Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.     

No role for bacterially produced salicylic Acid in rhizobacterial induction of systemic resistance in Arabidopsis

ABSTRACT The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell(-1) for WCS417r to >25 fg cell(-1) for WCS374r. Addition of 200 muM FeCl(3) to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28 degrees C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36 degrees C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis- related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.     

The enhanced disease susceptibility phenotype of ethylene-insensitive tobacco cannot be counteracted by inducing resistance or application of bacterial antagonists

In an attempt to overcome the enhanced disease susceptibility phenotype that is typical for transgenic ethylene-insensitive tobacco (Tetr), Tetr plants were treated with chemical agents that induce resistance or with antagonistic rhizobacteria. Treatments with β-aminobutyric acid (BABA), benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), methyl jasmonate (MeJA), or salicylic acid (SA) induced PR-genes generally to a lesser extent than in non-transformed plants and did not reduce wilting symptoms upon infection with Pythium sp., except for a marginal effect of SA. In Tetr lines overexpressing PR-1g, PR-5c, or both, no significant reduction in disease development was apparent. Also treatment of Tetr plants with the antagonistic rhizobacteria Bacillus cereus UW85, Pseudomonas aeruginosa 7NSK2, Pseudomonas fluorescens WCS417r or Q8r-196, Pseudomonas putida WCS358r, or antibiotic-producing derivatives of WCS358r, did not reduce symptoms caused by Pythium.     

Suppressive effect of abscisic acid on systemic acquired resistance in tobacco plants

Recent studies have indicated that the phytohormone abscisic acid (ABA), induced in response to a variety of environmental stresses, plays an important role in modulating diverse plant–pathogen interactions. In Arabidopsis thaliana, we previously clarified that ABA suppressed the induction of systemic acquired resistance (SAR), a plant defense system induced by pathogen infection through salicylic acid (SA) accumulation. We investigated the generality of this suppressive effect by ABA on SAR using tobacco plants. For SAR induction, we used 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) that activate upstream and downstream of SA in the SAR signaling pathway, respectively. Wild-type tobacco plants treated with BIT or BTH exhibited enhanced disease resistance against Tobacco mosaic virus (TMV) and tobacco wildfire bacterium, Pseudomonas syringae pv. tabaci (Pst), however, which was suppressed by pretreatment of plants with ABA. Pretreatment with ABA also suppressed the expression of SAR-marker genes by BIT and BTH, indicating that ABA suppressed the induction of SAR. ABA suppressed BTH-induced disease resistance and pathogenesis-related (PR) gene expression in NahG-transgenic plants that are unable to accumulate SA. The accumulation of SA in wild-type plants after BIT treatment was also suppressed by pretreatment with ABA. These data suggest that ABA suppresses both upstream and downstream of SA in the SAR signaling pathway in tobacco.     

Signal Transduction in Resistance to Plant Viruses

Salicylic acid is part of a signal transduction pathway that induces resistance to viruses, bacteria and fungi. In tobacco and Arabidopsis the defensive signal transduction pathway branches downstream of salicylic acid. One branch induces PR-1 proteins and resistance to bacteria and fungi, while the other triggers induction of resistance to RNA and DNA viruses. This virus-specific branch can be activated using antimycin A and cyanide, or inhibited with salicylhydroxamic acid, suggesting a role for alternative oxidase in resistance to viruses. The virus-specific defensive pathway activates multiple resistance mechanisms. In tobacco, salicylic acid induces resistance to systemic movement of cucumber mosaic virus but has no effect on its replication or cell-to-cell movement. However, in the case of tobacco mosaic virus in tobacco, salicylic acid appears to induce interference with the synthesis of viral RNA.     

Induction of plant defense gene expression by plant activators and Pseudomonas syringae pv. tomato in greenhouse-grown tomatoes

Plant activators provide an appealing management option for bacterial diseases of greenhouse-grown tomatoes. Two types of plant activators, one that induces systemic acquired resistance (SAR) and a second that activates induced systemic resistance (ISR), were evaluated for control of Pseudomonas syringae pv. tomato and effect on plant defense gene activation. Benzothiadiazole (BTH, SAR-inducing compound) effectively reduced bacterial speck incidence and severity, both alone and in combination with the ISR-inducing product. Application of BTH also led to elevated activation of salicylic acid and ethylene-mediated responses, based on real-time polymerase chain reaction analysis of marker gene expression levels. In contrast, the ISR-inducing product (made up of plant growth-promoting rhizobacteria) inconsistently modified defense gene expression and did not provide disease control to the same level as did BTH. No antagonism was observed by combining the two activators as control of bacterial speck was similar to or better than BTH alone.     

Virus-specific expression of systemic acquired resistance in tobacco mosaic virus- and tobacco necrosis virus-infected ‘Samsun NN’ and ‘Samsun’ tobacco

Systemic acquired resistance induced in intact ‘Samsun NN’ tobacco plants by either tobacco mosaic virus (TMV) or tobacco necrosis virus (TNV) was more effective against challenge inoculation with the same than with the other of the two viruses. However, in trimmed ‘Samsun NN’ plants resistance induced by either of these viruses was stronger against TMV than against TNV. In intact ‘Samsun’ tobacco plants TNV induced a systemic resistance against itself identical to the one expressed in ‘Samsun NN’. Moreover, it induced systemic resistance against TMV as based on a decrease in TMV content in challeng-inoculated leaves. These observations indicate that systemic acquired resistance is not limited to combinations in which both inducing and challenging virus give rise to a hypersensitive reaction, and further point to virus-specific factors regulating the extent of resistance expressed.     

β-Aminobutyric acid-mediated enhancement of resistance in tobacco to tobacco mosaic virus depends on the accumulation of salicylic acid

A number of chemical and biological agents are known as inducers of systemic acquired resistance (SAR) to tobacco mosaic virus (TMV) in tobacco plants. In the present study, a local spray application of the non-protein amino acid DL-β-aminobutyric acid (BABA) was effective in enhancing resistance to TMV in tobacco plants containing the N gene. In contrast, the isomer α-aminobutyric acid (AABA) showed a much lower activity whereas γ-aminobutyric acid (GABA) was completely inactive, indicating a strong isomer specificity of aminobutyric acid in triggering enhanced virus resistance.Rapid cell death was detected in tobacco leaf tissues after foliar application of BABA, subsequently resulting in the development of macroscopically visible, necrotic lesions. BABA-induced cell death was associated with the rapid generation of superoxide and hydrogen peroxide. As further consequences, the occurrence of lipid peroxidation in treated tissues, a local and systemic increase of salicylic acid (SA) levels and the expression of PR-1a, a molecular marker of SAR in tobacco, could be observed. None of these responses was detectable after treatment with GABA.Enhancement of virus resistance by BABA was found to be strictly dependent on SA-mediated signal transduction since it could not be detected in salicylate hydroxylase (nahG) expressing transgenic tobacco plants. These findings suggest that in tobacco, primary processes triggered by foliar application of BABA, resemble those initiated by microbes during a hypersensitive response (HR) that result in SAR activation.     

Induced systemic protection against tomato late blight elicited by plant growth-promoting rhizobacteria

ABSTRACT Two strains of plant growth-promoting rhizobacteria (PGPR), Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, elicited systemic protection against late blight on tomato and reduced disease severity by a level equivalent to systemic acquired resistance induced by Phytophthora infestans or induced local resistance by chemical inducer beta-amino butyric acid (BABA) in greenhouse assays. Germination of sporangia and zoospores of P. infestans on leaf surfaces of tomato plants treated with the two PGPR strains, pathogen, and chemical BABA was significantly reduced compared with the noninduced control. Induced protection elicited by PGPR, pathogen, and BABA were examined to determine the signal transduction pathways in three tomato lines: salicylic acid (SA)-hydroxylase transgenic tomato (nahG), ethylene insensitive mutants (Nr/Nr), and jasmonic acid insensitive mutants (def1). Results suggest that induced protection elicited by both bacilli and pseudomonad PGPR strains was SA-independent but ethylene- and jasmonic acid-dependent, whereas systemic acquired resistance elicited by the pathogen and induced local resistance by BABA were SA-dependent. The lack of colonization of tomato leaves by strain 89B61 suggests that the observed induced systemic resistance (ISR) was due to systemic protection by strain 89B61 and not attributable to a direct interaction between pathogen and biological control agent. Although strain SE34 was detected on tomato leaves, ISR mainly accounted for the systemic protection with this strain.     

Possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea

The possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea was studied. Cucumber plants were inoculated with tobacco necrosis virus on the cotyledons and the level of endogenous salicylic acid in the first true leaf was determined by gas chromatography. Salicylic acid increased continously from the second day after virus inoculation to the fifth day, when the same leaf was inoculated withSphaerotheca fuliginea. In healthy plants, the efficiency of exogenous salicylic acid in inducing resistance was assayed by applying aqueous solutions at different times beforeSphaerotheca fuliginea inoculation. To evaluate the level of induced resistance, the following parameters were examined by light microscopy: percentage of conidial germination, length of the hyphae derived from single conidia, number of haustoria, percentage of epidermal cells with lignified walls and of necrotic cells underlying fungal hyphae. In treated plants conidial germination was reduced, the total length of the hyphae was shorter, the number of haustoria was lower and the haustorium-containing epidermal cells had more frequently lignified walls. Moreover, an evident increase in callose deposition was observed leading to the formation of oversized papillae around the penetration pegs. These results indicate that the application of salicylic acid before inoculation withSphaerotheca fuliginea reduces the intensity of the infectious process and that salicylic acid is involved in the expression of systemic resistance in cucumber challenged by the biotrophic pathogenSphaerotheca fuliginea.     

Rhizobacteria-mediated Induced Systemic Resistance: Triggering, Signalling and Expression

Selected strains of rhizosphere bacteria reduce disease by activating a resistance mechanism in the plant named rhizobacteria-mediated induced systemic resistance (ISR). Rhizobacteria-mediated ISR resembles pathogen-induced systemic acquired resistance (SAR) in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface. In other cases, rhizobacteria trigger a different signalling pathway that does not require SA. The existence of a SA-independent ISR pathway has been demonstrated in Arabidopsis thaliana. In contrast to pathogen-induced SAR, ISR induced by Pseudomonas fluorescens WCS417r is independent of SA accumulation and pathogenesis-related (PR) gene activation but, instead, requires responsiveness to the plant hormones jasmonic acid (JA) and ethylene. Mutant analyses showed that ISR follows a novel signalling pathway in which components from the JA and ethylene response are successively engaged to trigger a defensive state that, like SAR, is controlled by the regulatory factor NPR1. Interestingly, simultaneous activation of both the JA/ethylene-dependent ISR pathway and the SA-dependent SAR pathway results in an enhanced level of protection. Thus combining both types of induced resistance provides an attractive tool for the improvement of disease control. This review focuses on the current status of our research on triggering, signalling, and expression of rhizobacteria-mediated ISR in Arabidopsis.     

Control of Tobacco mosaic virus by PopW as a result of induced resistance in tobacco under greenhouse and field conditions

In a previous study, we isolated a new harpin protein, PopW, from the bacterium Ralstonia solanacearum ZJ3721 that can induce a hypersensitive response in tobacco, Nicotiana tabacum, leaves. In the current study, we demonstrate that, in a greenhouse experiment, PopW induced tobacco-acquired resistance against the Tobacco mosaic virus (TMV) with a biocontrol efficacy of 80.9 to 97.4% at a concentration as low as 25 μg/ml in both PopW-treated and neighboring leaves. The resistance induced by PopW is systemic acquired resistance mediated by salicylic acid, which was certified by the development of resistance being accompanied by the expression of the pathogenesis-related-1 gene (PR1) 8 h after PopW was sprayed onto the tobacco leaves. In addition, hydrogen peroxide began to accumulate 10 h after PopW spraying, peaking at 24 h with a maximum concentration of 1.97 μM/g fresh weight. The activities of phenylalanine ammonia lyase (EC4.3.1.5), polyphenoloxidase (EC1.14.18.1), and peroxidase (EC1.11.1.7) also increased, peaking at different times in the PopW-treated tobacco leaves. PopW also reduced the level of TMV disease in field trials with a biocontrol efficacy of 45.2%. Furthermore, PopW both increased tobacco yield (by 30.4 more than in control plants) and improved tobacco foliar quality, with an increase of 50.2% in the number of first-class tobacco leaves from treated compared with untreated plants. All of these results indicate that the new harpin protein PopW has the potential to be an effective biocontrol agent against TMV in tobacco.     

3,4-二氯异噻唑-5-甲酰胺衍生物的合成及生物活性

异噻唑类杂环化合物具有广泛的生物活性。为了扩大先导结构的发现范围,以3,4-二氯异噻唑-5-甲酸为原料合成了10个未见文献报道的3,4-二氯异噻唑-5-甲酰胺衍生物;其结构经核磁共振氢谱、红外光谱和元素分析确证。初步生物测试结果表明,化合物Ⅲe对9种病原真菌表现出了广谱的抑菌活性,在50 μg/mL下其抑制率均超过50%;在100 μg/mL下,大部分化合物展现了较好的抗烟草花叶病毒(tobacco mosaic virus,TMV)或诱导烟草抗TMV活性,其中化合物Ⅲa和Ⅲi的诱导活性分别为41.88%和42.92%。     

Salicylic Acid Suppresses Potato virus Y Isolate N:O-Induced Symptoms in Tobacco Plants

ABSTRACT The effects of salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on the systemic development of symptoms induced by a severe isolate of Potato virus Y group N:O (PVY(N:O)) in tobacco were investigated. Upon inoculation, the systemic development of symptoms in tobacco plants could be divided into three stages: virus incubation stage, rapid symptom-progress stage, and partial recovery and symptom-shifting stage. Treatment of seedlings with SA delayed the virus-induced necrosis in stems by 1 to 2 days. SA, not ACC, also significantly suppressed the symptom severity in stems. However, neither SA nor ACC treatment affected the partial recovery phenotype exhibited in the latterly emerged upper parts of the plants. Further analysis indicated that the accumulation of PVY was retarded by SA at the early stage of infection, and the effects were more profound in stems than leaves. Peroxidase (POX) activity and pathogenesis-related (PR) genes PR-1a and PR-1b were enhanced by PVY infection. SA not only increased POX activity in stems and PR genes in stems and leaves of mock-inoculated plants, but also elevated the activity of POX in both leaves and stems and the expression of PR-1a in leaves of PVY-infected plants. Together, the results suggest that systemic acquired resistance plays a key role in suppressing PVY(N:O)-induced symptom development through SA-mediated and ethylene-independent pathways. The symptom suppression was correlated with reduced replication/ accumulation of virus at the early stage of infection. The results also suggest that neither SA nor ethylene plays a role in the recovery phenotype.     

心叶烟NPR1基因全长cDNA序列的克隆及表达分析

 NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。     

WRKY转录因子在烟草受CMV侵染和激素处理中的表达模式初步分析

WRKY转录因子在调控植物抵抗病原菌的侵染中发挥重要作用,但是,目前为止还没有WRKY转录因子在烟草抗CMV中调控机理的研究。本研究检测了感病品种‘K326’和抗病品种‘TT8’在接种CMV后其WRKY1、WRKY2、WRKY4和WRKY11的表达情况。结果显示,CMV侵染后‘TT8’中4个WRKY转录因子表达量均有大幅上升;而‘K326’中WRKY2、WRKY4和WRKY11的表达量与CMV侵染前无显著差异,WRKY1表达量则大幅下降。幼苗经外源激素(水杨酸、茉莉素、乙烯)处理后用RT-PCR检测,结果表明,‘K326’和‘TT8’中WRKY转录因子对外源激素的诱导响应比较复杂多样,但总体表现为在‘TT8’中WRKY1、WRKY2和WRKY4会受到乙烯和茉莉酸的诱导,但在‘K326’中转录因子的表达未受明显诱导。     

Study of Bacterial Determinants Involved in the Induction of Systemic Resistance in Bean by Pseudomonas putida BTP1

The ability of Pseudomonas putida BTP1 to induce resistance in bean to Botrytis cinerea was demonstrated in soil experiments on plants pre-inoculated at the root level with the bacteria before challenge with the leaf pathogen. As a first step to characterize the molecules from BTP1 responsible for induction of systemic resistance in bean, heat-killed cells and supernatant from culture in an iron-limited medium were tested for their protective effect. Most of the resistance-eliciting activity of the strain was retained in the crude cell-free culture fluid. In vivo assays with samples from successive fractionation steps of the BTP1 supernatant led, (i) to the conclusion that salicylic acid, pyochelin and pyoverdin, previously identified as Pseudomonas determinants for induced systemic resistance (ISR), were not involved in systemic resistance triggered by BTP1, and (ii) to the isolation of fractions containing one main metabolite that retained most of the resistance-inducing activity in bean. Although this molecule remains to be structurally characterized, its isolation is an addition to the range of determinants from plant growth-promoting rhizobacteria (PGPR) known to stimulate plant defences.     

嵌合蛋白MoSlp1-AtCERK1作为抗病激发子的研究

本研究通过DNA体外重组技术,将稻瘟病菌MoSlp1基因和拟南芥几丁质寡糖受体基因AtCERK1的跨膜结构域和胞内结构域基因融合,构成嵌合基因MoSlp1-AtCERK1。在烟草瞬时表达试验中,MoSlp1-AtCERK1嵌合蛋白诱导烟草产生以过敏性坏死为表现形式的过敏反应。以带有MoSlp1-AtCERK1蛋白的农杆菌注射烟草,可增强烟草对烟草花叶病毒的抗病性。同时提高烟草的PR-1基因转录表达水平,表明MoSlp1-AtCERK1嵌合蛋白基因通过水杨酸信号传导的途径激活烟草系统获得性抗病。