Binding sites for two novel phosphoproteins, 3AF5 and 3AF3, are required for rbcS-3A expression.

Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding sites for the nuclear factor GT-1 within the -166 deleted promoter of the ribulose-1,5-bisphosphate carboxylase-3A (rbcS-3A) gene, suggested that GT-1 might act in concert with an additional protein to confer light-responsive rbcS-3A expression. In this work, S1 analysis of RNA isolated from transgenic tobacco plants carrying mutant rbcS-3A constructs led to the identification of two short sequences located at the 5' and 3' ends of box III that are required for expression. These two sequences serve as binding sites for two novel proteins, 3AF5 and 3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and 3AF3 showed that complexes with faster mobilities were formed using nuclear extracts prepared from dark-adapted plants compared with those from light-grown tobacco plants. Phosphatase treatment of extracts from light-grown plants resulted in the formation of complexes with faster mobility. Although the binding of 3AF3 to its target site is dependent upon phosphorylation, the binding of 3AF5 does not appear to be affected by its phosphorylation state. These results suggest that the phosphorylated forms of both 3AF5 and 3AF3 are required for -166 rbcS-3A expression but that the mechanisms differ by which phosphorylation regulates the activities of 3AF5 and 3AF3.     

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.     

Molecular dissection of GT-1 from Arabidopsis.

We isolated and characterized an Arabidopsis cDNA encoding the DNA binding protein GT-1. This protein factor, which contains 406 amino acids, is highly homologous to the previously described tobacco DNA binding protein GT-1a/B2F but is 26 amino acids longer. Recombinant Arabidopsis GT-1, which was obtained from in vitro translation, bound to probes consisting of four copies of pea small subunit of ribulose bisphosphate carboxylase rbcS-3A box II and required the same GGTTAA core binding site as the binding activity of an Arabidopsis nuclear protein preparation. However, unlike the truncated tobacco GT-1a prepared from Escherichia coli extracts, the full-length Arabidopsis GT-1 bound to pea rbcS-3A box III and Arabidopsis chlorophyll a/b binding protein CAB2 light-responsive elements, both of which contain GATA motifs. Deletion and mutational analyses suggested that the predicted trihelix region of GT-1 is essential for DNA binding. Moreover, GT-1 binds to target DNA as a dimer, and its C-terminal region contains a putative dimerization domain that enhances the binding activity. Transient expression of the GT-1::beta-glucuronidase fusion protein in onion cells revealed the presence of a nuclear localization signal(s) within the first 215 amino acids of GT-1.     

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.     

Plant nuclear factor ASF-1 binds to an essential region of the nopaline synthase promoter

We have characterized a tobacco nuclear factor that binds to the -118 region of the nopaline synthase (nos) promoter from the Ti plasmid of Agrobacterium tumefaciens. The binding site for this factor, identified by DNase I footprinting, encompasses the region from -138 to -103 of the nos promoter. This region, which contains a potential Z-DNA-forming sequence, was previously shown to be essential for nos promoter activity in transgenic tobacco. A synthetic 21-base pair sequence from the protected region (from -131 to -111), designated as nos-1, was sufficient for factor recognition in vitro. In transgenic tobacco, a tetramer of nos-1 can confer leaf and root expression when fused upstream of a truncated 35 S promoter from the cauliflower mosaic virus. Mutations at the two TGACG-like motifs in nos-1 abolish factor binding while preserving the potential for Z-DNA formation. A tetramer of the nos-1 mutant sequence has no significant activity above background when tested in transgenic tobacco. Competition experiments with activation sequence factor (ASF)-1 binding sites from the 35 S promoter of cauliflower mosaic virus (as-1) and the wheat histone H3 promoter (hex-1) demonstrate that ASF-1 is the factor that binds to nos-1.     

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.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection

Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We divided this cDNA expression library into multiple pools and transfected mammalian cells with the individual pools. Following transfection, we tested the expressed proteins for DNA-binding activity by agarose thin-layer electrophoretic gelshift assay. After we identified a single expression poolfor the presence of a DNA-binding protein, the corresponding cDNA pool was further divided into smaller aliquots. Then, the cDNA expression and gelshift clone selection was repeated until a single clone was isolated In contrast to traditional polyacrylamide gels, the agarose thin-layer is significantly faster and resolves larger DNA-protein complexes. This method can be widely used for the cDNA cloning of DNA- and RNA-binding proteins from various mammalian host cells.