NahG Transgenic Plants Research Articles

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.

Cloning and identification of the promoter of the tobacco Sar8.2b gene, a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as- 1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.

Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants.

In order to identify genes that are involved in the defense reaction against pathogen attack, we screened for examples that are regulated during the hypersensitive response (HR) to infection by tobacco mosaic virus (TMV) of tobacco ( Nicotiana tabacum cv. Xanthi nc) carrying the N gene, which confers resistance to TMV. Among seven genes initially identified by fluorescent differential display, one clone was further characterized because its transcripts accumulated rapidly and transiently after the onset of HR. Its full-length cDNA of 1346 bp encoded a polypeptide consisting of 258 amino acids. The deduced protein contained a single WRKY domain, a Cys(2)His(2) zinc-finger motif and a leucine-zipper motif, showing high similarity to WIZZ, a member of the family of WRKY transcription factors in tobacco. The gene was thus designated TIZZ. A GFP-TIZZ fusion protein was found to localize to the nucleus upon introduction into epidermal cells of onion. Bacterially expressed TIZZ was able to bind to the W-box (TTGAC) element that is recognized by other WRKY proteins, but transactivation assays showed it to be unable to activate reporter gene expression by itself. TIZZ transcripts were induced in TMV-infected nahG transgenic tobacco plants, in which salicylic acid fails to accumulate. Neither exogenously applied salicylic acid nor mechanical wounding induced TIZZ transcript accumulation. These results indicate the presence of salicylic acid-independent pathways for HR signal transduction, in which a novel type of WRKY protein(s) may play a critical role for the activation of defense.

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.

Environmentally sensitive, SA‐dependent defense responses in the cpr22 mutant of Arabidopsis

To investigate the signaling pathways through which defense responses are activated following pathogen infection, we have isolated and characterized the cpr22 mutant. This plant carries a semidominant, conditional lethal mutation that confers constitutive expression of the pathogenesis-related (PR) genes PR-1, PR-2, PR-5 and the defensin gene PDF1.2. cpr22 plants also display spontaneous lesion formation, elevated levels of salicylic acid (SA) and heightened resistance to Peronospora parasitica Emco5. The cpr22 locus was mapped to chromosome 2, approximately 2 cM telomeric to the AthB102 marker. By analyzing the progeny of crosses between cpr22 plants and either NahG transgenic plants or npr1 mutants, all of the cpr22-associated phenotypes except PDF1.2 expression were found to be SA dependent. However, the SA signal transducer NPR1 was required only for constitutive PR-1 expression. A cross between cpr22 and ndr1-1 mutants revealed that enhanced resistance to P. parasitica is mediated by an NDR1-dependent pathway, while the other cpr22-induced defenses are not. Crosses between either coi1-1 or etr1-1 mutants further demonstrated that constitutive PDF1.2 expression is mediated by a JA- and ethylene-dependent pathway. Based on these results, the cpr22 mutation appears to induce its associated phenotypes by activating NPR1-dependent and NPR1-independent branches of the SA pathway, as well as an ethylene/JA signaling pathway. Interestingly, the SA-dependent phenotypes, but not the SA-independent phenotypes, are suppressed when cpr22 mutants are grown under high humidity.

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action.

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

SummaryProbenazole (PBZ; 3‐allyloxy‐1,2‐benzisothiazole‐1,1‐dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2‐benzisothiazol‐3 (2H)‐one 1,1‐dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild‐type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis‐related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1‐1 and coi1‐1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR‐1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1‐1 and coi 1‐1 mutant plants. Thus SA and NPR1 appear to be required for PBZ‐ and BIT‐mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1‐mediated defense signaling pathway upstream of SA.

Fumonisin B1–Induced Cell Death in Arabidopsis Protoplasts Requires Jasmonate-, Ethylene-, and Salicylate-Dependent Signaling Pathways

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.

Salicylic acid has a role in regulating gene expression during leaf senescence.

Leaf senescence is a complex process that is controlled by multiple developmental and environmental signals and is manifested by induced expression of a large number of different genes. In this paper we describe experiments that show, for the first time, that the salicylic acid (SA)-signalling pathway has a role in the control of gene expression during developmental senescence. Arabidopsis plants defective in the SA-signalling pathway (npr1 and pad4 mutants and NahG transgenic plants) were used to investigate senescence-enhanced gene expression, and a number of genes showed altered expression patterns. Senescence-induced expression of the cysteine protease gene SAG12, for example, was conditional on the presence of SA, together with another unidentified senescence-specific factor. Changes in gene expression patterns were accompanied by a delayed yellowing and reduced necrosis in the mutant plants defective in SA-signalling, suggesting a role for SA in the cell death that occurs at the final stage of senescence. We propose the presence of a minimum of three senescence-enhanced signalling factors in senescing leaves, one of which is SA. We also suggest that a combination of signalling factors is required for the optimum expression of many genes during senescence.

Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent.

Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA.

Cell Wall-Degrading Enzymes from Erwinia carotovora Cooperate in the Salicylic Acid-Independent Induction of a Plant Defense Response

The virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a large variety of plant cell wall-degrading enzymes, including several pectinases and cellulases. Treatment of tobacco plants with culture filtrates (CFs) from the pathogen (containing the secreted cell wall-degrading enzymes) induces both local and systemic expression of genes involved in plant defense response. We have characterized the contribution of individually produced cell wall-degrading enzymes from E. carotovora subsp. carotovora in their ability to trigger local and systemic induction of a defense-related gene (coding for a basic β-1,3-glucanase) as well as resistance to this pathogen in tobacco. Our results show that mainly pectic enzymes and to some extent one cellulase induce expression of the β-1,3-glucanase gene. Cellulases appear to act cooperatively in the defense gene induction when added in combination with pectic enzymes. Similarly, pectinases and cellulases cooperate in triggering systemic resistance to the pathogen. Salicylic acid (SA) does not appear to be involved in this process, as systemic resistance was induced similarly in transgenic NahG plants that overproduce a salicylate hydroxylase and cannot accumulate SA and in nontrans-formed control plants. The lack of SA requirement for the induced resistance against E. carotovora subsp. carotovora suggests the presence of a different signal transduction pathway involved in this plant-pathogen interaction.

Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco Plants1

Abstract The tobacco (Nicotiana tabacum) cultivar Xanthi-nc (genotype NN) produces high levels of salicylic acid (SA) after inoculation with the tobacco mosaic virus (TMV). Gaseous methyl salicylate (MeSA), a major volatile produced in TMV-inoculated tobacco plants, was recently shown to be an airborne defense signal. Using an assay developed to measure the MeSA present in tissue, we have shown that in TMV-inoculated tobacco plants the level of MeSA increases dramatically, paralleling increases in SA. MeSA accumulation was also observed in upper, noninoculated leaves. In TMV-inoculated tobacco shifted from 32 to 24°C, the MeSA concentration increased from nondetectable levels to 2318 ng/g fresh weight 12 h after the temperature shift, but subsequently decreased with the onset of the hypersensitive response. Similar results were observed in plants inoculated withPseudomonas syringae pathovarphaseolicola, in which MeSA levels were highest just before the hypersensitive response-induced tissue desiccation. Transgenic NahG plants unable to accumulate SA also did not accumulate MeSA after TMV inoculation, and did not show increased resistance to TMV following MeSA treatment. Based on the spatial and temporal kinetics of its accumulation, we conclude that tissue MeSA may play a role similar to that of volatile MeSA in the pathogen-induced defense response.

Protein dephosphorylation mediates salicylic acid‐induced expression of PR‐1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis‐related (PR) proteins such as the acidic PR‐1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco ‐ elevated H2O2 levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA‐effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR‐1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA‐mediated induction of PR‐1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K‐252a and staurosporine induced PR‐1 gene expression. PR‐1 gene induction by K‐252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K‐252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR‐1 gene induction. The SA‐inducible acidic PR‐2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia‐derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis‐related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia, and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia. This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild‐type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA‐mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia. The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA‐mediated response.