Is the High Basal Level of Salicylic Acid Important for Disease Resistance in Potato?

Potato (Solanum tuberosum) plants contain a high basal level of salicylic acid (SA), the role of which in disease resistance is currently unclear. Here we report that, in spite of a drastic reduction in total SA levels in transgenic potato plants expressing the bacterial salicylate hydroxylase gene (nahG), there was no significant increase in disease severity when infected by Phytophthora infestans. Therefore, the high basal level of SA does not lead to constitutive resistance in healthy potato plants. However, in contrast to control plants, arachidonic acid failed to induce systematic acquired resistance (SAR) in nahG plants against P. infestans, indicating an essential role of SA in potato SAR. These results suggest that in potato the development of SAR against P. infestans may involve increased sensitivity of the plant to SA.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

Identification of a Soluble,High-Affinity Salicylic Acid-Binding Protein in Tobacco

Salicylic acid (SA) is a key component in the signal transduction pathway(s), leading to the activation of certain defense responses in plants after pathogen attack. Previous studies have identified several proteins, including catalase and ascorbate peroxidase, through which the SA signal might act. Here we describe a new SA-binding protein. This soluble protein is present in low abundance in tobacco (Nicotiana tabacum) leaves and has an apparent molecular weight of approximately 25,000. It reversibly binds SA with an apparent dissociation constant of 90 nM, an affinity that is 150-fold higher than that between SA and catalase. The ability of most analogs of SA to compete with labeled SA for binding to this protein correlated with their ability to induce defense gene expression and enhanced resistance. Strikingly, benzothiadiazole, a recently described chemical activator that induces plant defenses and disease resistance at very low rates of application, was the strongest competitor, being much more effective than unlabeled SA. The possible role of this SA-binding protein in defense signal transduction is discussed.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.     

Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

Chloroisonicotinamide derivative induces a broad range of disease resistance in rice and tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR in dicotyledonous plants such as tobacco and Arabidopsis has been partially elucidated and is mediated by salicylic acid (SA). However, the SAR mechanism of monocotyledonous rice plants remains to be clarified, although some similarities between SAR mechanisms in both types have been reported. Here we have characterized N-cyanomethyl-2-chloroisonicotinamide (NCI) as an effective SAR inducer in both plant species. Soil drench application of NCI induces a broad range of disease resistance in tobacco and rice and, more specifically, PR gene expression in tobacco. Both SA measurements in wild-type NCI-treated tobacco and pathogenic infection studies using NahG transgenic tobacco plants indicate that NCI-induced resistance enhancement does not require SA. Therefore, it is suggested that NCI induces SAR by triggering signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid. The fact that all of these chemicals are effective in rice and tobacco suggests that several common components function in disease resistance in both plant species.     

利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因

以晚疫病病原菌混合小种接种处理48h的马铃薯水平抗性材料(R-gene-free)叶片为目的材料,以未处理材料作为对照,用抑制差减杂交技术构建了一个富集晚疫病抗性相关基因的差减文库。应用反向Northern技术对840个克隆进行斑点杂交筛选,筛选出150个病原诱导后信号明显增强的克隆。26个片段测序结果表明：部分片段基因功能与抗病性明显相关。7个差异表达片段与GenBank EST数据库中已有晚疫病原诱导马铃薯叶片得到的EST有很高同源性(达95％～100％);部分片段核苷酸或氨基酸序列分别与番茄、烟草、拟南芥等的EST序列或氨基酸序列有较高同源性;另有4个基因片段在GenBank EST数据库中未找到明显的同源序列,可能为新发现的基因片段。     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco

Most commercially grown apple cultivars are susceptible to fungal diseases. Malus hupehensis has high resistance to many diseases affecting apple cultivars. Understanding innate defence mechanisms would help to develop disease-resistant apple crops. Non-expressor of pathogenesis-related genes 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). MhNPR1 cDNA, corresponding to genomic DNA and its 5' flanking sequences, was isolated from M. hupehensis. Sequence analysis showed that the regulatory mechanism for oligomer-monomer transition of the MhNPR1 protein in apple might be similar to that of GmNPR1 in soybean, but different from that of AtNPR1 in Arabidopsis. No significant differences in MhNPR1 expression were found in M. hupehensis after infection with Botryosphaeria berengeriana, showing that MhNPR1 might be regulated by pathogens at the protein level, as described for Arabidopsis and grapevine. SA treatment significantly induced MhNPR1 expression in leaves, stems and roots, while methyl jasmonate (MeJA) treatment induced MhNPR1 expression in roots, but not in leaves or stems. The expression of MhNPR1 was highly increased in roots, moderately in leaves, and did not change in stems after treatment with 1-aminocyclopropane-1-carboxylic acid (ACC). SAR marker genes (MhPR1 and MhPR5) were induced by SA, MeJA and ACC in leaves, stems and roots. Overexpression of MhNPR1 significantly induced the expression of pathogenesis-related genes (NtPR1, NtPR3 and NtPR5) in transgenic tobacco plants and resistance to the fungus Botrytis cinerea, suggesting that MhNPR1 orthologues are a component of the SA defence signalling pathway and SAR is induced in M. hupehensis.     

Pyrazolecarboxylic acid derivative induces systemic acquired resistance in tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through asalicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA's effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.     

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.     

Signaling of systemic acquired resistance in tobacco depends on ethylene perception

The hypersensitive interaction between Tobacco mosaic virus (TMV) and tobacco results in accumulation of salicylic acid (SA), defense gene expression, and development of systemic acquired resistance (SAR) in uninfected leaves. The plant hormones SA and ethylene have been implicated in SAR. From a study with ethylene-insensitive (Tetr) tobacco, we concluded that ethylene perception is required to generate the systemic signal molecules in TMV-infected leaves that trigger SA accumulation, defense gene expression, and SAR development in uninfected leaves. Ethylene perception was not required for the responses of the plant to the systemic signal that leads to SAR development.     

A benzothiadiazole derivative induces systemic acquired resistance in tobacco

Systemic acquired resistance (SAR) is a pathogen-induced disease resistance response in plants that is characterized by broad spectrum disease control and an associated coordinate expression of a set of SAR genes. Benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a novel synthetic chemical capable of inducing disease resistance in a number of dicotyledenous and monocotyledenous plant species. In this report, the response of tobacco plants to BTH treatment is characterized and the fact that it controls disease by activating SAR is demonstrated. BTH does not cause an accumulation of salicylic acid (SA), an intermediate in the SAR signal transduction pathway. As BTH also induces disease resistance and gene expression in transgenic plants expressing the nahG gene, it appears to activate the SAR signal transduction pathway at the site of or downstream of SA accumulation. BTH, SA and TMV induce the PR-1a promoter using similar cis-acting elements and gene expression is blocked by cycloheximide treatment. Thus, BTH induces SAR based on all of the physiological and biochemical criteria that define SAR in tobacco.     

N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.     

Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H₂O₂-degrading enzyme and thus elevate H₂O₂ levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 ( cat1 ) or catalase 2 ( cat2 ) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.