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)

Elicitins are a family of small proteins secreted by species of Phytophthora. They are thought to be major determinants of the resistance response of tobacco against these oomycetes, since purified elicitins, alone and at low concentrations, can induce vigorous defense responses in tobacco (i.e., hypersensitive cell death and resistance against subsequent pathogen attack), and in vitro elicitin production by Phytophthora isolates is strongly negatively correlated with their pathogenicity on tobacco plants. A

The H-box (CCTACC(N)7CT(N)4A), which occurs three times within the -154 to -42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.

A cDNA library from Chlamydomonas reinhardti, constructed in the phage expression vector Agtll, was probed with antiserum directed against the nuclear-encoded y subunit of the chloroplast H+-transporting ATP synthase [ATP phosphohydrolase (H+-transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti-ysubunit serum to yield a product that coelectrophoresed with the immunoprecipitated product from in vitro-translated polyadenylylated RNA. These proteins were larger than the mature y subunit, either immunoprecipitated as chloroplast coupling factor 1 or as the individual subunit. Thus, the y subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii. Furthermore, the precursor protein encoded by the cDNA is imported into pea chloroplasts and processed to a lower molecular weight polypeptide that coelectrophoreses with mature C. reinhardtii y subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (--1900 bases long) and probably contains the entire coding region. Southern blot analyses revealed restriction fragment length polymorphisms and that the y subunit is probably encoded by an intron-containing single-copy gene.enzyme (11), and the mechanism of protein import into the chloroplast.Chlamydomonas reinhardtii is a genetically malleable green alga (12) that contains one large chloroplast per cell. It is possible to isolate intact chloroplasts (13-15) that can import precursor proteins (16). Precursors of the nuclearencoded subunits are difficult to detect in vivo since their half-lives are very short (9, 10). The study of the import of these subunits and their assembly into the complex is most easily accomplished by using isolated chloroplasts; thus, it becomes essential to synthesize these proteins in vitro.Toward this end, we have constructed a cDNA library from C. reinhardtii in the chimeric protein expression vector Agtll and cloned a cDNA for the precursor to the y subunit of CFo and CF1. While this work was in progress, Tittgen et al. (17) published work describing the isolation of a cDNA for the y subunit of the spinach ATP synthase. The two subunits probably fulfill similar roles in their respective complexes. Regardless of the degree of amino acid sequence homology, their nucleotide sequences should be markedly different since the nuclear DNA of C. reinhardtii uses codons biased toward a guanine or cytosine in the second and third positions (18-21).Proton-transporting ATP synthases [ATPases; ATP phosphohydrolase (H + -transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] are a class of multisubunit enzymes found in energy-transducing membranes. The composition of these coupling factors 0 and 1 (F0-F1) type enzymes varies. For instance, the number of subunit types found in the Escherichia coli enzyme is less than the number of subunit types found in the chloroplast ATP synthase, which...