植物抗病反应的信号传导网络

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应(HR)并延伸到远处组织的系统抗性或获得性抗性(SAR),受制于一种信号传导网络的调控。这个信号系统由抗病蛋白和病原菌非毒性蛋白在一种配体-受体的互作模式下激发,并由信号分子H2O2,NO和系统信号分子SA,JA和乙烯和通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生有类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发。这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

花魔芋抗软腐病植株的鉴定及其抗性机理的初步研究

通过筛选获得了对魔芋(Amorphophallus konjac)软腐病具有较强抗性的花魔芋抗病植株, 经核型分析发现, 其染色体数目与普通植株一致, 均为2n=26。抗病实验结果表明, 该抗病植株对魔芋软腐病的抗性较普通植株强。采用酶联免疫吸附法, 测定了软腐病病原菌接种30小时内的抗病与普通植株叶片中内源植物激素(SA与JA)的含量变化。结果显示, 抗病和普通植株叶片中SA和JA含量变化的总体趋势明显不同。主要表现在, 抗病和普通植株叶片中SA和JA积累的时间不同; 此外, 积累的量也有明显差异。推测魔芋抗病植株可能存在与目前大多数植株不同的抗病机制。     

植物抗病反应的信号传导网络

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应（HR）并延伸到远处组织的系统抗性或获得性抗性（SAR）,受制于一种信号传导网络的调控,这个信号系统由抗病蛋白和病原菌非毒性蛋白在一种配体-受体的互作模式下激发,并由信号分子H2O2,NO和系统信号分子SA,JA和乙烯和通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生有类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发,这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

植物系统获得性抗性的信号转导

坏死病原菌（necrotizingpathogen）的侵染或者一些化学因子的处理能诱导植物的非侵染或非处理部位产生对多种病原再侵染产生抗性,即系统获得性抗性（systemicacquiredresistance,SAR）。获得系统抗性的组织中SAR基因产物的累积和防卫反应的潜在诱导增强（potentiation）是其两类抗病机制。SAR至少有通过水杨酸（salicylicacid,SA）或茉莉酸（jasmonicacid,JA）、乙烯（ethylene）为系统信号分子的两类信号转导途径。遗传分析已用于SAR产生的信号转导过程的分析,一些与SAR信号转导相关的基因已经和正在克隆,这些基因具有明显提高植物广谱抗性的潜能。     

丛枝菌根菌丝桥介导的番茄植株根系间抗病信号的传递

菌根菌丝桥是植物间在地下进行物质交流的通道,但它能否作为植物间地下化学通讯的通道来传递抗病信号则缺乏研究.本文利用丛枝菌根真菌(AMF)摩西球囊霉在供体与受体番茄植株间建立菌丝桥,对供体植株接种早疫病病原菌茄链格孢菌,研究供体与受体番茄植株根系间是否存在抗病信号的传递.荧光定量PCR检测表明,AMF侵染后的供体番茄植株再接种病原菌,其根系中苯丙氨酸解氨酶基因(PAL)、脂氧合酶基因(LOX)和几丁质酶基因(PR3)的转录水平显著高于仅接种病原菌、未接种病原菌和AMF,以及只接种AMF的番茄植株.更重要的是,与供体有菌丝桥连接的受体番茄根系中PAL、LOX和PR3的基因的表达量也显著高于无菌丝桥连接、菌丝桥连接被阻断以及有菌丝桥连接但供体植物未接种病原菌的处理,3个基因最高转录水平达到无菌丝桥连接对照受体植物的4.2、4.5和3.5倍.此外,供体植株根系启动防御反应的时间(18和65 h)比受体(100和140 h)早.表明病原菌诱导番茄供体根系产生的抗病信号可以通过菌丝桥传递到受体根系.     

外源茉莉酸诱导的菜豆叶片生化抗性及其对西花蓟马体内保护酶和解毒酶活性的影响

为探讨外源茉莉酸诱导的菜豆叶片抗性及对西花蓟马体内酶活性的影响,在室内对菜豆植株分别喷施1、0.1、0.01和0.001 mmol·L^-1 4个浓度的茉莉酸,以健康植株为对照,分别于处理后1、5和10 d测定菜豆叶片营养物质及次生物质的含量.另在同样处理叶片上分别接西花蓟马2龄若虫,分析其体内保护酶和解毒酶活性的变化.结果表明: 不同浓度茉莉酸处理1 d后,菜豆叶片的蛋白质含量和健康植株没有明显差异,但在5和10 d时显著低于健康植株;菜豆叶片游离氨基酸含量在茉莉酸处理1 d后显著高于健康植株,之后逐渐降低;茉莉酸处理下菜豆叶片可溶性糖含量显著低于健康植株,并随着茉莉酸浓度的升高和处理时间的延长而进一步下降;叶绿素含量在处理1 d后显著降低,随着处理时间的增加逐渐升高.叶片单宁、黄酮和总酚的含量在不同浓度茉莉酸和处理时间下均显著高于对照.蓟马取食导致菜豆叶片生化物质含量的变化与外源茉莉酸诱导的相似.西花蓟马取食茉莉酸处理的菜豆植株24 h后,体内保护酶系(超氧化物歧化酶、过氧化氢酶、过氧化物酶)和解毒酶系(谷胱甘肽S-转移酶、羧酸酯酶、乙酰胆碱酯酶)均明显高于健康植株,但茉莉酸浓度与处理时间对其影响程度不同.取食虫害菜豆叶片后西花蓟马体内酶活性的变化与取食外源茉莉酸诱导的叶片相似.说明外源茉莉酸处理可诱导菜豆植株的抗性,西花蓟马取食处理后的菜豆叶片可产生明显的反防御来适应寄主植物的变化.     

植物系统获得性抗性的分子机理

系统获得性抗性(systemic acquired resistance, SAR)是水杨酸(salicylic acid,SA)介导的植物对病原物的广谱抗病反应,NPR1和WRKY是SA信号传递过程中的重要转录因子.SAR的发生需要可移动信号(mobile signal)由局部到系统的长途运输,水杨酸甲酯(methyl salicylate, MeSA)和茉莉酸(jasmonic acid,JA)是两种可能的可移动信号.     

香蕉幼苗三类有机小分子溶质对尖孢镰刀菌侵染的生理响应

为阐明香蕉枯萎病发病机制,研究了尖孢镰刀菌侵染后,香蕉植株中几种对尖孢镰刀菌生长有显著作用的物质(氨基酸、有机羧酸、酚酸)种类和含量的变化。结果表明:(1)病原菌侵染后,伤害逐渐加剧,株高和生物量显著下降。(2)病原菌侵染后,叶片氨基酸总量显著升高,其中丝氨酸、缬氨酸、组氨酸、异亮氨酸和亮氨酸增幅较大,病原菌侵染16 d,其含量分别为侵染前的7.1、6.2、4.4、3.5和2.3倍;而根氨基酸总量开始显著降低,差异逐渐变小。(3)叶片有机羧酸酸含量在病原菌侵染后显著增加,而在根中显著降低。侵染植株叶片中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株叶片的2.6、1.6、1.9、1.8和2.3倍;根中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株的81%、42%、44%、28%和59%。(4)病原菌侵染后,植株叶片和根中酚酸含量都显著升高。叶片中阿魏酸、肉桂酸和水杨酸含量分别是未侵染叶片的2.9、1.7和2.9倍;而根中对羟基苯甲酸和丁香酸含量分别是未侵染根的4.3和1.5倍。研究结果表明,尖孢镰刀菌侵染后,植物与病原菌的相互作用使得植物体内抑菌物质和促菌物质都会相应的增加,植株对病害有一定的抗性,但促菌物质种类和含量较高最终使得感病植株发病。     

生长调节物质和可溶性糖含量对丹参中丹酚酸类物质积累的影响

采用盆栽实验研究了生长调节物质水杨酸（SA）、茉莉酸甲酯（MeJA）、赤霉素（GA3）对不同生长时期丹参植株中非结构糖含量、碳/氮比及根中丹酚酸类物质积累的影响;并进一步测定培养基中不同浓度蔗糖、葡萄糖、果糖对丹参毛状根中丹酚酸类物质积累的影响,对盆栽实验的结论进行了验证。结果显示,SA处理的丹参幼苗及花后期植株中蔗糖含量有增加趋势,而MeJA处理的丹参幼苗及花后期植株及GA3处理的丹参花后期植株中蔗糖积累均有降低趋势;且SA、MeJA和GA3处理对花后期植株地上和地下部分碳/氮比的影响不同。然而,SA和MeJA处理的丹参幼苗及花后期植株地上部分和根中还原糖含量、GA3处理的花后期植株根中还原糖含量均显著增加;同时,SA和MeJA处理的丹参幼苗根中迷迭香酸含量,以及SA、MeJA、GA3处理的花后期植株根中迷迭香酸含量和丹酚酸类总量显著增加。毛状根培养结果进一步证明,葡萄糖促进毛状根中迷迭香酸的产生,增加丹酚酸类总量,毛状根中迷迭香酸、丹酚酸B的积累及丹酚酸类总量与培养基中蔗糖浓度不相关。可见,丹参（植株）根中丹酚酸类物质的产生和积累受SA、MeJA和GA3的诱导,其与碳/氮比及植株中蔗糖含量没有相关性,推测植株中葡萄糖含量的增加促进根中丹酚酸类物质的积累。     

微生物诱导的植物系统抗性

综述了由植物病原菌和非病原性的根际促生菌诱导产生的两种植物系统抗性:系统获得性抗性(SAR)和系统诱导抗性(ISR),比较了两类系统抗性的诱导、信号分子和机理的异同点,阐述了信号分子水杨酸在系统获得性抗性诱导过程中的作用及茉莉酸和乙烯在系统诱导抗性产生过程中的作用。     

Salicylic Acid Enhances Resistance to Fusarium oxysporum f. sp. phaseoli in Common Beans (Phaseolus vulgaris L.)

Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli (Fop) is one of the most serious diseases of common bean (Phaseolus vulgaris L.) and is especially prevalent in China. In this study, we demonstrated that exogenous application of 2 mM salicylic acid (SA) by leaf spraying could induce resistance against Fop in common beans. Accumulation of free and conjugated SA in roots was detected by HPLC analysis and compared. After 168 h of daily SA treatment, the free SA level in roots was eight times higher than in control plants. However, the conjugated SA level reached a peak at 72 h of SA treatment, which was nine times higher than in control plants, and then sharply declined at 168 h. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidases (POX, EC 1.11.1.7) in roots were 9.4 and 6.3 times higher than in control plants after 168 h of SA treatment, respectively. H₂O₂ and O₂ ^? levels reached 2.6 and 13.6 times higher, respectively, than in the control plants at 168 h after SA treatment. Host reactions of SA-treated plant roots infected by Fop observed in microscopy included the deposition of electron-dense materials along the secondary walls. However, untreated inoculated plants showed marked cell wall degradation and total cytoplasm disorganization of root cells. These results indicated that SA applied to foliar tissue is capable of enhancing the systemic acquired resistance of common bean roots to infection by Fop.     

<Emphasis Type="Italic">Fusarium sambucinum</Emphasis> isolate FS-94 induces resistance against fusarium wilt of tomato via activation and priming of a salicylic acid-dependent signaling system

The wheat rhizosphere-inhabiting nonpathogenic Fusarium sambucinum isolate FS-94 protected tomato from Fusarium wilt (F. oxysporum f. sp. lycopersici) in laboratory experiments. Seed soaking or immersion of seedling roots in a FS-94 spore suspension prior to inoculation with the pathogen delayed the appearance of wilt symptoms and significantly reduced disease severity in plants of a susceptible tomato cultivar. Quantification of fungal ergosterol in infected tomato showed that protection against wilt agent was related to limitation of the pathogen growth in plants exposed to FS-94. Incubation of tomato seedlings in a FS-94 spore suspension for 48 or 72 h led to plant protection and increased the salicylic acid (SA) concentration in their roots, suggesting that this isolate was involved in a plant-mediated mode of action and induced resistance. Soaking tomato seeds in the spore suspension did not induce SA accumulation in seedling roots, but nevertheless resulted in a significant reduction in wilt severity when the seedlings were challenged with the pathogen. In response to pathogen attack, the SA content in susceptible seedlings grown from FS-94-treated seeds started to increase within 1 day and remained elevated for 72 h. This suggests that F. sambucinum isolate FS-94 primed a SA-dependent signaling system in tomato.     

Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1

Salicylic acid (SA) plays important roles in plants, most notably in the induction of systemic acquired resistance (SAR) against pathogens. A non-destructive in situ assay for SA would provide new insights into the functions of SA in SAR and other SA-regulated phenomena. We assessed a genetically engineered strain of Acinetobacter sp. ADP1, which proportionally produces bioluminescence in response to salicylates including SA and methylsalicylate, as a reporter for salicylate accumulation in the apoplast of plant leaves. SA was measured quantitatively in situ in NN genotype tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves inoculated with tobacco mosaic virus (TMV). The biosensor revealed accumulation of apoplastic SA before the visible appearance of hypersensitive response (HR) lesions. When the biosensor was infiltrated into TMV-inoculated leaves displaying HR lesions at 90 and 168 h post-inoculation, salicylate accumulation was detected predominantly in tissues surrounding the lesions and in veins adjacent to HR lesions. These images are consistent with previous data demonstrating that SA accumulation occurs prior to and following the onset of visible HR lesions. We also used the biosensor to observe apoplastic SA accumulation in tobacco leaves inoculated with virulent and HR-eliciting strains of the bacterial plant pathogen Pseudomonas syringae. The work demonstrates that the Acinetobacter sp. ADP1 biosensor is a useful new tool to non-destructively assay salicylates in situ and to map their spatial distribution in plant tissues.     

Systemic acquired resistance in Cavendish banana induced by infection with an incompatible strain of Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fact that there are no economically viable biological, chemical, or cultural measures of controlling the disease in an infected field leads to search for alternative strategies involving activation of the plant's innate defense system. The mechanisms underlying systemic acquired resistance (SAR) are much less understood in monocots than in dicots. Since systemic protection of plants by attenuated or avirulent pathogens is a typical SAR response, the establishment of a biologically induced SAR model in banana is helpful to investigate the mechanism of SAR to Fusarium wilt. This paper described one such model using incompatible Foc race 1 to induce resistance against Foc tropical race 4 in an in vitro pathosystem. Consistent with the observation that the SAR provided the highest level of protection when the time interval between primary infection and challenge inoculation was 10 d, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL, EC 4.3.1.5), peroxidase (POD, EC 1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), and superoxide dismutase (SOD, EC 1.15.1.1) in systemic tissues also reached the maximum level and were 2.00–2.43 times higher than that of the corresponding controls on the tenth day. The total salicylic acid (SA) content in roots of banana plantlets increased from about 1 to more than 5 μg g⁻¹ FW after the second leaf being inoculated with Foc race 1. The systemic up-regulation of MaNPR1A and MaNPR1B was followed by the second up-regulation of PR-1 and PR-3. Although SA and jasmonic acid (JA)/ethylene (ET) signaling are mostly antagonistic, systemic expression of PR genes regulated by different signaling pathways were simultaneously up-regulated after primary infection, indicating that both pathways are involved in the activation of the SAR.     

一株内生砖红镰刀菌促进烟草生长和增强青枯病抗性

课题组前期报道了一株对马铃薯具有促生防病作用的内生砖红镰刀菌Fusarium lateritium (FL617)。为拓展该菌株的应用范围,本研究以同为茄科作物的烟草为研究对象,探究了砖红镰刀菌对其生长和抗病的影响。结果表明,与对照组相比,处理组叶表面积、主根数、叶片数、叶绿素a和叶绿素b含量分别提高了5.0、3.9、1.4、1.3和1.3倍;该结果表明砖红镰刀菌对烟草具有促生作用。生测结果表明,砖红镰刀菌增强了烟草对青枯病的抗病性,其青枯病病情指数下降约30%。植物激素合成相关基因表达模式分析,发现处理组植物激素合成相关基因表达显著上调(1.6-39.9倍);用青枯病菌Ralstonia solanacearum感染寄主植物后分析其水杨酸(SA)、茉莉酸(JA)和R基因信号相关基因的转录模式,发现与对照组相比,处理组SA、JA相关基因均显著上调(1.2-8.3倍),仅有一个R基因显著下调(50%)。进一步用GFP标记的菌株进行荧光定殖观察,发现植物根系周围簇生着带有绿色荧光信号的真菌菌丝,表明砖红镰刀菌可以定殖于烟草根系。综上所述,推测砖红镰刀菌F. lateritium能够通过定殖于烟草根系介导植物激素、免疫防御相关基因的表达从而影响植株的生长发育和抗病性。     

Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.     

Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signaling pathway in tobacco systemic acquired resistance.

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.