Evaluation of the Use of the Tobacco PR-1a Promoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

Transient assay system for the analysis of PR-1a gene promoter in tobacco BY-2 cells

In order to develop a rapid and versatile assay system suitable for the analysis of regulated expression of tobacco pathogenesis-related protein 1a (PR-1a) gene, we investigated the use of the transient gene expression system in tobacco BY-2 cells by microprojectile bombardment. Using dual luciferase assay as a reporter gene expression detection system, we observed significant induction of PR-1a promoter activity by salicylic acid (SA) treatment. On the other hand, treatment with 4-hydroxybenzoic acid (4-HBA) resulted in no detectable increase in luciferase activity. Co-expression of a trans-acting factor, the NPR1/NIM1 protein of Arabidopsis, resulted in the induction of higher expression levels of the PR-1a promoter. These results suggest that the assay system is applicable for the analysis of factors involved in the regulated expression of SA-inducible defense-related genes.     

A jasmonate-responsive element within the A. thaliana vsp1 promoter

The vsp1 gene of Arabidopsis thaliana encodes a storage protein that accumulates in vegetative organs. Transgenic plants expressing a vsp1 promoter-gus (beta-glucuronidase) gene fusion were found to contain high GUS activity when challenged with jasmonate, a volatile plant hormone. The induction of vsp1-gus expression by jasmonate could be measured in tobacco mesophyll protoplasts, after transient expression. A number of deletions were operated in the vsp1 promoter in order to locate its jasmonate-responsive element. A 41 bp sequence taken approximately 150 bp upstream of the vsp1 TATA box could confer jasmonate responsiveness upon a short CaMV 35S promoter. Whereas the deletion of a CAAT box-like element within the 41 bp sequence did not affect promoter activity, mutation of a short palindromic motif completely abolished jasmonate responsiveness. This motif shows no homology with the jasmonate-responsive elements of other promoters.     

Interspecies compatibility of NAS1 gene promoters.

Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying the regulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-acting elements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genes and understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous (Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiency induced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS induced strong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned in Arabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plants and Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On the other hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency. In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 and OsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compatible with some shoot-specific Fe deficiency-related trans-factors in tobacco.     

Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis

Proteins belonging to the newly identified Cerato-platanin (CP) family have been shown to have elicitor activity in inducing disease resistance responses in various plants. In this study, we characterized a gene, MgSM1 , from Magnaporthe grisea , encoding a putative small protein belonging to the CP family. MgSM1 was constitutively expressed not only in different fungal growth stages but also during its infection process in rice plants. Agrobacterium-mediated transient expression of MgSM1 in Arabidopsis resulted in hypersensitive response in the infiltrated local leaves and enhanced disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato ( Pst ) DC3000 in upper leaves of plants, accompanyed by up-regulated expression of defense genes ( PR-1 , PR-5 and PDF1.2 ). Transgenic Arabidopsis plants expressing MgSM1 under control of a dexamethasone (DEX)-inducible promoter were generated. Expression of MgSM1 in transgenic plants was induced by exogenous application of DEX. MgSM1- expressing plants showed normal growth with application of <10 μ m DEX. After DEX induction, the MgSM1 -expressing plants showed enhanced disease resistance against B. cinerea , Alternaria brassicicola and Psto DC3000 as well as up-regulated expression of some of defense genes. Moreover, accumulation of reactive oxygen species was observed in MgSM1 -expressing plants. These results collectively suggest that ectopic expression of MgSM1 in transgenic plants confers broad-spectrum resistance against different types of pathogens. Our study also provides a novel strategy to generate environment-friendly crops with enhanced broad-spectrum resistance through ectopic expression of microbe-derived disease resistance-inducing proteins.     

Chemically induced virus resistance in Arabidopsis thaliana is independent of pathogenesis-related protein expression and the NPR1 gene

Salicylic acid (SA) treatment triggers inhibition of replication or movement of several positive-sense RNA plant viruses in tobacco. This resistance can also be stimulated by nonlethal concentrations of cyanide and antimycin A (AA) without triggering induction of pathogenesis-related PR-1 protein genes. In two ecotypes of Arabidopsis thaliana (Columbia and N?ssen), SA-induced resistance to a tobamovirus, Turnip vein clearing virus (TVCV), was also induced by nonlethal concentrations of cyanide and AA without concomitant induction of PR-1 gene expression. Furthermore, chemically induced resistance to TVCV, as well as the induction of the plant mitochondrial alternative oxidase (a potential target for the chemicals), was independent of NPR1, a gene that plays a key role downstream of SA in the induction of PR proteins. The chemically induced resistance to TVCV appeared to be due to inhibition of replication at the site of inoculation. Taken together, these results show that in Arabidopsis, as in tobacco, resistance to viruses can be induced via a distinct branch of the defensive signal transduction pathway. This suggests that the existence of this virus-specific branch may be widespread among plants.     

Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo

Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (–902 to –656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.     

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.     

Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.     

Jasmonate response of the Nicotiana tabacum agglutinin promoter in Arabidopsis thaliana

NICTABA is a carbohydrate-binding protein (also called lectin) that is expressed in several Nicotiana species after treatment with jasmonates and insect herbivory. Analyses with tobacco lines overexpressing the NICTABA gene as well as lines with reduced lectin expression have shown the entomotoxic effect of NICTABA against Lepidopteran larvae, suggesting a role of the lectin in plant defense. Until now, little is known with respect to the upstream regulatory mechanisms that are controlling the expression of inducible plant lectins. Using Arabidopsis thaliana plants stably expressing a promoter-β-glucuronidase (GUS) fusion construct, it was shown that jasmonate treatment influenced the NICTABA promoter activity. A strong GUS staining pattern was detected in very young tissues (the apical and root meristems, the cotyledons and the first true leaves), but the promoter activity decreased when plants were getting older. NICTABA was also expressed at low concentrations in tobacco roots and expression levels increased after cold treatment. The data presented confirm a jasmonate-dependent response of the promoter sequence of the tobacco lectin gene in Arabidopsis. These new jasmonate-responsive tobacco promoter sequences can be used as new tools in the study of jasmonate signalling related to plant development and defense.     

An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.     

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.     

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.