Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.     

Development of insect resistant transgenic cotton lines expressing cry1EC gene from an insect bite and wound inducible promoter

Transgenic cotton lines were developed for high-level expression of a synthetic cry1EC gene from a wound inducible promoter. The tobacco pathogenesis related promoter PR-1a was modified by placing CaMV35S promoter on its upstream in reverse orientation. The resultant chimeric promoter CaMV35S(r)PR-1a expressed constitutively and was further up-regulated at the site of feeding by insects. It was induced more rapidly by treatment with salicylic acid (SA). The CaMV35S(r)PR-1a cry1EC expressing transgenic lines of cotton showed 100% mortality of Spodoptera litura larvae. The tightly regulated low-level expression of PR-1a was modified to a highly expressing constitutive expression by CaMV35S placed in reverse orientation. Salicylic acid treatment and wounding enhanced the expression further by the chimeric promoter. The leaves expressed more δ-endotoxin around the sites of insect bites. The levels of expression and induction varied among different transgenic lines, suggesting position effect. Some of the transgenic lines that expressed Cry1EC from the chimeric promoter at a low level also showed 100% mortality when induced with salicylic acid. A highly expressing insect bite and wound inducible promoter is desirable for developing insect resistant transgenic 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.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

Constitutive expression of the neutral PR-5 (OLP, PR-5d) gene in roots and cultured cells of tobacco is mediated by ethylene-responsive cis-element AGCCGCC sequences

The constitutive accumulation of tobacco neutral PR-5 (osmotin-like protein; OLP, PR-5d) in roots and cultured cells was studied in transgenic tobacco plants harboring the OLP promoter::GUS gene. This construct showed strong β-glucuronidase expression in vascular tissues and cortex of roots as well as in cultured cells. Analysis using a mutated promoter showed that ethylene-responsive elements (AGCCGCC) were necessary for constitutive expression in roots and cultured cells. An electrophoretic mobility shift assay indicated that ERF3 (EREBP3), an ethylene-responsive-element-binding factor that was reported to be expressed in roots and in cultured cells as well as in ethephon-treated leaves, could bind to the AGCCGCC sequences of the OLP gene. These findings suggest that AGCCGCC sequences and ERFs mediate the constitutive expression of the OLP gene in roots and cultured cells of tobacco. Received: 14 November 1997 / Revision received: 29 May 1998 / Accepted: 8 July 1998     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5' end of a reporter gene, beta-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

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.     

Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco

A 1.5 kb promoter fragment from the rice (Oryza sativa L.) RCH10 gene, which encodes a basic endochitinase inducible by wounding and fungal elicitor, was translationally fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation. Wounding of leaves induced GUS activity from low basal levels, and addition of fungal elicitor to the wounded tissue caused a further marked activation of the gene fusion. During vegetative development high levels of GUS activity were observed in roots and moderate levels in stems. Histochemical analysis indicated that the promoter was active in vascular and epidermal tissue, and the root apical tip. In flowers, high levels of GUS activity were observed in stigmas, ovaries and pollen-containing anthers, but only low levels in sepals and petals. The promoter 5′-deleted to ?160 exhibited the same patterns of expression in floral organs, and was also strongly induced by wounding and elicitor, but GUS activity was markedly reduced in vegetative organs. More detailed 5′ deletions showed that a cis-element required for floral expression was located between ?160 and ?74, and a cis element sufficient for stress induction was located 3′ of ?74. This proximal region 3′ of ?74 was also sufficient for expression in transfected rice protoplasts derived from suspension cultured cells. These data indicate that the complex developmental and environmental regulation of RCH10 promoter activity involves several distinct cis-elements for vegetative expression, floral expression and stress induction, and that signal pathways for wound and elicitor induction are conserved between monocotyledonous and dicotyledonous plants.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

Functional organization of the cassava vein mosaic virus (CsVMV) promoter

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of -glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.