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.     

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.     

Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103-1 and Nt103-35

We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes. All active deletion constructs tested showed expression of the reporter gene -glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots. Auxin treatment greatly enhanced the level of expression. The Nt103-1 promoter region –370/–276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of –370. The region –651/–370 contains sequence information necessary for uninduced expression. The Nt103-35 promoter manifested its auxin-responsiveness within the –504/–310 region. Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position –560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site. A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element. The as103 element is present in both promoters and positioned around –360, so within the region determined to be indispensable for the response to auxin. A third factor was found binding to the –276/–190 region of both promoters. Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes. The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response.     

Effects of promoter,intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA -glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5 transcribed region decreased expression strength.