Portal de Periódicos da CAPES

Rapid stimulation of a soybean protein-serine kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses

1997; Springer Nature; Volume: 16; Issue: 4 Linguagem: Inglês

10.1093/emboj/16.4.726

1460-2075

Wolfgang Dröge‐Laser, Annette Kaiser, William P. Lindsay, Barbara Ann Halkier, Gary J. Loake, Peter Doerner, Richard A. Dixon, Chris Lamb,

Plant tissue culture and regeneration

Article15 February 1997free access Rapid stimulation of a soybean protein-serine kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses Wolfgang Dröge-Laser Wolfgang Dröge-Laser Search for more papers by this author Annette Kaiser Annette Kaiser Search for more papers by this author William P. Lindsay William P. Lindsay Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Barbara Ann Halkier Barbara Ann Halkier Search for more papers by this author Gary J. Loake Gary J. Loake Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Peter Doerner Peter Doerner Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Richard A. Dixon Richard A. Dixon Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Chris Lamb Corresponding Author Chris Lamb Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Wolfgang Dröge-Laser Wolfgang Dröge-Laser Search for more papers by this author Annette Kaiser Annette Kaiser Search for more papers by this author William P. Lindsay William P. Lindsay Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Barbara Ann Halkier Barbara Ann Halkier Search for more papers by this author Gary J. Loake Gary J. Loake Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Peter Doerner Peter Doerner Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Richard A. Dixon Richard A. Dixon Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA Search for more papers by this author Chris Lamb Corresponding Author Chris Lamb Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Wolfgang Dröge-Laser2, Annette Kaiser3, William P. Lindsay4, Barbara Ann Halkier5, Gary J. Loake4,6, Peter Doerner1, Richard A. Dixon4 and Chris Lamb 1 1Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA 2Universität Bielefeld, Lehrstuhl für Genetik, 33501 Bielefeld, Germany 3Institüt für Pharmazeutische Biologie, 38106 Braunschweig, Germany 4Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73402 USA 5Royal Veterinary and Agricultural University, Department of Plant Biology, Copenhagen, Denmark 6University of Edinburgh, Institute of Cellular and Molecular Biology, Edinburgh, UK The EMBO Journal (1997)16:726-738https://doi.org/10.1093/emboj/16.4.726 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The G-box (CACGTG) and H-box (CCTACC) cis elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal salicylic acid as early responses to pathogen attack. We have isolated a soybean cDNA encoding a novel bZIP protein, G/HBF-1, which binds to both the G-box and adjacent H-box in the proximal region of the chalcone synthase chs15 promoter. While G/HBF-1 transcript and protein levels do not increase during the induction of phenylpropanoid biosynthetic genes, G/HBF-1 is phosphorylated rapidly in elicited soybean cells, almost exclusively on serine residues. Using recombinant G/HBF-1 as a substrate, we identified a cytosolic protein-serine kinase that is rapidly and transiently stimulated in cells elicited with either glutathione or an avirulent strain of the soybean pathogen Pseudomonas syringae pv. glycinea. Phosphorylation of G/HBF-1 in vitro enhances binding to the chs15 promoter and we conclude that stimulation of G/HBF-1 kinase activity and G/HBF-1 phosphorylation are terminal events in a signal pathway for activation of early transcription-dependent plant defense responses. Introduction Plants respond to pathogen avirulence signals by the elaboration of inducible defenses including oxidants, phytoalexins, cell wall modifications and deployment of pathogenesis-related (PR) proteins, such as chitinase, with antimicrobial activities (Briggs, 1995). Defense induction associated with the expression of localized hypersensitive resistance is observed in the early stages of incompatible interactions following attempted infection by a non-pathogen or avirulent strain of a pathogen (Lamb et al., 1989). In some cases, these responses are also observed in distant tissue, associated with the expression of systemic acquired resistance in which immunity to a broad range of normally virulent pathogens gradually develops throughout the plant (Ryals et al., 1994). Defense responses can also be induced by defined molecules including microbial glycan and peptide elicitors, or metabolites such as arachidonic acid, glutathione and salicylic acid (Ebel and Cosio, 1994). With the exception of callose production and oxidative responses including cross-linking of cell wall structural proteins, induction of this battery of defenses involves a massive switch in host gene expression (Dixon et al., 1994), and there is a temporal and spatial hierarchy of defense gene activation with some genes exhibiting rapid, localized activation, whereas many, but not all, PR protein genes undergo slower activation, both locally and then systemically throughout the plant (Ryals et al., 1994; Hahlbrock et al., 1995). Prominent among the class of rapidly induced defense genes are those encoding enzymes of phenylpropanoid biosynthesis involved in the synthesis of lignin precursors and a number of phytoalexins (Dixon and Paiva, 1995). In addition, the phenylpropanoid-derived metabolite salicylic acid is a signal potentiating the expression of both localized hypersensitive resistance and systemic acquired resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusarenko, 1996). The early activation of phenylpropanoid biosynthetic genes is correlated with the expression of hypersensitive resistance in tissues challenged with an avirulent pathogen or non-pathogen (Dixon and Paiva, 1995; Hahlbrock et al., 1995), and in parsley and bean cell suspension cultures transcription of these genes is transiently activated within 10–20 min of elicitor treatment, with maximum rates of transcription after ∼1 h and maximum accumulation of transcripts after 3–4 h (Chappell et al., 1984; Cramer et al., 1985; Lawton and Lamb, 1987). Delineation of the cis elements and cognate trans factors underlying the rapid activation of phenylpropanoid biosynthetic genes provides the basis for characterizing the terminal stages of a signal transduction pathway involved in the deployment of early transcription-dependent defenses and the generation of salicylic acid as a secondary signal. Two cis elements, the G-box (CACGTG) and H-box (CCTACC), are found in the proximal region of the promoters of a number of genes encoding phenylpropanoid biosynthetic enzymes including phenylalanine ammonia-lyase (pal) and 4-coumarate:CoA ligase, which catalyze the first and third steps respectively in the central, common pathway of phenylpropanoid biosynthesis, and chalcone synthase (chs), which catalyzes the first step in a branch pathway specific for the synthesis of flavonoids and isoflavonoid-derived pterocarpan phytoalexins (Lois et al., 1989; Ohl et al., 1990). These cis elements are the sites of elicitor-inducible in vivo footprints in the parsley pal1 promoter (Lois et al., 1989) and likewise an H-box in the chs15 promoter is the location of an elicitor-inducible DNase I-hypersensitive site in chromatin from bean cells (Lawton et al., 1990). G- and H-box functions in chs15 expression have been confirmed by functional analysis of the effects of promoter mutations on reporter gene expression in electroporated protoplasts (Dron et al., 1988; Loake et al., 1992), stably transformed cells and transgenic plants (O.Faktor, R.A.Dixon and C.Lamb, unpublished data), as well as by in vitro transcription assays (Arias et al., 1992; W.P.Lindsay, R.A.Dixon and C.Lamb, unpublished data). The G-box functions in the regulation of diverse genes by developmental cues, abscisic acid, light, UV irradiation and wounding as well as pathogen signals, and a family of bZIP proteins that bind G-box sequences has been described (Foster et al., 1994; Menkens et al., 1995). Functional specificity appears to be determined by DNA-binding affinities governed by nucleotides immediately flanking the G-box ACGT core and by combinatorial interactions with other cis element–trans factor systems (Williams et al., 1992; Izawa et al., 1993; Menkens et al., 1995). However, only the in vivo function of opaque2, as a regulator of zein expression, has been fully established (Schmidt et al., 1992), and while transgenic manipulation of CPRF-1 disrupts light induction of parsley chs (Feldbrügge et al., 1994), there is no information on the function of G-box factors in elicitor or pathogen activation of phenylpropanoid biosynthetic genes. Likewise, a flower-specific Myb transcription factor, which stimulates pal2 transcription through binding to the H-box, appears to function in developmental regulation and there is no report of its involvement in elicitor or pathogen induction (Sablowski et al., 1994, 1995). A novel factor from parsley, BFP-1, binds to an AC-rich element related to the H-box that is also in vivo footprinted in elicited parsley cells (da Costa e Silva et al., 1993). BFP-1 transcripts are induced in elicited cells and infected tissues, consistent with a function in defense gene regulation, although it is not clear whether the activity of this factor is directly modulated by elicitor or pathogen signals for the rapid initial stimulation of early defense genes. Polypeptides of 97 and 56 kDa, designated KAP-1 and KAP-2 respectively, which bind to H-boxes in the bean chs15 promoter, have been purified by DNA affinity chromatography (Yu et al., 1992). Elicitation 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 in the induction of chs15 and related defense genes. While these studies have identified a number of trans factors which might function in the elicitor or pathogen induction of phenylpropanoid biosynthetic genes, no picture has yet emerged of how the functional activities of factors interacting with the G-box and H-box cis elements are regulated in the initial stimulation of early defense gene transcription. In the present study, we describe a novel bZIP protein, G/HBF-1, which binds to the G-box and the adjacent H-box in the proximal region of the chs15 promoter. While G/HBF-1 transcript and protein levels do not increase during the induction of pal and chs transcription, G/HBF-1 is phosphorylated rapidly in elicited cells. Using recombinant G/HBF-1 as a substrate, we identify a cytosolic protein-serine kinase activity that is rapidly and transiently stimulated in elicited cells and demonstrate that G/HBF-1 phosphorylation enhances binding to the chs15 promoter. These observations delineate a terminal event in a signal pathway for activation of early transcription-dependent defense responses and indicate that in plants, as in animals, stimulus-dependent transcription factor phosphorylation contributes to the selective regulation of gene expression. Results Isolation of a soybean cDNA encoding G/HBF-1, a novel bZIP DNA-binding protein Two soybean (Glycine max) cDNA libraries were constructed in λgt11 and λZAP-II vectors using respectively poly(A)+ RNA from control soybean cell suspension cultures and a mixture of poly(A)+ RNA from control cells and cells 4 h after elicitor treatment. A total of 3×105 plaques from each library were probed with the −80 to −42 fragment of the bean chs15 elicitor-inducible promoter. This fragment contains both the G-box and adjacent, TATA-proximal H-box (H-box III). A clone, designated λG/HBF-1, expressing a protein that strongly bound this promoter fragment, was isolated from the λgt11 library. λG/HBF-1 contained a 1.4 kb insert, and repeated screening of the λgt11 and λZAP-II cDNA libraries with this insert as a probe failed to identify hybridizing clones with larger inserts. The λG/HBF-1 cDNA contains a single long open reading frame of 1134 bp encoding a protein of 41 kDa (Figure 1). The putative ATG start codon is flanked by a nucleotide sequence optimal for translation initiation in plants (Lütke et al., 1987). The deduced protein product shows characteristic features of a bZIP transcription factor including a highly basic, putative DNA-binding domain and a leucine zipper domain in which every seventh amino acid residue is leucine or another small hydrophobic residue (Figure 1). G/HBF-1 also contains two domains rich in proline and acidic amino acids respectively. Figure 1.Nucleotide sequence of the λG/HBF-1 cDNA. The deduced amino acid sequence of the longest open reading frame is given below. The basic domain is shown in bold and the heptameric repeats of the leucine zipper are underlined. Download figure Download PowerPoint Figure 2.Comparison of the amino acid sequences of the bZIP transcription factors G/HBF-1, CPRF-2, OHP1 and OHP2. Conserved amino acid residues are indicated as dots, except those constituting the putative leucine zipper, which are shown in bold. The conserved domains D1–D4 and the basic region are enclosed in boxes and the peptides used for antibody production are marked by dotted lines. Download figure Download PowerPoint Protein sequence alignments reveal substantial similarities in the bZIP region to the equivalent regions of other plant bZIP proteins such as RITA-1 (Izawa et al., 1994) or opaque2 (Schmidt et al., 1992). However, while the C-terminal half of the basic region is highly conserved among G/HBF-1 and many other plant bZIP factors binding G-box or related motifs, the N-terminal part of the basic region of G/HBF-1 only exhibits a high degree of similarity to the common plant regulatory factor 2 (CPRF-2) from parsley (Weisshaar et al., 1991). Four other domains, designated D1–D4, are highly conserved among G/HBF-1, CPRF-2 (Weisshaar et al., 1991) and the maize opaque2 heterodimerizing proteins OHP1 and OHP2 (Pysh et al., 1993). D1, which is located half way between the N-terminus and the basic domain, comprises a peptide predicted to form a helix, and D2, which is located just N-terminal of the basic domain, is relatively rich in acidic residues. D3, which is adjacent to the bZIP region, is similar to a domain also present in some animal transcription factors including the helix–loop–helix protein MyoD (Scales et al., 1990). D4, located near the C-terminus, shows no distinctive structural features. Overall, G/HBF-1 showed a high degree of similarity to CPRF-2 (67% identity in 361 amino acids) and OHP1 and OHP2 (48% identity in 339 amino acids), and these sequence comparisons define a sub-family of plant G-box-binding bZIP proteins. Southern blots of soybean genomic DNA probed with G/HBF-1 sequences at high stringency showed only one or two hybridizing bands in a range of restriction endonuclease digestions (data not shown), indicating that G/HBF-1 is likely to be encoded by a single copy gene. G/HBF-1 binding to the chs15 G-box and H-box III DNA binding by G/HBF-1 was examined in experiments using radiolabeled oligonucleotides to probe plaque lawns of λG/HBF-1 (Table I) and by gel retardation assays with purified recombinant G/HBF-1 (Table I and Figure 3). G/HBF-1 cDNA was fused to the T7 promoter in the vector pET-28a (Novagene), and the recombinant G/HBF-1 carrying an N-terminal hexameric histidine peptide tag, G/HBF-1(His6), was purified from Escherichia coli extracts by immobilized Ni affinity chromatography. Gel retardation assays with the recombinant factor demonstrated binding to the −80 to −42 chs15 promoter sequence containing both the G-box (−72 to −67) and TATA-proximal H-box (H box III, −59 to −53) and to each of these two cis elements when tested separately (Figure 3A). The major binding complex formed with the −80 to −42 sequence had a similar electrophoretic mobility to the complexes formed with either the chs15 G-box sequence CACGTG (−74 to −69) or the extended H-box III sequence TCACCTACCCTA (−65 to −53) when tested separately, suggesting that G/HBF-1 binding was mainly at one location when both cis elements were in close proximity. Incubation of G/HBF-1 with the −80 to −42 sequence also generated a second, low abundance complex with increased electrophoretic mobility not observed with either cis element alone. Figure 3.Gel retardation assay of G/HBF-1 binding to specific cis elements. (A) G/HBF-1(His6) binding to the −80 to −42 region of chs15 and indicated oligonucleotide sequences within this region. (B) DNA-binding specificity monitored by competition between test oligonucleotides and labeled wild-type −80 to −42 chs15 sequence for binding to recombinant G/HBF-1(His6). Download figure Download PowerPoint Table 1. DNA-binding activity of the G/HBF-1 protein Box Oligonucleotides Binding Pizza −80/−42 GTG TTG CAC GTG ATA CTC ACC TAC CCT ACT TCC TAT CCA ++ + −80/−42 (MI) GTG TTG Cgt acG ATA CTC ACC TAC CCT ACT TCC TAT CCA + n.d. −80/−42 (MII) GTG TTG CAC GTG ATA CTC Aaa TAa aCT ACT TCC TAT CCA ++ n.d. −80/−42 (MIII) GTG TTG Cgt acG ATA CTC Aaa TAa aCT ACT TCC TAT CCA − n.d. HI AGA AAC TCC TAC CTC ACG AAC TAG GA − − HII T TGC ACT GCC TAC CAT GTC TGC TTC CT − − HIII AGA CTC ACC TAC CCT ACT TCC TAT CC ++ + HIII (MI) AGA CTC ACC atC CCT ACT TCC TAT CC + − HIII (MII) AGA CTC Aaa TAa aCT ACT TCC TAT CC − − HIII (MIII) AGA CTC ACg TAC CCT ACT TCC TAT CC n.d. + HIII (MIV) AGA CTC AgC TAC CCT ACT TCC TAT CC − − HIII (MV) AGA CTC ACC TAt tCT ACT TCC TAT CC + + H consensus CGA CTC ACC TAC CTG ACA TGC TAC GCA G − n.d. G GCT TTG GTG TTG CAC GTG ATA CT ++ + G (MI) GCT TTG GTG TTG Cgt acG ATA CT − n.d. boxII-s C TTA TTC CAC GTG GCA G − n.d. boxII-1 GAT CTC TTA TTC CAC GTG GCC ATC CGG ATC C + n.d. as-1 GAT ATC TCC ACT GAC GTA AGG GAT GAC GTT AAC − n.d. Summary of 'Pizza blot' and gel retardation experiments monitoring the binding of recombinant G/HBF-1 to specific oligonucleotides. The H-box core sequence is single underlined and the G-box is double underlined. Mutated bases are printed in lower case. The H-box consensus sequence was derived by Yu et al. (1992). Nucleotide sequence requirements for G/HBF-1 binding were analyzed by direct binding of test oligonucleotides to λG/HBF-1 plaque lawns and by competition with the −80 to −42 chs15 promoter fragment for binding to recombinant G/HBF-1(His6) in gel retardation assays. Mutation of the G-box sequence GCACGTGA to GCgtacGA abolished binding when tested in isolation from H-box III. However, the core ACGT was not sufficient for G/HBF-1 binding since the as-1 sequence TGACGTT was not recognized. Likewise, the parsley chs G-box, CCACGTGG, involved in light regulation (Weisshaar et al., 1991), was not recognized, indicating that not only the ACGT core but also immediately flanking nucleotides appear to be important for G/HBF-1 binding. Interestingly, G/HBF-1 bound to a larger fragment of the parsley chs promoter containing not only the G-box but an immediately downstream sequence with an AC-rich cis element resembling the H-box. Mutation of the H-box core motif CCTACC to CCatCC or aaTAaa severely impaired binding to the H-box III cis element when tested in isolation from the G-box, whereas binding was observed with the sequence CCTAtt. However, no binding was observed with upstream fragments of the chs15 promoter-containing H-box I (−159 to −135) or H-box II (−139 to −113) in which the CCTACC motif is embedded in different flanking sequences compared with the TATA-proximal H-box III (Yu et al., 1992). Thus, sequences in addition to the core H-box motif are important for G/HBF-1 binding to H-box III and, when immediately flanking sequences are taken into account, a single mutation in H-box III, CACCTACC to CACgTACC, generates an almost perfect second version of the chs15 G-box (CACGTG). The CACGTACC sequence, intermediate between the G-box and H-box III, was also bound by G/HBF-1. G/HBF-1 regulation during chs induction G/HBF-1 sequences hybridized at high stringency to a single transcript in total cellular RNA isolated from uninduced soybean cells (Figure 4). The size of the transcript was 1.4 kb, consistent with the size of the full-length G/HBF-1 cDNA. Elicitation of soybean cells with reduced glutathione caused little change in the level of G/HBF-1 transcripts, whereas chs transcripts rapidly accumulated from low basal levels (Figure 4A). Figure 4.G/HBF-1 transcripts and protein levels do not change during chs induction. Soybean cells were elicited with 0.5 mM glutathione. (A) chs and G/HBF-1 transcript levels. Northern blots of total RNA were hybridized with chs1 or G/HBF-1 cDNA probes. (B) G/HBF-1 protein levels. Western blots of whole cell extracts were probed with α-G/HBF-1(P2). Download figure Download PowerPoint To monitor G/HBF-1 regulation at the protein level, we generated a panel of polyclonal antisera, including two peptide antibodies, α-G/HBF-1(P2) and α-G/HBF-1(P4), to two internal peptides showing little similarity to the peptide sequences found in the corresponding regions of otherwise closely related bZIP transcription factors such as CPRF-2 (Figure 2). α-G/HBF-1(P2) bound to a single protein in Western blots of total cellular protein (Figures 4 and 5A), whereas α-G/HBF-1(P4) and a cross-reacting antibody, α-OHP, raised against the maize OHP C-terminal region, which shows very strong peptide sequence identity to the corresponding region of G/HBF-1, bound to two protein species exhibiting almost identical electrophoretic mobilities (Figure 5A). The protein species recognized by these antibodies were found in both cytosolic and nuclear fractions and neither their overall abundance nor distribution between cytosol and nucleus changed appreciably during glutathione induction of chs transcription. However, antibody supershift gel retardation experiments indicated that G/HBF-1 was involved in a chs15 promoter–nuclear protein binding complex induced in elicited cells (Figure 5B). The major binding complex formed by incubation of soybean nuclear extracts with the −80 to −42 region of the chs15 promoter was neither elicitor regulated nor supershifted by incubation with α-G/HBF-1(P4) prior to gel retardation analysis. In contrast, the other prominent complex was observed only with nuclear extracts from elicited cells, and pre-incubation with α-G/HBF-1(P4) caused a marked reduction in the electrophoretic mobility of this complex. This supershifted DNA-binding complex co-migrated with the DNA-binding complex formed with recombinant G/HBF-1 in the presence of α-G/HBF-1(P4). Figure 5.Elicitor induces formation of a chs15 promoter–nuclear protein complex containing G/HBF-1. (A) Cellular localization of G/HBF-1. Cytosolic and nuclear extracts, respectively, of uninduced soybean cells and cells at various times after elicitation with 0.5 mM glutathione were analyzed by probing Western blots with α-G/HBF-1(P2) and α-G/HBF-1(P4). The latter antibody recognizes two forms of G/HBF-1, labeled a and b respectively. (B) Antibody supershift analysis of the involvement of G/HBF-1 in DNA-binding complexes with the −80 to −42 region of the chs15 promoter. The effects of pre-treatment of binding complexes with pre-immune serum (lanes 2, 4 and 5) or α-G/HBF-1(P4) (lanes 3, 6 and 7) on electrophoretic mobility were analyzed in gel retardation assays with nuclear extract from cells 2 h after elicitation with Psg(avrA) (lanes 5 and 7) and nuclear extract from equivalent control cells (lanes 4 and 6). Electrophoretic mobilities were compared with those of binding complexes formed with no added proteins (lane 1), recombinant G/HBF-1 alone (lane 8), recombinant G/HBF-1 plus pre-immune serum (lane 2) or recombinant G/HBF-1 plus α-G/HBF-1(P4) (lane 3). A, α-G/HBF-1(P4)-supershifted elicitor-regulated complexes; B, major complex not α-G/HBF-1(P4)-supershifted or elicitor regulated; C, complex with recombinant G/HBF-1; D, elicitor-regulated complex. Download figure Download PowerPoint Phosphorylation of G/HBF-1 Incorporation of G/HBF-1 into a chs15 promoter-binding complex in elicited cells implied post-translational regulation of G/HBF-1, and we next investigated whether G/HBF-1 was phosphorylated in vivo by labeling soybean cells with [32P]phosphate. Immunoprecipitation of G/HBF-1 from extracts prepared after exposure of cells to [32P]phosphate resulted in the incorporation of radioactivity into a single protein of the appropriate electrophoretic mobility (Figure 6B). Moreover, several fold greater incorporation of 32P into immunoprecipitable G/HBF-1 was observed in [32P]phosphate-labeled cells after treatment with 0.5 mM glutathione than in equivalent pulse-labeled control cells. Phosphoamino acid analysis of 32P-labeled, immunoprecipitable G/HBF-1 demonstrated that phosphorylation was almost exclusively on serine residues, with little detectable phosphothreonine (1–5%) and no phosphotyrosine (Figure 6C). Figure 6.Phosphorylation of G/HBF-1. (A) Effect of alkaline phosphatase treatment on G/HBF-1 reactivity in Western blots probed with α-G/HBF-1(P2). (B) Elicitor stimulation of G/HBF-1 phosphorylation in vivo. Cells were pre-labeled with [32P]phosphate for 15 min. Incorporation of 32P into immunoprecipitable G/HBF-1 was compared in cells then elicited with 0.5 mM glutathione for 30 min and in equivalent pulse-labeled control cells. (C) Phosphoamino acid analysis of in vivo 32P-labeled G/HBF-1. The hydrolysate of immunoprecipitated G/HBF-1 was analyzed by two-dimensional thin layer chromatography and radiolabeled residues detected by autoradiography. (D) Two-dimensional mapping of G/HBF-1 phosphopeptides. Recombinant G/HBF-1(His6) was incubated with [γ-32P]ATP and a cytosolic extract from elicited cells. After immunoprecipitation, the trypsin digest of in vitro phosphorylated G/HBF-1(His6) was analyzed by two-dimensional electrophoresis and autoradiography. (E) Relationship between G/HBF-1 phosphorylation and α-G/HBF-1(P4) immunoreactivity. Lanes 1–4: recombinant G/HBF-1 was pre-incubated with [γ-32P]ATP and extracts from either control cells (lane 1) or cells 2 h after inoculation with Psg(avrA) (lanes 2–4). The protein kinase reaction in lane 3 contained 5 μM K252A and 5 μM staurosporine and in lane 4 contained 25 mM EDTA. The upper panel is an autoradiogram of 32P labeling of recombinant G/HBF-1 and the lower panel is a Western blot analysis of α-G/HBF-1(P4) immunoreactivity of the same reaction products. Lane 5: Western blot analysis of the immunoreactivity of recombinant G/HBF-1 with α-G/HBF-1(P4) (upper panel) and α-G/HBF-1(P2) (lower panel). Download figure Download PowerPoint α-G/HBF-1(P4) gave strong immunoreactivity with recombinant G/HBF-1, whereas α-G/HBF-1(P2) did not react with the bacterially expressed protein (Figure 6E). However, incubation of recombinant G/HBF-1 with soybean whole cell extracts in the presence of ATP resulted in the appearance of strong α-G/HBF-1(P2) immunoreactivity in vitro (Figure 6E). Inhibition of the appearance of α-G/HBF-1(P2) immunoreactivity by inclusion of either EDTA or K252A and staurosporine in the protein kinase reactions closely followed the effects of these protein kinase inhibitors on the labeling of recombinant G/HBF-1 with 32P from [γ-32P]ATP in the same reactions (Figure 6E). Moreover, alkaline phosphatase treatment of soybean cytosolic extracts resulted in the loss of α-G/HBF-1(P2) immunoreactivity with native G/HBF-1, indicating that α-G/HBF-1(P2) recognizes a phosphorylation-dependent conformation of G/HBF-1 (Figure 6A). Elicitor and pathogen activate G/HBF-1 kinase To investigate further the phosphorylation control of G/HBF-1, Ni affinity-purified, recombinant factor was incubated with [γ-32P]ATP and cytosolic or nuclear extracts prepared from soybean cells at various times after elicitation with glutathione (Figure 7A). No in vitro phosphorylation of recombinant G/HBF-1 was observed following incubation with nuclear extracts from control or elicited cells, although the nuclear extract from cells 30 min after elicitation gave substantial phosphorylation of an endogenous substrate of apparent Mr ∼33 kDa. In contrast, in vitro phosphorylation of recombinant G/HBF-1 was observed following incubation with the cytosolic fraction, together with phosphorylation of an endogenous substrate of the same electrophoretic mobility as the endogenous nuclear substrate. Figure 7.Stimulation of extractable G/HBF-1 kinase activity in elicited soybean cells.