detectable as constitutive expression determined immediately after treatment. Comparison indicated that HpaG_(10-42) was markedly stronger than HpaGxooc in the induction of NPR1 and PR1 expression analyzed at 48 hpt. HpaG_(10-42) was more active than HpaG_(Xooc) in inducing expression of several genes that regulate rice defence and growth processes, which may explain the greater beneficial effects observed. We found also that specific fragments are more effective in activating certain signaling pathways than HpaG_(Xooc). Overall, our results suggest that a particular fragment of HpaG_(Xooc), HpaG_(10-42), holds promise for practical agricultural use to enhance disease resistance and growth of rice.
2. Plant growth and expansin gene expression regulated by ethylene and gibberellin in response to a bacterial typeⅢeffector
Expansin proteins (EXPs) act to loose cell walls and modulate growth of the cell and plant under mediation by some hormones. It is not clear whether a hormone is specific and distinct hormones interact to regulate EXP activity. Here we report plant growth enhancement (PGE) and expansin gene (EXP) expression in response to HrpN_(Ea), a bacterial type-Ⅲeffector, and roles of ethylene (ET) and gibbrellin (GA) in both responses.
Arabidopsis root growth at 15 dpt and weight of 20-d seedlings revealed 89% and 67% increases following soaking seeds and spaying treatment by HrpN_(Ea) vs. EVP. Treating tomato seeds with a HrpN_(Ea) solution promoted seed germination and subsequent growth of roots and plants; Plants grew better evidently in treatment with HrpN_(Ea) vs. EVP, as observed at 12 and 30 dpt; Similarly, 46% and 52% increases were found in height and weight of 12-d tomato plants. The percentages were 32% and 50% for height and weight of 40-d tobacco plants, 56% and 48% for height and weight of 12-d rice plants treating with a HrpN_(Ea) solution. These results suggest that HrpN_(Ea) similarly stimulates growth of the different plants. Besides, HrpN_(Ea) treatment resulted in not only increase in leaf size but also production of 2-4 more leaves per plants compared to control. For example, Arabidopsis and tobacco plants had 2-3 and 3-4 additional leaves in treatment with HrpN_(Ea) vs. EVP when plants were surveyed at 30 and 40 dpt. Total nitrogen content was increased by 28%, 36.4%, 28.9% and 25.4%, respectively, in Arabidopsis, tomato, tobacco and rice plants following treatment with HrpN_(Ea) in contrast to EVP. The increase was consistent with PGE levels in the plants. Levels of chlorophylls a and b, total levels either, increased evidently in the fours plants responding to HrpN_(Ea) vs. EVP, as determined at 10 d after treated imbibed seeds. Moreover, marked increases were seen in proportion of chlorophyll a to b. Therefore, HrpN_(Ea) intensifies the biosynthetic and productive pathways through affecting the basic physiological responses.
We conducted RT-PCR protocols using EF1αas a standard to test EXP expression in Arabidopsis, tomato and tobacco plants following treatment with HrpN_(Ea). Expression of AtEXP7 in Arabidopsis leaves and stems was not evident but showed high levels in roots. In contrast, LeEXP5 preferred to express in stems but not leaves and roots, whereas the other 8 EXPs, including AtEXP10, LeEXP2 and AtEXP2, were expressed greater in leaves compared to roots and stems. The induced expression of AtEXP 16 and NtEXP1 was quite similar to that of AtEXP10 and NtEXP2, respectively. AtEXP10, LeEXP2 and NtEXP2 were leaf-specific in expression accumulated transcripts at lower levels in stems and roots vs. leaves. When expressed in the preferential organs, patterns and time course of expression varied depending on genes. AtEXP2, LeEXP18 and NtEXP2 exhibited a similar expression pattern; they increased transcripts gradually with time in 72 or 96 hpt; the other EXPs maintained high expression levels from 6 hpt through 72 hpt.
To relate an ET signal with EXPs and PGE, we studied a transcriptional coincidence of ET-response genes with EXPs and PGE. ETR1 was induced by the protein and increased levels with time. EIN2 was constitutive and was enhanced quickly at 6 hpt with HrpNEa but not change evidently thereafter. The PDF1.2 and PR-3b were expressed after induction, suggesting an activation of the pathway. Consistently, LeETR1 and NtETR1, which encode ET receptor homologs in tomato and tobacco, showed to be induced by HrpN_(Ea) applied to 20-d plants. We found that the Arabidopsis etr1-1 mutant markedly compromised AtEXP10 expression, compared to WT, in 20-d seedlings sprayed with a HrpN_(Ea) solution. PGE in tomato, tobacco and rice was impaired markedly by 1-MCP present in HrpN_(Ea) treatment, a ethylene sensitivity inhibitor. Growth of plants subsequent to soaking seeds in a solution of HrpN_(Ea) and 1-MCP was evidently decreased relative to that in treatment with only HrpN_(Ea). Therefore, ET sensing is critical to the induction of EXP expression and PGE by HrpN_(Ea) in the four plants.
We compared HrpN_(Ea) and GA_3 in the effects on OsEXPs, which increase expression in rice plants treated with GA_3, an important form of GA. When applied to 20-d rice plants, HrpN_(Ea) acted similarly as did GA_3 in inducing expression of OsEXP2, OsEXP4 and 0sEXP16. However,HrpN_(Ea) seemed to function slower and less effectively than GA_3; gene expression became evident earlier and expression levels also were greater in response to GA_3 vs. HrpN_(Ea). In particular, OsEXP2 and OSEXP16 were expressed at high extents successively since 6 hpt with GA_3 but increased expression levels gradually with time since 12 hpt with HrpN_(Ea). OsEXP4 markedly accumulated transcript at 12 hpt with HrpN_(Ea) or GA_3 and thereafter remained a moderate level of expression. We addressed if EXP expression and PGE require a GA signal by determining effects of the GA synthesis inhibitor pp333 on EXPs and PGE. The expression of OsEXP4 was greatly compromised when pp333 was present in HrpN_(Ea) treatment. In WT tomato, the expression of LeEXP2 induced by the application of HrpN_(Ea) was inhibited by pp333 supplied to HrpN_(Ea) treatment. NtEXP2 showed evident constitutive expression and was enhanced markedly to increase expression level by HrpN_(Ea) applied alone; pp333 eliminated the effect. In consistence, pp333 evidently impaired the effect of HrpN_(Ea) in promoting growth of the three plants. Based on these results, a GA signal is required for the induction of EXP expression and PGE.
3. Cloning and function analysis of tobacco cDNA involved in constitutive expression of systemic acquired resistance.
Tobacco constitutive expresser of SAR1-1 and ces2-1 mutants have been generated from the variety NC89. The three genotypes varied greatly in growth and resistance to Alternaria alternata. Primers used for rapid amplification of cDNA 5'- and 3'- ends (5'-RACE and 3'-RACE) were designed on the basis of the mRNA differential display fragment. 5- terminal fragment cloned by 5'-RACE and 3'- terminal fragment amplified by 3'-RACE were pieced together integrally and the complete sequence was validated by homologous Blast and DNAStar software.
The newly identified cDNA fragment was 759 bp in length which included the complete ORF or CDS of CES1 from 88 bp to 459 bp coding 123 amino acid (aa), and nominated CES1. Beside the ORF region 372 bp in length, 87 bp upstream form initiation codon ATG and 300 bp downstream from stop codon TGA were also amplified by 5'-RACE and 3'-RACE, respectively.
The online blast result indicated that CES1 shared 89% identities with the auxin-repressed protein gene (APR1) in a 123 aa region. The predicted protein contained a Glycosaminoglycan attachment site, two Protein kinase C phosphorylation sites, two Casein kinaseⅡphosphorylation sites and three N-myristoylation sites. A sequence similarity search in public database showed that the ARP gene has homologs in various higher plants including monocots and dicots. The deduced amino acid sequences are highly conserved among these homologs (up to 89% identity).
The semi-quantitative RT-PCR assay showed the development and defense-related genes were strongly induced by treated with 2,4-D and hrpN_(Ea) and the plants were enhanced to resistance to Alternaria alternate. Transient expression of the CES1-GFP protein in onion epidermal cell showed that CES1 was localized in cell nuclei. These indicated that the CES1 gene positively regulates SAR but negatively regulates growth by regulating associating gene and activating different signaling pathway.
4. Ethylene and abscisic acid signaling synergizes to regulate the phloem-related defense and insect repellency in plants responding to HrpN_(Ea)
Plant phloem-related defense (PRD) functions against attacks by sap-sucking insects. Known signaling pathways are implicated in PRD induction by biotic elicitors, such as HrpN_(Ea) (a bacterial type-Ⅲprotein), but how the pathways interact to regulate PRD is unclear. Here we show that abscisic acid (ABA) and ethylene (ET) signaling synergism modulated by transcription factor(s) controls HrpNEa-induced PRD and insect repellency in Arabidopsis and we have screened 13 transcription factors up-regulated by HrpN_(Ea). Aphid repellency and depressed phloem-feeding activities were attributed to PRD configuration by phloem protein and callose deposition in the phloem. Aphid Repellency Is Affected by Feeding Activity and Both Events Require Plant EIN2/ABI2 Synergism in Response to HrpN_(Ea).
5. Summary remarks
Results obtained from studies described above have provided us with further understanding on action mechanism of defense and growth in plants responding to Harpin proteins. First, we identify and test nine functional fragments of HpaG_(Xooc), and these proteins have different effects on Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Among them, HpaG_(62-137) and HpaG_(10-42) 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, HpaG_(Xooc) Second, plant growth enhancement was induced by HrpN_(Ea) concomitantly with induced expression of EXPs gene, a cell wall loose protein, and both inductions require GA and ET in plants responding to HrpN_(Ea). The results show that inhibiting Arabidopsis, tomato and tobacco plants to synthesize GA or sense ET compromised EXP expression and PGE phenotypes. Third, abscisic acid and ethylene signaling controls HrpNEa-induced PRD and insect repellency in Arabidopsis, and aphid repellency requires plant EIN2/ABI2 synergism in response to HrpN_(Ea). Moreover, we have screened 13 transcription factors based on their gene transcripts up-regulated by HrpN_(Ea).
革兰氏阴性植物病原细菌产生的蛋白质激发子harpins可诱导植物多种反应，如抗病，抗虫，促生长和抗旱等。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，harpins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，它们相互之间有没有交叉对话，如何交叉对话、以及harpins如何实现这些功能等多个问题成为了研究的重点。
1．不同HpaG_(Xooc)功能片段的分离鉴定及对植物的诱导抗病和促生长作用
Harpins是由革兰氏阴性植物病原细菌产生的一类蛋白质激发子，他们的共同特点是：富含甘氨酸、热稳定、对蛋白酶敏感，注射到产生菌的非寄主植物叶片细胞间隙后，可以诱导过敏性细胞死亡(hypersensitive cell death,HCD)，诱导植物产生防卫反应、促进植物生长。Harpin蛋白的不同效应，有些是对植物有益的，而这些效应的发生可能是依赖于蛋白质不同的功能域实现的。分离、鉴定蛋白质有益和有害的功能区域，更有利于我们将harpin相关蛋白应用于农作物，而不产生诸如细胞坏死之类的负面效应。
水稻细菌性条斑病菌(Xanthomonas oryzae pv．oryzicola)Hpa G_(Xooc)作为Harpin蛋白家族中的一员，包含两个富含甘氨酸的结构域(glycine-rich motif,GRM)，一个半胱氨酸残基(cysteine)。GRM是Harpin类蛋白质共有的特征，而半胱氨酸则是其他Harpin类蛋白质所没有的。本研究根据HpaG_(Xooc)不同功能域和区段，基于PCR的体外诱变技术产生九个HpaG_(Xooc)的蛋白质功能片段，分别为HpaG_(1-105)、HpaG_(1-94)、HpaG_(1-61)、HpaG_(1-47)、HpaG_(7-61)、HpaG_(62-137)、HpaG_(10-42)、HpaG_(95-137)、HpaG_(84-94)，右下角的数字分别代表了蛋白质所包括的HpaG_(Xooc)相应的氨基酸区域．将这九个片段体外表达，产生的蛋白质施用于烟草(Nicotiana tabaccum)和水稻(Oryza sativa)能产生不同的效应。HapG_(1-94)、HpaG_(95-137)、HpaG_(84-94)和HpaG_(Xooc)诱导细胞死亡的能力相近，HpaG_(1-105)、HpaG_(1-61)、HpaG_(1-47)、HpaG_(7-62)及△GRM诱导细胞死亡的能力高于HpaG_(Xooc)63％以上；HpaG_(62-137)诱导细胞死亡的能力是HpaG_(Xooc)的2倍，但是HpaG_(10-42)诱导细胞死亡的能力低于其他蛋白约90％以上。
同HpaG_(Xooc)全长蛋白相比较，HpaG_(62-137)能在烟草上诱导更强的HCD，而HpaG_(10-42)不表现明显的细胞死亡，但都能在水稻上能激发更强的防卫反应和促进植株更快的生长。在检测的9个片段及全长蛋白质中，对水稻白叶枯病菌(Xanthomonasoryzae pv.oryzae)的抗性诱导效应中，HpaG_(10-42)诱导效应最强，HpaG_(1-61)和HpaG_(7-61)次之，HpaG_(62-137)的诱导抗病性效应表现为最低；而对稻瘟病菌(Magnaporthe grisea)的诱导抗性效应中，HpaG_(62-137)处理的叶片上仅能看到侵染点，没有明显的坏死斑；而HpaG_(10-42)处理的叶片上甚至连坏死斑都无法看见。因此，HpaG_(10-42)能同时抑制细菌和真菌病原的致病性。在对水稻的促生长效应中，以HpaG_(Xooc)为对照，HpaG_(1-105)、HpaG_(1-62)、HpaG_(1-47)、HpaG_(62-137)和HpaG_(84-94)的促生长效应较弱；但是△GRM、HpaG_(1-94)、HpaG_(7-61)及HpaG_(10-42)处理后根系分别增长了15％、55％、20％和48％。因此HpaG_(1-94)和HpaG_(10-42)在促进水稻生长的效应最强。
在蛋白粗提液实验中HpaG_(1-94)、HpaG_(10-42)、HpaG_(62-137)和△GRM对植物的作用明显强于HpaG_(Xooc)，因此我们将这几种蛋白质进行镍柱纯化，并进一步检测、验证他们的生物活性以确证上面的实验结果。从结果可以看出不管是粗蛋白提取液还是纯化蛋白质均表现相同的效应。
由于在检测的所有蛋白质中HpaG_(62-137)和HpaG_(10-42)表现为更高水平的HCD诱导活性及所有其他有益效应，因此我们使用HpaG_(62-137)和HpaG_(10-42)同HpaG_(Xooc)相比较，来检测他们在诱导植物HCD和激活生长防卫相关信号传导通路的活性。HpaG_(62-137)处理后HCD标志基因hsr203和hin1基因在处理6小时后就开始积累转录，并随着处理时间的推移叶片中的表达量也逐渐增加；处理48小时后，HpaG_(62-137)处理的叶片中这两个基因的表达量明显强于HpaG_(Xooc)的处理。此外HpaG_(10-42)处理6个小时后防卫反应基因NPR1和PR1开始被诱导表达；特别是PR1基因的表达明显地依赖于诱导，因为在处理0小时时没有明显的组成性表达。从HpaG_(10-42)和HpaG_(Xooc)处理48小时后NPR1和PR1基因表达的结果可以看出，HpaG_(10-42)的诱导表达效应明显强于HpaG_(Xooc)。由于HpaG_(10-42)的诱导活性明显强于HpaG_(Xooc)全长蛋白质的活性，因此可以解释我们所观察到的植物上有益效应的表型。我们同时发现某些特异性片段相比较全长蛋白更能有效地激活某些信号通路。总之，本研究结果阐释了一个HpaG_(Xooc)的特异性片段HpaG_(10-42)能促进水稻生长和诱导抗病性，具有潜在的农业应用价值。
2．HrpN_(Ea)诱导植物生长和膨胀素基因表达及乙烯与赤霉素的调控作用
膨胀素(Expansin)是植物细胞生长期间释放的一种能使细胞壁松弛的蛋白质，是细胞壁的关键调节剂，对细胞生长有重要作用。有研究表明植物激素介导了膨胀素的功能，但是植物激素是特异性作用还是共同起作用及作用机制仍然不是很清楚。本文报道了HrpN_(Ea)促进植物生长和诱导expansin基因表达所启动的不同信号通路。
拟南芥浸种处理15天后在MS平板上可以看见，HrpN_(Ea)可以明显的促进根系生长，根系增长89％，幼苗鲜重增加67％；同样盆播幼苗在HrpN_(Ea)喷雾处理15天和30天后也明显的促进的植株地上部分的生长。HrpN_(Ea)浸种番茄后可以促进种子萌发、根系生长及后期的营养生长，HrpN_(Ea)处理12天后植株高度增加46％，鲜重增加52％。烟草在HrpN_(Ea)处理20天后可以看见促生长作用，到40天促生长作用更明显，高度和鲜重分别增加32％和50％，到处理60天营养生长后期，与对照相比，地上部分生长明显加快，茎节粗壮，叶片肥大，长势旺盛。HrpN_(Ea)处理水稻后极大的促进了种子萌发和根系生长，处理12天后可以明显观察到促生长作用，高度和鲜重分别增加56％和48％，直接促进了幼苗的生长．此外与对照相比，HrpN_(Ea)不仅增加了植株叶面积，而且还增加了叶片数量。HrpN_(Ea)明显增加植物体内全氮含量，其提高幅度分别比对照高28％(拟南芥)，36．4％(番茄)，28．9％(烟草)和25．4％(水稻)。HrpN_(Ea)浸种处理四种植物10天后，叶绿素a和叶绿素b及叶绿素总量均明显增加。此外，HrpN_(Ea)处理后，叶绿素a／b比值也显著高于对照处理。当HrpN_(Ea)喷雾处理10天与20天的植株地上部分后，叶片全氮量和叶绿素含量同样增加。因此，HrpN_(Ea)通过影响基本生理过程来强化植物的生物合成功能和增加植物生物学产量．
我们检测了HrpN_(Ea)处理四种不同植物后对生长相关基因的诱导表达。HrpN_(Ea)处理后拟南芥AtEXP7在叶片和茎中表达不明显，而在根中表达量较高；LeEXP2和LeEXP5在茎中表达而在叶片和根中不表达；另外8个EXPs，包括AtEXP10和AtEXP2在叶片中表达量高，而在根和茎中表达量较低。同时AtEXP16和NtEXP2的诱导表达分别相似于AtEXP10和NtEXP2，在叶片中特异性表达的EXPs如AtEXP10、LeEXP2和NtEXP2，在根和茎中转录水平明显降低。在不同的器官、不同的处理模式及诱导时间的不同，基因表达水平也不相同。AtEXP2、LeEXP18和NtEXP2表达模式相同，在处理72小时和96小时内随着处理时间的推移表达量逐渐增加。相反地，其他的EXPs基因在处理6小时后直到72小时始终保持高水平表达量。
为了阐释HrpN_(Ea)诱导EXPs基因表达和促生长作用同乙烯信号通路的相关性，我们测定了HrpN_(Ea)处理后乙烯信号通路中关键基因的表达情况及同EXPs表达的关系。拟南芥中ETR1的表达受HrpN_(Ea)诱导，并且随着诱导时间表达量逐渐增强；EIN2表达是组成性的，并且在HrpN_(Ea)处理6小时后表达量快速增加达到顶峰，随后不再增加。HrpN_(Ea)处理后乙烯信号通路的标志基因PDF1．2和PR-3b被诱导表达，说明HrpN_(Ea)激活乙烯信号通路。同样地，在番茄和烟草中编码乙烯受体类的基因LeETR1和NtETR1在HrpN_(Ea)处理后也诱导表达，并且在对照和处理0小时时没有明显的组成性表达。HrpN_(Ea)喷雾处理20天拟南芥野生型和突变体etr1-1幼苗后，突变体中的AtEXP10的表达量相比较野生型极大的降低，处理2天后，突变体中基因表达水平仅是野生型的5％。当用HrpN_(Ea)和乙烯抑制剂1-MCP混合处理番茄、烟草和水稻幼苗后，LeEXP2、NtEXP2和OsEXP4基因的表达量比HrpN_(Ea)单独处理时有明显的下降。HrpN_(Ea)和1-MCP共同处理番茄、烟草和水稻后，植株的生长明显受到抑制。因此，在供试的四种植物中，乙烯的接受在HrpN_(Ea)促生长和诱导EXP基因的表达过程中是关键的。
我们比较了HrpN_(Ea)和GA_3对水稻OsEXPs基因的影响。水稻中EXPs基因均能被HrpN_(Ea)和GA_3诱导增强表达。水稻中HrpN_(Ea)诱导OsEXP2、OsEXP4和OsEXP16表达增强，作用类似于GA_3。但HrpN_(Ea)的作用明显滞后于GA_3，并且诱导效应低于GA_3。尤其是OsEXP2和OsEXP16，在GA_3处理6小时后开始持续增强表达，而HrpN_(Ea)处理12小时后表达水平才逐渐增强。OsEXP4基因在处理12小时后表达开始明显增强，然后一直保持在正常表达水平。我们使用了赤霉素合成抑制剂pp333来验证HrpN_(Ea)在诱导EXP基因的表达和促生长作用是否需要赤霉素信号传导。HrpN_(Ea)处理水稻幼苗后OsEXP4被极大地诱导增强表达，HrpN_(Ea)和抑制剂pp333共处理后，OsEXP4的表达却被抑制了。在番茄和烟草中，pp333同样抑制了HrpN_(Ea)对LeEXP2和NtEXP2诱导表达效应。以上结果可知，HrpN_(Ea)诱导EXPs基因的表达和促生长效应需要赤霉素信号通路。为了检测拟南芥中赤霉素是否参与了HrpN_(Ea)促生长和诱导AtEXP基因过程，研究使用了野生型(Col-0)植株和赤霉素合成突变体ga5-1。HrpN_(Ea)诱导AtEXP7基因在根中增强表达，而AtEXP10基因在叶中被诱导增强表达；同野生型相比，突变体ga5-1在HrpN_(Ea)处理后这两个基因均没有明显的表达。浸种试验中，HrpN_(Ea)不能诱导ga5-1的促生长效应。因此，阻断赤霉素合成同样消弱了HrpN_(Ea)的促生长和诱导EXPs基因表达的作用。
本研究结果说明HrpN_(Ea)促生长和诱导EXPs基因表达需要乙烯和赤霉素信号通路。
3．烟草诱导防卫与生长调控相关基因CES1的克隆和功能初步分析
本实验室以前使用病毒TMV N14和CMV SV52分别诱导处理NC89后，通过体细胞无性系繁殖得到两个突变体ces1-1、ces2-1，三者在抗病性和长势上存在明显差异，在幼苗期ces2-1的长势最强，ces1-1的长势最弱；5-6叶期之后NC89的长势最强，而ces2-1的长势减弱，ces1-1的长势最弱。5-6叶期接种烟草赤星病菌(Alternaria alternata)发现，ces1-1、ces2-1的抗病性较NC89明显强，且ces2-1较ces1-1强。
本研究中利用差显片段设计特异性引物，RACE法克隆到全长基因，CES1基因ORF长372 bp碱基，翻译出的蛋白质一级机构包括124个氨基酸，命名为CES1(constitutive expresser of systemic acquired resistancel)。通过功能分析发现，此蛋白质在53-69 bp之间有一个富含脯氨酸/苏氨酸区域(Pro／Thr-rich domain)、含有一个氨基葡聚糖结合位点、两个蛋白激酶C磷酸化位点、两个酪氨酸蛋白激酶Ⅱ磷酸化位点以及三个N端酰基化位点。在线预测该蛋白质发现有2个α-螺旋、3个β-片、6个翻转。由于此蛋白质研究较少，没有预测到此蛋白质的空间结构。二级结构预测没有发现信号肽。通过序列比对发现该基因是在高等植物中是保守的，并且在N末端和C末端高度保守。2,4-D和HrpN_(Ea)处理后均增强了突变体的抗病性，并诱导增强防卫相关基因的表达。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明：CES1定位于细胞核内。通过抗病性分析发现，CES1正向调节抗病性，负向调节生长。
4．乙烯与脱落酸信号共同调控HrpN_(Ea)诱导的韧皮部防卫反应与昆虫驱避
植物韧皮部相关防卫(plant phloem-related defense，PRD)能够抵抗刺吸式昆虫的取食。已知HrpN_(Ea)类生物激发子能够激发抗虫信号传导途径，可能参与了韧皮部防卫反应的诱导，但是抗虫途径如何调节PRD反应仍是未知的。本章初步研究了拟南芥ABA和ET信号传导通路协同作用参与了HrpN_(Ea)介导的PRD反应及抗虫驱避效应，并且初步筛选了13个受HXpN_(Ea)上调并且参与调控ABA和ET信号通路的转录调节因子。原位杂交及化学发光实验结果证明HrpN_(Ea)处理后可以增加在韧皮部产生韧皮部相关蛋白的表达及沉积胼胝质，两者共同构成了PRD反应，并且有利于驱避蚜虫、减少蚜虫韧皮部取食活动；实验结果证明HrpN_(Ea)诱导的蚜虫驱避效应需要EIN2扣ABI2因子的协同作用，同时蚜虫的取食活动同驱避效应有关。
对拟南芥生态型Col-0和Ler-0在HxpN_(Ea)处理后蚜虫趋避效应以及蚜虫刺吸电子信号分析，结果表明蚜虫更喜好取食Col-0型植株；同时对照处理中，蚜虫驱避效应主要发生在20-40分钟和3-12小时两个阶段；而在HrpN_(Ea)处理中，蚜虫驱避主要集中在1-12小时。此外，HrpN_(Ea)处理增加了蚜虫对叶片的试探性的刺吸，而韧皮部取食活动明显降低，说明处理后降低了蚜虫对植株的取食喜好。
为了确定HrpN_(Ea)处理后ET和ABA信号传导途径是否影响蚜虫的取食活动，我们使用了拟南芥ET和ABA信号通路相关的突变体。HrpN_(Ea)处理后，突变体ein5-1和abi1-1在HrpN_(Ea)处理后，同野生型处理一样降低了蚜虫取食的喜好。相反地，突变体ein2-1和abi2-1处理后这种降低效应不明显，蚜虫喜好没有明显降低，对蚜虫的活动没有明显的影响。因此，HrpN_(Ea)诱导植株的蚜虫趋避效应需要EIN2和ABI2的协同作用，主要作用在蚜虫韧皮部取食活动阶段，降低蚜虫对植物的喜好。
遗传学和化学药物抑制双重实验结果说明，ET和ABA信号通路交叉调控HrpN_(Ea)诱导的抑制蚜虫繁殖力的效应，植株ET和ABA的产生及接受能力对于蚜虫趋避是很关键的。化学抑制ABA不敏感突变体abi2-1中ET的接受以及化学抑制乙烯不敏感突变体ein2-1中ABA生物合成均抑制了HrpN_(Ea)的诱导效应。
同时罗丹明B及胼胝质沉积的化学发光实验也证明HrpN_(Ea)处理后降低了蚜虫对植株的取食喜好，增加了韧皮部胼胝质沉积，并且这一过程需要EIN2和ABI2的协同作用。通过分析ET和ABA信号通路中关键基因和标志基因以及韧皮部相关基因的表达谱，表明HrpN_(Ea)处理后，ET和ABA信号途径协同作用，并且EIN2作用于ABI2的上游。通过对花柱顶端横切面的原位杂交结果可以看出，AtPP2-A1和AtPP2-A2基因定位在韧皮部，并且在HrpN_(Ea)处理的植株中杂交信号强于对照植株。
5．总结
通过以上研究结果，我们对harpins诱导防卫和调控生长的作用机理有了更进一步的认识。第一，本研究根据HpaG_(Xooc)不同功能域和区段，分离、鉴定了九个HpaG_(Xooc)的蛋白质功能片段，并对不同的功能片段进行了生物活性测定，结果显示不同的功能片段在植物上的效应也不相同，其中功能片段HpaG_(62-137)能在烟草上诱导更强的HCD，而HpaG_(10-42)不表现明显的细胞死亡，但他们都能在水稻上激发更强的防卫反应和促进植株更快的生长。第二，HrpN_(Ea)促进植物生长过程中，诱导了细胞壁松弛蛋白基因EXPs基因的表达，并且两者需要通过乙烯和赤霉素信号通路。在番茄、烟草和拟南芥中，抑制赤霉素合成和乙烯接受同样也抑制了HrpN_(Ea)诱导EXP基因表达和促生长效应。第三，本论文初步研究了拟南芥脱落酸(ABA)和乙烯(ET)信号传导通路参与了HrpN_(Ea)介导的PRD反应及抗虫趋避效应，结果证明HrpN_(Ea)诱导的蚜虫趋避效应需要EIN2(ETHYLENE INSENSITIVE 2)和ABI2(ABA INSENSITIVE 2)因子的协同作用，原位杂交及化学发光实验结果证明HrpN_(Ea)处理后可以增加在韧皮部产生韧皮部相关蛋白的表达及沉积胼胝质，并且初步筛选了13个受HrpN_(Ea)上调并且参与调控ABA和乙烯信号通路的转录调节因子。
The activation of defense responses in plants is initiated by host recognition of pathogen-encoded molecules called elicitors, e.g., microbial proteins, small peptides, and oligosaccharides, etc. The interaction of pathogen elicitors with host receptors (many of which may be encoded by R genes) likely activates a signal transduction cascade that may involve protein phosphorylation, ion fluxes, reactive oxygen species (ROS), and other signaling events. Subsequent transcriptional and/or posttranslational activation of transcription factors eventually leads to the induction of plant defense genes. In addition to eliciting primary defense responses, pathogen signals may be amplified through the generation of secondary plant signal molecules such as SA. Both primary pathogen elicitors and secondary endogenous signals may activate a diverse array of plant protectant and defense genes. So it is ubiquitous for these pathways to crosstalk with each other, depending on which signaling components are recruited into the pathway in response to different stimuli. Such signaling components are the keys to link different pathways and construct an efficient signaling network for plant to confront challenges encountered.
1. Identification of specific fragments of HpaG_(Xooc), a Harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in rice
Harpin 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. Identification of beneficial and deleterious regions might facilitate the advantageous use of harpin-related proteins on crops without causing negative effects like cell death. HpaG_(Xooc) 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 that contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Here we report the identification and testing of nine functional fragments of HpaGxooc using PCR-based mutagenesis. These specific proteins named hpaG_(1-105), hpaG_(1-94), hpaG_(1-61), hpaG_(1-47), hpaG_(7-61), hpaG_(62-137), hpaG_(10-42), hpaG_(95-137) and hpaG_(84-94) which span the indicated amino acid residues of the HpaG sequence, which caused different responses following their application to Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Cell death levels were close when induced by HapG_(1-94), HpaG_(95-137), HpaG_(84-94), and HpaG_(Xooc), over 63% greater in response to HpaG_(1-105), HpaG_(1-16), HpaG_(1-47), or HpaG_(7-62), and AGRM, but 2-fold greater when induced by HpaG_(62-137) compared to HpaG_(Xooc)- Noticeably, HpaG_(10-12) was over 90% less active than other proteins in the effect. Fragments HpaG_(62-137) and HpaG_(10-42) 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, HpaG_(Xooc).
When the 10 variants of HpaG_(Xooc) were used to induce resistance to Xanthomonas oryzae pv. oryzae, which causes bacterial leaf blight of rice, HpaG_(1-61) and HpaG_(7-61) were much more active than others except HpaG_(10-42), whereas HpaG_(10-42) was most effective, HpaG_(62-137) surprisingly provided the lowest level of disease resistance. Of the nine fragments and full-length HpaG_(Xooc) tested to induce the resistance to Magnaporthe grisea, which causes rice blast, blast symptoms shown as tissue necrosis appeared on several sites of leaves treated with EVP, but were limited on HpaG_(Xooc)-treated leaves. On leaves treated with HpaG_(62-137), infection signs were found but necrosis was not evident, while even infection signs were not evident on leaves treated with HpaG_(10-42) So HpaG_(10-42) was consistent in impeding pathogenicity by the bacterial and fungal pathogens.
We studied rice root growth by soaking seeds separately in solutions of cell-free preparations of the 12 proteins. Compared to control, all the proteins tested supported better growth of roots as observed at 6 dpt. When compared with HpaG_(Xooc), HpaG_(1-105), HpaG_(1-62), HpaG_(1-47), HpaG_(62-137), and HpaG_(84-94) were less active; in contrast,△GRM, HpaG_(1-94), HpaG_(7-61), and HpaG_(10-42) increased root length by 15%, 55%, 20%, and 48%, respectively. Clearly, HpaG_(1-94) and HpaG_(10-42) were robust in promoting rice growth.
Because HpaG_(1-94), HpaG_(10-42), HpaG_(62-137), and AGRM markedly preponderated over HpaG_(Xooc) to affect plants in assays with cell-free protein preparations, purified forms of these proteins were tested for bioactivities to corroborate results shown above. We obtained the same results in testing both cell-free and purified proteins. Because HpaG_(62-137) and HpaG_(10-42) tested versus other proteins provided higher levels of HCD and all the defined beneficial effects, respectively, both variants were compared to HpaG_(Xooc) in activating plant signaling events associated with HCD and associated with plant defense and growth.
In leaves treated with HpaG_(62-137), hsr203 and hin1 both were induced to accumulate transcripts since 6 hpt and expression leaves were increased with time. When compared at 48 hpt, markedly greater transcripts of both genes were detected in leaves treated with HpaG_(62-137) than HpaG_(Xooc). We studied responses of NPR1 and PR1 to HpaG_(10-42) tested in comparison with HpaG_(Xooc), both genes were expressed at levels increased with time after treatment with HpaG_(10-42), as monitored at intervals in 48 hpt. The low levels of expression in 6 hpt suggested that both genes were induced for transcription. Especially, the expression of PR1 seemed dependent of induction because little transcripts were detectable as constitutive expression determined immediately after treatment. Comparison indicated that HpaG_(10-42) was markedly stronger than HpaGxooc in the induction of NPR1 and PR1 expression analyzed at 48 hpt. HpaG_(10-42) was more active than HpaG_(Xooc) in inducing expression of several genes that regulate rice defence and growth processes, which may explain the greater beneficial effects observed. We found also that specific fragments are more effective in activating certain signaling pathways than HpaG_(Xooc). Overall, our results suggest that a particular fragment of HpaG_(Xooc), HpaG_(10-42), holds promise for practical agricultural use to enhance disease resistance and growth of rice.
2. Plant growth and expansin gene expression regulated by ethylene and gibberellin in response to a bacterial typeⅢeffector
Expansin proteins (EXPs) act to loose cell walls and modulate growth of the cell and plant under mediation by some hormones. It is not clear whether a hormone is specific and distinct hormones interact to regulate EXP activity. Here we report plant growth enhancement (PGE) and expansin gene (EXP) expression in response to HrpN_(Ea), a bacterial type-Ⅲeffector, and roles of ethylene (ET) and gibbrellin (GA) in both responses.
Arabidopsis root growth at 15 dpt and weight of 20-d seedlings revealed 89% and 67% increases following soaking seeds and spaying treatment by HrpN_(Ea) vs. EVP. Treating tomato seeds with a HrpN_(Ea) solution promoted seed germination and subsequent growth of roots and plants; Plants grew better evidently in treatment with HrpN_(Ea) vs. EVP, as observed at 12 and 30 dpt; Similarly, 46% and 52% increases were found in height and weight of 12-d tomato plants. The percentages were 32% and 50% for height and weight of 40-d tobacco plants, 56% and 48% for height and weight of 12-d rice plants treating with a HrpN_(Ea) solution. These results suggest that HrpN_(Ea) similarly stimulates growth of the different plants. Besides, HrpN_(Ea) treatment resulted in not only increase in leaf size but also production of 2-4 more leaves per plants compared to control. For example, Arabidopsis and tobacco plants had 2-3 and 3-4 additional leaves in treatment with HrpN_(Ea) vs. EVP when plants were surveyed at 30 and 40 dpt. Total nitrogen content was increased by 28%, 36.4%, 28.9% and 25.4%, respectively, in Arabidopsis, tomato, tobacco and rice plants following treatment with HrpN_(Ea) in contrast to EVP. The increase was consistent with PGE levels in the plants. Levels of chlorophylls a and b, total levels either, increased evidently in the fours plants responding to HrpN_(Ea) vs. EVP, as determined at 10 d after treated imbibed seeds. Moreover, marked increases were seen in proportion of chlorophyll a to b. Therefore, HrpN_(Ea) intensifies the biosynthetic and productive pathways through affecting the basic physiological responses.
We conducted RT-PCR protocols using EF1αas a standard to test EXP expression in Arabidopsis, tomato and tobacco plants following treatment with HrpN_(Ea). Expression of AtEXP7 in Arabidopsis leaves and stems was not evident but showed high levels in roots. In contrast, LeEXP5 preferred to express in stems but not leaves and roots, whereas the other 8 EXPs, including AtEXP10, LeEXP2 and AtEXP2, were expressed greater in leaves compared to roots and stems. The induced expression of AtEXP 16 and NtEXP1 was quite similar to that of AtEXP10 and NtEXP2, respectively. AtEXP10, LeEXP2 and NtEXP2 were leaf-specific in expression accumulated transcripts at lower levels in stems and roots vs. leaves. When expressed in the preferential organs, patterns and time course of expression varied depending on genes. AtEXP2, LeEXP18 and NtEXP2 exhibited a similar expression pattern; they increased transcripts gradually with time in 72 or 96 hpt; the other EXPs maintained high expression levels from 6 hpt through 72 hpt.
To relate an ET signal with EXPs and PGE, we studied a transcriptional coincidence of ET-response genes with EXPs and PGE. ETR1 was induced by the protein and increased levels with time. EIN2 was constitutive and was enhanced quickly at 6 hpt with HrpNEa but not change evidently thereafter. The PDF1.2 and PR-3b were expressed after induction, suggesting an activation of the pathway. Consistently, LeETR1 and NtETR1, which encode ET receptor homologs in tomato and tobacco, showed to be induced by HrpN_(Ea) applied to 20-d plants. We found that the Arabidopsis etr1-1 mutant markedly compromised AtEXP10 expression, compared to WT, in 20-d seedlings sprayed with a HrpN_(Ea) solution. PGE in tomato, tobacco and rice was impaired markedly by 1-MCP present in HrpN_(Ea) treatment, a ethylene sensitivity inhibitor. Growth of plants subsequent to soaking seeds in a solution of HrpN_(Ea) and 1-MCP was evidently decreased relative to that in treatment with only HrpN_(Ea). Therefore, ET sensing is critical to the induction of EXP expression and PGE by HrpN_(Ea) in the four plants.
We compared HrpN_(Ea) and GA_3 in the effects on OsEXPs, which increase expression in rice plants treated with GA_3, an important form of GA. When applied to 20-d rice plants, HrpN_(Ea) acted similarly as did GA_3 in inducing expression of OsEXP2, OsEXP4 and 0sEXP16. However,HrpN_(Ea) seemed to function slower and less effectively than GA_3; gene expression became evident earlier and expression levels also were greater in response to GA_3 vs. HrpN_(Ea). In particular, OsEXP2 and OSEXP16 were expressed at high extents successively since 6 hpt with GA_3 but increased expression levels gradually with time since 12 hpt with HrpN_(Ea). OsEXP4 markedly accumulated transcript at 12 hpt with HrpN_(Ea) or GA_3 and thereafter remained a moderate level of expression. We addressed if EXP expression and PGE require a GA signal by determining effects of the GA synthesis inhibitor pp333 on EXPs and PGE. The expression of OsEXP4 was greatly compromised when pp333 was present in HrpN_(Ea) treatment. In WT tomato, the expression of LeEXP2 induced by the application of HrpN_(Ea) was inhibited by pp333 supplied to HrpN_(Ea) treatment. NtEXP2 showed evident constitutive expression and was enhanced markedly to increase expression level by HrpN_(Ea) applied alone; pp333 eliminated the effect. In consistence, pp333 evidently impaired the effect of HrpN_(Ea) in promoting growth of the three plants. Based on these results, a GA signal is required for the induction of EXP expression and PGE.
3. Cloning and function analysis of tobacco cDNA involved in constitutive expression of systemic acquired resistance.
Tobacco constitutive expresser of SAR1-1 and ces2-1 mutants have been generated from the variety NC89. The three genotypes varied greatly in growth and resistance to Alternaria alternata. Primers used for rapid amplification of cDNA 5'- and 3'- ends (5'-RACE and 3'-RACE) were designed on the basis of the mRNA differential display fragment. 5- terminal fragment cloned by 5'-RACE and 3'- terminal fragment amplified by 3'-RACE were pieced together integrally and the complete sequence was validated by homologous Blast and DNAStar software.
The newly identified cDNA fragment was 759 bp in length which included the complete ORF or CDS of CES1 from 88 bp to 459 bp coding 123 amino acid (aa), and nominated CES1. Beside the ORF region 372 bp in length, 87 bp upstream form initiation codon ATG and 300 bp downstream from stop codon TGA were also amplified by 5'-RACE and 3'-RACE, respectively.
The online blast result indicated that CES1 shared 89% identities with the auxin-repressed protein gene (APR1) in a 123 aa region. The predicted protein contained a Glycosaminoglycan attachment site, two Protein kinase C phosphorylation sites, two Casein kinaseⅡphosphorylation sites and three N-myristoylation sites. A sequence similarity search in public database showed that the ARP gene has homologs in various higher plants including monocots and dicots. The deduced amino acid sequences are highly conserved among these homologs (up to 89% identity).
The semi-quantitative RT-PCR assay showed the development and defense-related genes were strongly induced by treated with 2,4-D and hrpN_(Ea) and the plants were enhanced to resistance to Alternaria alternate. Transient expression of the CES1-GFP protein in onion epidermal cell showed that CES1 was localized in cell nuclei. These indicated that the CES1 gene positively regulates SAR but negatively regulates growth by regulating associating gene and activating different signaling pathway.
4. Ethylene and abscisic acid signaling synergizes to regulate the phloem-related defense and insect repellency in plants responding to HrpN_(Ea)
Plant phloem-related defense (PRD) functions against attacks by sap-sucking insects. Known signaling pathways are implicated in PRD induction by biotic elicitors, such as HrpN_(Ea) (a bacterial type-Ⅲprotein), but how the pathways interact to regulate PRD is unclear. Here we show that abscisic acid (ABA) and ethylene (ET) signaling synergism modulated by transcription factor(s) controls HrpNEa-induced PRD and insect repellency in Arabidopsis and we have screened 13 transcription factors up-regulated by HrpN_(Ea). Aphid repellency and depressed phloem-feeding activities were attributed to PRD configuration by phloem protein and callose deposition in the phloem. Aphid Repellency Is Affected by Feeding Activity and Both Events Require Plant EIN2/ABI2 Synergism in Response to HrpN_(Ea).
5. Summary remarks
Results obtained from studies described above have provided us with further understanding on action mechanism of defense and growth in plants responding to Harpin proteins. First, we identify and test nine functional fragments of HpaG_(Xooc), and these proteins have different effects on Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Among them, HpaG_(62-137) and HpaG_(10-42) 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, HpaG_(Xooc) Second, plant growth enhancement was induced by HrpN_(Ea) concomitantly with induced expression of EXPs gene, a cell wall loose protein, and both inductions require GA and ET in plants responding to HrpN_(Ea). The results show that inhibiting Arabidopsis, tomato and tobacco plants to synthesize GA or sense ET compromised EXP expression and PGE phenotypes. Third, abscisic acid and ethylene signaling controls HrpNEa-induced PRD and insect repellency in Arabidopsis, and aphid repellency requires plant EIN2/ABI2 synergism in response to HrpN_(Ea). Moreover, we have screened 13 transcription factors based on their gene transcripts up-regulated by HrpN_(Ea).
引文
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