Interaction of tobacco nuclear protein with an elicitor-responsive element in the promoter of a basic class I chitinase gene

The expression of tobacco class I chitinase genes is effectively induced by a fungal elicitor in suspension-cultured cells. A putative cis-acting elicitor-responsive element (ElRE) was identified previously in the promoter of the class I chitinase gene, CHN50. To confirm that the ElRE sequence directly mediates the regulation of gene expression by the elicitor, I constructed a deleted promoter that controlled a reporter gene for -glucuronidase (gus) and examined expression of the construct in transgenic tobacco calli. Both expression and responsiveness to the elicitor disappeared, when the region of the promoter that included the ElRE sequence had been deleted. To define the specific sequence within the ElRE that interacts with nuclear factor(s), a gel mobility shift assay was performed with wild-type and mutated elements. Results of binding and competition experiments revealed that the nuclear factor(s) bound specifically to the sequence motif, ^-534GGTCANNNAGTC^-523, and that both of the repeated sites were involved in the binding of the nuclear factors. Moreover, the binding was influenced by the distance between the two repeated sites. In addition, the elicitor-inducible activity of the binding to this motif was reduced in nuclear extracts prepared from the cells that had been treated with cycloheximide or staurosporine.     

Expression of DNA binding proteins in carrot somatic embryos that specifically interact with a cis regulatory element of the French bean phaseolin gene

Nuclear proteins from bean (Phaseolus vulgarus) embryos bind specifically to a 55 bp DNA sequence located upstream of the seed storage protein gene phaseolin. This sequence is capable of elevating gene expression in transgenic tobacco plants by as much as 150-fold when fused to a chimeric -glucuronidase reporter gene. Results presented in this paper demonstrate that nuclear extracts from carrot embryos bind to a phaseolin DNA sequence that includes a phaseolin activator sequence. This specific DNA binding activity is modulated during somatic embryogenesis. Two separable protein species react specifically with the labeled phaseolin DNA fragment (58.0 and 51.7 dDa). These results suggest that the cis- and trans-acting elements controlling gene expression have been highly conserved during evolution.Abbreviations bp base pairs - CAMV cauliflower mosaic virus - GUS -glucuronidase - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT–AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.     

A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter

Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.