Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

Mechanisms of Phosphate-induced Disease Resistance in Cucumber

Certain phosphate salts are known inducers of systemic acquired resistance (SAR). In the present study, a local spray application of dipotassium hydrogenphosphate (K₂HPO₄) was effective in inducing a high level of systemic protection in cucumber plants against anthracnose caused by Colletotrichum lagenarium. Resistance induction by K₂HPO₄ was associated with localized cell death in cucumber leaves treated with the phosphate salt. The cell death observed, subsequently resulted in the appearance of macroscopically visible, necrotic spots. Appearing lesions resembled those provoked by tobacco necrosis virus (TNV) during a hypersensitive response (HR) that leads to pathogen-induced activation of SAR. Phosphate-mediated cell death was preceeded by a rapid generation of superoxide and hydrogen peroxide. As a further consequence of phosphate application, a local and systemic increase in free and conjugated salicylic acid (SA) levels was detected. The phosphate-induced responses were also identified with a similar time range in cucumber leaves that had been pre-inoculated with TNV. In contrast, none of these responses was triggered by application of the commercial plant activator benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (BTH), which nevertheless was highly effective in inducing SAR in cucumber against anthracnose. In conclusion, the chemical SAR inducer K₂HPO₄ and the biological inducer TNV share some common early steps in signal transduction leading to SAR in cucumber, which differ from those involved in BTH-mediated SAR.     

The salicylic acid-dependent defence pathway is effective against different pathogens in tomato and tobacco

The role of salicylic acid (SA) was investigated in basal defence and induced resistance to powdery mildew ( Oidium neolycopersici ) and grey mould ( Botrytis cinerea ) in tomato ( Lycopersicon esculentum ) and tobacco ( Nicotiana tabacum ). A comparison of NahG transgenic tomato and tobacco (unable to accumulate SA) to their respective wild types revealed that in tomato, SA was not involved in basal defence against O. neolycopersici but NahG tobacco showed an enhanced susceptibility to O. neolycopersici infection, the effect becoming more obvious as the plants grew older. In contrast, SA played no role in the basal defence of tobacco against B. cinerea , but seemed to contribute to basal defence of tomato against B. cinerea. Activation of the SA-dependent defence pathway via benzothiadiazole (BTH) resulted in induced resistance against O. neolycopersici in tobacco but not in tomato. Microscopic analysis revealed that BTH treatment could prevent penetration of the Oidium germ tube through tobacco leaves, whereas penetration was successful on tomato leaves irrespective of BTH treatment. In contrast, soil or leaf treatment with BTH induced resistance against B. cinerea in tomato but not in tobacco. It is concluded that the SA-dependent defence pathway is effective against different pathogens in tomato and tobacco.     

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.     

Plant immunisation: from myth to SAR

The idea that plants might be able to develop a form of acquired immunity to infection following exposure to a pathogen has been current ever since discovery of the animal immune system in the later years of the nineteenth century. Early attempts to demonstrate a comparable system in plants focused on the detection of precipitating antibodies and hence were doomed to failure. Nevertheless, largely anecdotal evidence for plant immunisation continued to accumulate, culminating in the discovery of phytoalexins in the 1940s. Convincing evidence for systemic changes in plant resistance following an inducer inoculation was not available until 20 years later, when pioneering work on tobacco infected with blue mould (Peronospora tabacina) or tobacco mosaic virus (TMV) showed that tissues remote from the inoculation site were altered in disease reaction type. Increased resistance was expressed as a reduction in lesion numbers and size, and a reduced rate of pathogen reproduction. Systemic acquired resistance (SAR) has now been demonstrated in at least 20 plant species in at least six plant families, although detailed genetic or molecular analysis has mainly been confined to a few models, such as tobacco, cucumber and Arabidopsis. SAR is associated with the coordinate induction of genes encoding defence proteins which can be used as molecular markers of the response. The availability of Arabidopsis mutants altered in the induction and expression of SAR is now providing new insights into the signal transduction pathway(s) involved, and will enable comparison with the molecular mechanisms operating in other plant taxa. Important unresolved questions concern the nature of the translocated signal, the mechanism of defence ‘priming’, efficacy of the response against different pathogens, and practical exploitation of SAR in crop protection. The first generation of chemical plant defence activators is now commercially available and optimal use of these SAR inducers in integrated disease control requires further evaluation. The prospects for engineering transgenic crops altered in the regulation or expression of SAR is also a subject for further investigation. © 1999 Society of Chemical Industry     

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.     

Induction of systemic resistance to<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> in bean by a benzothiadiazole derivative and rhizobacteria

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10⁻³ to 10⁻⁷ M BTH 3 days before the challenge inoculation. At a high concentration (10⁻³ M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg⁻¹ BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.     

拟南芥转录因子IDD4不同突变体对灰葡萄孢菌的抗性分析

为明确IDD家族IDD4基因在拟南芥Arabidopsis thaliana抵抗灰葡萄孢菌Botrytis cinerea侵染过程中的作用,通过统计病情指数检测拟南芥野生型（wild type,WT）植株、过表达植株IDD4-OE和缺失突变体idd4植株感染灰葡萄孢菌情况,利用组织染色检测叶片细胞死亡和H₂O₂的积累情况,采用实时荧光定量PCR（real-time quantitative-PT-PCR,qRT-PCR）技术分析灰葡萄孢菌肌动蛋白基因Bc. ACTIN在3种植株叶片中的表达情况,并施加0.1 mmol/L外源水杨酸（salicylic acid,SA）后测定IDD4-OE植株的病情指数。结果显示,不同株系对灰葡萄孢菌的抗性由高到低依次为idd4＞WT＞IDD4-OE,IDD4-OE植株中病原菌感染部位的寄主细胞死亡程度比idd4植株严重。染色结果表明,病原菌侵染拟南芥后4 h,接种部位已有H₂O₂积累。qRT-PCR反应结果显示,Bc. ACTIN在IDD4-OE中比在idd4植株中的表达水平更高,表明灰葡萄孢菌在IDD4-OE植株中的繁殖速率更快。对IDD4-OE植株外源施加SA后,其病情指数、Bc. ACTIN表达量与WT植株间均无显著差异,说明SA能将感病植株的抗性提高至WT植株的水平,表明IDD4作为负调控因子参与了拟南芥对灰葡萄孢菌的抗性调控,SA在其中发挥着重要作用。     

Determination of the role of salicylic acid and Benzothiadiazole on physico-chemical alterations caused by <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tomato

Shoe-string disease caused by Cucumber mosaic virus (CMV) is one of the major threats to tomato production worldwide. The alteration in some biochemical parameters in leaves of the susceptible tomato genotype (Nagina) associated with CMV infection and the effect of exogenous application of salicylic acid (SA) and benzothiadiazole (BTH) were studied in this paper. Results showed that exogenous treatment with SA and BTH not only led to plants which gave significantly more yield than diseased controls (DC), but also delayed symptom expression and reduced disease severity. Analysis of biochemical parameters indicated that exogenous application of elicitors and viral infection, significantly affected the activity of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Compared to the DC plants, minimum disease severity and maximum number of fruit were recorded after a single dose of SA + BTH. Maximum plant height was recorded after weekly application of SA and maximum fruit yield per plant was gained with single dose of SA. Moreover, the activity of POD was significantly elicited many-fold after weekly application of SA + BTH, while higher amount of SOD was recorded with single dose of SA. The activity of CAT was also significantly accelerated after weekly application of SA + BTH while increased level of APX was noticed with single dose of BTH. In conclusion, the combined application of SA and BTH played an important role in induction of defense mechanism against CMV infection and can be useful in tomato disease management programs.     

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.     

Induced resistance in tomato plants to the toxin-dependent necrotrophic pathogen Alternaria alternata

A necrotrophic pathogen, the tomato pathotype of Alternaria alternata (Aa) causes Alternaria stem canker on tomato. Its pathogenicity depends on the production of host-specific AAL-toxin. Pre-inoculation with nonpathogenic Aa or pretreatment an elicitor prepared from Aa reduced disease symptoms by the pathogen. Salicylic acid (SA)- and jasmonic acid (JA)-dependent defense responses in tomato are not involved in the resistance to the pathogen induced by nonpathogenic Aa. The results suggest that an alternative and unknown signaling pathway independent of SA- and JA-signaling might modulate the induced resistance by activating the expression of the multiple defense genes.     

Characterization of Elicitor-inducible Tobacco Genes Isolated by Differential Hybridization

Inducible responses in plants against pathogen attack play a major role in resistance to disease. The defense responses are mostly associated with the expression of various kinds of inducible genes. We employed differential hybridization to isolate elicitor-inducible genes (EIGs) of tobacco (Nicotiana tabacum cv. Samsun NN) using the tobacco-fungal elicitor system. A cDNA library was constructed from tobacco leaves treated for 12 hr with hyphal wall components (HWC) prepared from Phytophthora infestans, and six EIGs were identified. Expression of all EIGs was induced after inoculation with the soybean pathogen Pseudomonas syringae pv. glycinea (nonpathogenic on tobacco) or treatment with salicylic acid, and a variety of expression patterns of EIG mRNAs was observed. Sequence analysis of EIG cDNAs revealed similarities to genes for SAR8.2 (EIG-B39 and EIG-D14), glycine-rich protein (EIG-G7), extensin (EIG-I30), acyltransferase (EIG-I24) and unknown protein (EIG-J7). Possible roles of EIG products in disease resistance are discussed. Received 30 August 2000/ Accepted in revised form 30 November 2000     

Induced resistance to Verticillium longisporum in Brassica napus by β‐aminobutyric acid

A preinoculative soil drench application of 0·5 mm β‐aminobutyric acid (BABA) significantly inhibited colonization of oilseed rape (Brassica napus, susceptible cultivar Falcon) by Verticillium longisporum and also prevented plant stunting caused by the pathogen. To better understand the defence responses induced by BABA, the presence of occlusions in the plant hypocotyl, levels of salicylic acid (SA) and hydrogen peroxide (H₂O₂), phenylalanine ammonia lyase (PAL) activity and expression of PR‐1 and PDF1.2 genes were examined. Transverse sections through the hypocotyl region of BABA‐treated plants showed clear vessels surrounded by phenol‐storing cells, in contrast to numerous obstructed vessels in water‐treated plants, in response to the pathogen. A significant increase in SA levels was observed in the hypocotyls of both water‐ and BABA‐treated plants in response to the pathogen; however, SA levels were unrelated to disease resistance. The level of H₂O₂ decreased in both treatments in response to the pathogen. A significant increase in PAL activity was observed in hypocotyl tissues of BABA‐treated plants. The expression patterns of PR‐1 and PDF1.2 were similar in the two treatments in response to the pathogen, indicating no involvement of these genes in resistance. The results indicate a similar organ specificity of the plant hypocotyl for chemically induced internal resistance as for genotype‐related resistance, two phenomena which, however, are based on contrasting cytological responses in the vascular tissues. Nonetheless, evidence is provided that the activity of the phenylpropanoid pathway plays a crucial role in both types of resistance.     

Tobacco necrosis virus A overcomes local cell death response in Nicotiana tabacum

Tobacco necrosis virus A (TNVA) belongs to the genus Alphanecrovirus in the Tombusviridae family. Symptoms induced by TNVA include necrotic lesions on inoculated leaves in a wide host range. The necrotic cell death response to certain viruses is poorly understood. This study characterizes the mechanisms underlying the local cell death triggered by TNVA in Nicotiana tabacum, showing that it shares several components with the hypersensitive response (HR) mediated by resistance proteins. TNVA perception triggers the accumulation of local viral-derived small interference RNA and the regulation of biological processes related to pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), including hydrogen peroxide accumulation, cell wall reinforcement, the activation of unfolded protein response and the induction of salicylic acid (SA) and SA-dependent pathways. However, these antiviral defences do not prevent either local virus multiplication or systemic movement, leading to disease development. Nevertheless, SA-deficient NahG tobacco plants challenged with TNVA showed an increase in virus accumulation on noninoculated leaves, which was correlated with the development of systemic necrosis, highlighting the role of SA signalling in TNVA-induced defence. In addition, SA treatment enhanced the local defence response to TNVA infection and suppressed systemic necrosis in N. benthamiana. Taken together, our data suggest that TNVA induces an impaired plant defence response by modulating host factors to persist at low levels in distal tissues.     

Benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH) stimulates defense reactions in <Emphasis Type="Italic">Xanthosoma sagittifolium</Emphasis>

The root rot disease caused by Pythium myriotylum is responsible for about 70% of cocoyam production loss in Cameroon. The potential of benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH) to trigger resistance in cocoyam (Xanthosoma sagittifolium) plants against P. myriotylum was investigated. Under controlled conditions, BTH was an efficient elicitor of some defense reactions in cocoyam. Application of 0.2 mg ml⁻¹ of BTH on leaves 7 days before inoculation of roots with P. myriotylum enhanced the activities of peroxidase (Pox) and polyphenoloxidase (PPO) as well as the total phenolic content. This resistance was noted as a decrease in disease incidence and severity in BTH-treated plants. This increase in Pox activities was correlated with two new isoforms in a white (sensitive) cultivar inoculated after stimulation. In a yellow (resistant) cultivar, stimulation was characterized by the appearance of one isoform. Qualitative analysis of phenolic compounds by HPLC showed an increase of hydroxycinnamic and flavonoid derivatives after inoculation. We also observed the appearance of a new caffeoylshikimic acid derivative after stimulation followed by inoculation of both cultivars. The findings indicated that the pattern of induction is different and depends on the variety.