Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter.

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 fro the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of plant transcripts encoding protein kinase homologs.

Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences.

Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection.

Activation of plant defense genes was investigated by analysis of transcripts completed in vitro by isolated nuclei. Elicitor treatment of suspension-cultured bean (Phaseolus vulgaris L.) cells caused marked transient stimulation of transcription of genes encoding apoproteins of cell wall hydroxyproline-rich glycoproteins (HRGP) and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), concomitant with the onset of rapid accumulation of the respective mRNAs and hence expression of the phytoalexin (PAL, CHS), lignin (PAL), and HRGP defense responses. While there was a lag of 2 h prior to stimulation of HRGP gene transcription, induction of the transcription of PAL and CHS genes occurred within 5 min of elicitor treatment. Induction of transcription of PAL, CHS, and HRGP genes was also observed in wounded hypocotyls and in infected hypocotyls during race-cultivar-specific interactions with the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. Transcriptional activation occurred not only in directly infected tissue but also in distant, hitherto uninfected tissue, indicating intercellular transmission of an endogenous signal for defense gene activation. It is concluded that transcriptional activation of defense genes characteristically underlies induction of the corresponding defense responses and expression of disease resistance.

Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.

Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.