©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of cDNA Encoding a Protein That Binds a Palindromic Promoter Element Essential for Induction of Fungal Cutinase by Plant Cutin (*)

Daoxin Li , Pappachan E. Kolattukudy (§)

From the (1) Department of Neurobiotechnology, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

Previous studies showed that a palindromic sequence located at -159 base pairs is essential for induction of cutinase gene in Fusarium solani f. sp. pisi (Nectria haematococca mating type VI) by the hydroxy fatty acids from plant cutin and that a 50-kDa nuclear protein binds to a promoter that contains this element. Screening of a phage gt11 expression library with the concatenated palindromic sequence as the probe identified a cDNA encoding a palindrome-binding protein (PBP). Nucleotide sequence of this cDNA revealed an open reading frame that would code for PBP with a calculated molecular weight of 49,847. This PBP contains a putative nuclear localization signal and a zinc finger motif sharing homology with the zinc finger DNA binding domains of transcription factors from mammals, Saccharomyces cerevisiae, Neurospora crassa, and Ustilago maydis. A highly basic region immediately adjacent to the carboxyl side of the zinc finger was also observed. PBP expressed in Escherichia coli showed specific binding to the palindromic DNA fragment.


INTRODUCTION

Cutinase can assist fungal pathogens to breach the cuticular barrier of the host plant and thus plays a significant role in pathogenesis (1) . Cutinase gene was shown to be induced in fungal conidia by contact with the plant cuticle (2) . It was postulated that conidia of virulent fungi would sense the contact with plants via the unique cutin monomers that would be released by the small amount of cutinase carried on the conidia, and these monomers would then induce cutinase in the germinating conidia to assist in the penetration into the host (3) . Conidia from highly virulent strains of Fusarium solani f. sp. pisi were shown to carry cutinase, and contact with cutin was shown to cause induction of cutinase gene (2) . The unique cutin monomers were found to be the best inducer of cutinase (2, 4) . A promoter analysis with transformants generated with the 5`-flanking region of the cutinase gene and its deletion and substitution mutants fused to chloramphenicol acetyltransferase (cat) gene identified a positive-acting G-rich Sp1-like element at -310 bp,() a silencer at -249 bp, and a palindromic sequence at -159 bp that is absolutely essential for the inducible expression of cutinase gene (5) . Gel retardation analysis revealed the presence of nuclear protein(s) that specifically bound the palindromic promoter element, and methylation interference analysis confirmed the binding to this specific region of the promoter (5) . Cross-linking of the protein to the promoter by ultraviolet (UV) light followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the palindrome-binding nuclear protein is composed of 50-kDa monomers (5) . Previously a 50-kDa phosphorylated protein was detected in the fungal nuclear preparation that was induced to initiate cutinase gene transcription with cutin monomers, and indirect evidence was presented that this 50-kDa protein is involved in the induction of cutinase gene by the hydroxy fatty acid monomers (6) . However, such a transcription factor has not been available for direct studies on its involvement in transcription and the mechanism of its action. In fact, no transcription factor involved in plant-fungus interaction has been cloned. In this report we describe the cloning of PBP, compare the sequence of this 50-kDa transcription factor with characteristic features of other transcription factors, and demonstrate that PBP expressed in Escherichia coli specifically binds this palindromic promoter element of cutinase gene.


MATERIALS AND METHODS

Materials and Bacterial Strains-E. coli DH5 (Life Technologies, Inc. (7) ) was used for propagating all plasmids (8). Restriction and modification enzymes and Taq DNA polymerase were from Life Technologies, Inc., poly[d(I-C)]) from Boehringer Mannheim, and labeled nucleotides from either DuPont NEN or Amersham Corp. Chemicals were from Sigma or Amresco. Nitrocellulose filter circles were from Schleicher & Schuell. gt11 cDNA Library Construction-F. solani f. sp. pisi (T8) was grown in Roux bottles with 100 ml of minimal medium (9) containing 1% glucose at 26 °C until the glucose was depleted (9) . Cutin hydrolysate (80 µg/ml) was added to the culture, and after a period of 10-15 min mycelia were harvested and frozen with liquid nitrogen. Poly(A) RNAs were isolated (8) and cDNAs were synthesized with Invitrogen's Librarian cDNA kit with random hexamers. The resultant amplified library in gt11 had a titer of 7 10 plaque-forming units/ml.()

Gel Retardation and Southwestern Hybridization

Two single-stranded 37-mer oligonucleotides (AAT TCG AAA TGG ATC GCG AGC CGA GGC TCG ATT TCA G and AAT TCT GAA ATC GAG CCT CGG CTC GCG ATC CAT TTC G) were annealed to form a double-stranded cutinase gene promoter palindromic element (-159 to -178 bp) with flanking sequences of 5 bp at the 5` end and 7 bp at the 3` end (5) . External EcoRI sites at both ends of the palindrome fragment were used to concatenate this DNA fragment (10) . Gel retardation with the palindrome fragment and T8 nuclear extracts (5) was performed to optimize conditions for screening the above cDNA library as described (5) except with 5% (v/v) glycerol and a final concentration of 60, 80, 100, or 120 mM KCl. Gel retardation with 0.5 µg of partially purified recombinant PBP was done in the same buffer with 0.025 µg/µl of poly[d(I-C)].

Library Screening

The palindrome fragments were phosphorylated on both ends as described (11) and then concatenated to an average size of 738 bp or 20 copies of the palindrome fragment (5) . A total of 7 10 independent plaques of the T8 gt11 expression library was screened according to the method of Vinson et al.(10) containing 10 µg/ml calf thymus single-stranded DNA in the binding buffer (12) . All likely hybridizing plaques were eluted (8) and assigned into 9 groups for secondary screening.

Subcloning and Sequence Analysis

DNA inserts from gt11 clones were directly amplified from phage lysate by polymerase chain reaction (PCR) with gt11 forward and reverse primers and then subcloned into pCR II vector (Invitrogen Corporation). DNAs were prepared with the Magic Maxipreps DNA Purification System (Promega) and used directly for sequencing with Sequenase (U. S. Biochemical Corp.) or for automated sequencing with a model 373A sequencer from Applied Biosystems. The sequences were analyzed with DNA Strider 1.2. Protein homology searches were conducted with the Blast program from NCBI (13) .

Construction of PBP Expression Vector

Two single-stranded oligonucleotides (TAT GCA CCA TCA CCA TCA CCA TCA CAC TAG TGA GCT CTC GAG and GAT CCT CGA GAG CTC ACT AGT GTG ATG GTG ATG GTG ATG GTG CA) were annealed to produce a double-stranded DNA fragment containing a 5` end NdeI site, an internal fragment encoding a short peptide of 7 histidines, and a multiple cloning site for SpeI, SacI, XhoI, and BamHI. This fragment was then cloned into the NdeI and BamHI sites of pET-3a (14) to yield pEXP. The complete open reading frame encoding PBP in gt11 was amplified by PCR with Pfu (Stratagene), digested with SpeI and BamHI, and purified (15) with a Geneclean II kit (Bio 101). Cloning of PBP SpeI/BamHI fragment into pEXP generated pEXP/PBP. pEXP/PBP was introduced into E. coli BL21(DE3)pLysS (14) to express the cloned PBP.

Partial Purification of Recombinant PBP

Production of PBP was induced with -D-thiogalactopyranoside (IPTG) for 6 h at 25 °C (16) . Use of rich medium (17) and induction at a lower temperature (18) seemed to increase the solubility of the recombinant proteins. Induced bacterial cultures were processed as described (19) except that the supernatant was recentrifuged at 105,000 g for 90 min at 4 °C and to the supernatant phenylmethylsulfonyl fluoride was added to 1 mM. Partially purified PBP was obtained with a Ni-nitrilotriacetic acid- agarose (Qiagen) column (19) . Fractions were subjected to SDS-PAGE with a 13% polyacrylamide gel, and those that contained PBP and few minor components were pooled and concentrated with a Centricon 30 filtration unit (Amicon); the buffer was exchanged to 50 mM sodium phosphate (pH 8.0), 300 mM NaCl containing 1 mM phenylmethylsulfonyl fluoride and used for functional assays.

RESULTS

To identify and isolate the protein that binds the palindromic promoter element found to be essential for induction of cutinase gene transcription by hydroxy fatty acids, we used the recognition site probe cloning method originally developed by Singh et al. (20) and modified by Vinson et al.(10) . Optimal KCl concentration in the binding buffer was determined by gel retardation. The identical DNA-protein complex as described by Kämper et al.(5) was detected with a final KCl concentration of 60-120 mM (data not shown). Therefore a binding buffer containing 65 mM KCl, 25 mM HEPES (pH 7.9), 1 mM MgCl, 0.1 mM EDTA, and 0.6 mM dithiothreitol was employed for subsequent screening (10) . Initial screening yielded 547 clones, and two more rounds of plaque purification produced discrete clones. Among them two plaques that appeared to give the strongest hybridization signal were selected and were demonstrated to contain the same insert by PCR amplification and restriction enzyme digestion of the PCR products (data not shown). This phage clone was designated pbp. The insert as amplified above by PCR was subcloned into pCR II to yield the plasmid pPBP.

The nucleotide sequence of the insert in pPBP was determined by primer walking. pPBP contains a single open reading of 1371 bp encoding a putative acidic protein of 457 amino acids with a calculated pI of 5.77 and molecular weight of 49,847, with the first in-frame ATG as the translational initiation codon. Examination of PBP revealed that there are 11 potential consensus sequences for phosphorylation by casein kinase II (21) (boxed in Fig. 1). Three potential sites for phosphorylation by the cAMP-dependent protein kinase A (22) are also found (overbars in Fig. 1). Four potential phosphorylation sites for protein kinase C (23) are also identified (underlinearrows in Fig. 1). Seven potential asparagine-glycosylation sites (24) are also observed (brackets in Fig. 1). A plausible nuclear localization signal (25) , GDKKKKLK, is present at the C terminus of PBP. Homology searches identified that a zinc finger domain is located at the C terminus from amino acid residues 402 to 429 of PBP ( Fig. 1 and 2). A basic region (pI = 11.3) is localized to the carboxyl side of the zinc finger domain of PBP.


Figure 1: Nucleotide sequence of the cDNA and the deduced amino acid sequence of PBP. Structural motifs recognized include the first in-frame ATG codon (circled), consensus sequences for casein kinase II (boxed), protein kinase A (overbars), protein kinase C (underlinearrows) and potential N-glycosylation sites (brackets). The plausible nuclear localization signal is underlinedsingly. The zinc finger domain and the basic region at its carboxyl side are doublyunderlined.



To produce PBP in E. coli, we used the in vivo T7 RNA polymerase expression system (14) . The entire coding region of PBP was cloned downstream of the histidine tag in pEXP. After introduction of pEXP/PBP into BL21(DE3)pLysS cells, 16 randomly picked colonies were assayed for PBP production, and all were shown to produce recombinant PBP (data not shown). To test the solubility of the recombinant PBP expressed in E. coli, a randomly selected transformant was induced to produce PBP. Approximately 75% of the expressed PBP was found in the supernatant (Fig. 3, lane6).


Figure 3: Solubility of recombinant PBP expressed in E. coli. Total proteins corresponding to 100 µl of bacterial culture were resolved on a 13% SDS-PAGE gel. Lanes: 1, host cell protein; 2, non-induced transformant culture; 3, IPTG-induced transformant culture; 4, 10,000 g supernatant of the sonicated transformant cells; 5, pellet from 10,000 g centrifugation; 6, 105,000 g supernatant from the cell lysate as shown in lane4; 7, pellet of the 105,000 g centrifugation; M, molecular mass marker; numbers on the left indicate the sizes of the protein markers in kDa.



To test whether PBP specifically binds the palindromic sequence two approaches were used. Total proteins from six randomly selected transformants were subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. The bound proteins were subjected to denaturation/renaturation treatment and then exposed to P-labeled concatenated palindrome fragments. An autoradiogram showed that the labeled palindrome bound only to the 50-kDa protein that corresponded to the recombinant PBP (Fig. 4, lanes 2-7). Proteins from non-transformed bacterial host cells did not bind the concatenated palindrome fragment (Fig. 4, lane1). The partially purified PBP was used for gel retardation assay. A single retarded band of palindrome fragment-PBP complex was observed (Fig. 5). That this retarded band represented specific binding was shown by the preferential competition for binding to the labeled oligonucleotide by the unlabeled palindrome fragment when compared with a nonspecific DNA fragment.


Figure 4: Binding of the concatenated palindrome fragment to PBP expressed in E. coli. Six randomly selected transformants were induced with IPTG. Total proteins from 100 µl of bacterial culture were resolved on a 13% SDS-PAGE and transferred onto nitrocellulose. The proteins on the membrane were subjected to the same treatments as those for southwestern hybridization and probed with the concatenated palindrome fragment. A, SDS-PAGE analysis of total proteins from IPTG-induced cells of host bacterial non-transformant (lane1) and transformants (lanes 2-7). B, binding of the expressed PBP to the concatenated palindrome fragment. The description of lanes is the same as those in panelA.




Figure 5: Specific binding of the palindrome to PBP expressed in E. coli. Partially purified PBP (0.5 µg) was incubated with a P-labeled palindrome fragment in the presence of molar excesses of either nonspecific competitor DNA fragments or specific unlabeled palindrome DNA fragments. The letter C indicates the DNA-protein complex and the letterF free DNA probe.



DISCUSSION

In this report we describe the isolation of a cDNA clone that encodes a protein that binds a palindromic DNA element by the recognition site probe cloning method (20) and demonstrate that this protein, expressed in E. coli, binds to the palindrome of the minimal promoter essential for induction of the cutinase gene by plant cutin monomers (5) . The clone pbp encoding PBP was obtained by functional expression of the complementary DNA of the target gene. The first in-frame ATG codon, located 36 bp downstream of the 5` end, was in a reasonable context to function as an initiation codon for translation as it compared well with the eukaryotic consensus sequence, (GCC)GCC(A/G)CCATGG, for efficient translation initiation (26) . The presence of a putative nuclear localization signal near the carboxyl end of PBP suggested that this protein might be a nuclear protein. The plausible PBP C-terminal zinc finger motif shared homology to the zinc finger domains of several other regulatory proteins such as GLN3, DAL80, NIT2, Area, Urbs1, GATA-1, and GATA-3; dal80 gene encodes a negative regulator of multiple nitrogen catabolic genes of Saccharomyces cerevisiae(27) , and NIT2, GLN3, and Area are transcriptional activator proteins involved in nitrogen metabolism in Neurospora crassa(28) , S. cerevisiae(29) and Aspergillus nidulans(30) . Urbs1 of Ustilago maydis is a potential transcription factor for regulating the gene, sid1, that encodes the first committed enzyme for siderophore biosynthesis (31) . The erythroid-specific factors, GATA-1 and GATA-3, regulate gene expressions in mammalian erythroid cells (32-34). The target DNA binding sites for these reported transcription factors contain the core sequence GATAA (35) . PBP, however, binds to a palindromic sequence (5) . It is noteworthy that some transcription factors can bind multiple distinct DNA target sites or different types of DNA target sites. For example, the pathway-specific activator protein, ALCR, of A. nidulans that regulates the ethanol metabolic genes alcA and aldA and its own transcription can bind either a palindromic or a direct repeat DNA target site consisting of the core sequence of CCGCA (36) . Furthermore, the CCAAT-binding protein/enhancer-binding protein (C/EBP) and the octamer-binding protein OBP-100 can recognize multiple distinct DNA sequences (37) .

The basic region immediately adjacent to the carboxyl side of the PBP zinc finger domain was also found in yeast GAL4, PPR1, and LAC9 (38, 39) , although the PBP basic region does not share amino acid homology with those of GAL4, PPR1, and LAC9. Whether this basic region in PBP is involved in DNA binding, as reported in GAL4, PPR1, and LAC9 (40) , is yet to be elucidated. The presence of plausible nuclear localization signal and zinc finger DNA-binding domain in PBP and its specific binding to the palindromic promoter sequence suggest that PBP might be a DNA-binding protein that is involved in the regulation of the cutinase gene by the unique hydroxy fatty acids of plant cutin.

The presence of multiple plausible phosphorylation sites for casein kinase II and protein kinase C in PBP suggests that the function of PBP might be modulated by phosphorylation. In fact, experimental evidence had suggested the involvement of protein phosphorylation in the induction of cutinase gene by hydroxy fatty acids of plant cutin. Nuclear protein binding to a 360-bp 5`-flanking region of cutinase gene, containing the palindrome that is essential for activation of cutinase gene transcription, was found to be abolished by treatment of the protein with immobilized phosphatase (6) . In fungal nuclear preparations, cutinase gene transcription activation required a 30-min preincubation with cutin monomer and a fungal protein (4) ; phosphorylation of a 50-kDa protein occurred during this period. Kinase inhibitors prevented this phosphorylation and activation of cutinase gene transcription (6) , suggesting that a cutin-dependent phosphorylation of a transcription factor promotes the binding of this factor to the cutinase gene promoter to trigger transcription of the cutinase gene. Whether the 50-kDa PBP we cloned is identical to the 50-kDa protein phosphorylated in the nuclear preparations is not yet known.

The plausible phosphorylation site for protein kinase A might be functionally important. Cutinase gene shows a typical catabolite repression in that cutin monomers induce its expression only upon depletion of glucose in the culture medium (9) . cAMP could overcome this glucose effect in that exogenous cAMP caused cutinase gene expression prior to depletion of glucose and glucose depletion resulted in an almost 3-fold increase in cAMP levels in the fungus.() Even though these observations suggest possible involvement of protein kinase A, whether phosphorylation of the putative protein kinase A sites found in PBP is involved in this regulation is not yet known.

Even though expression of unique genes is known to be triggered in the process of plant-fungus interaction (41) , the molecular mechanisms involved remain unclear, and no transcription factor involved in such regulation has previously been isolated. Activation of the cutinase gene of F. solani f. sp. pisi by cutin monomers and suppression by glucose constitute a model system, which can be exploited to elucidate some of the molecular events underlying plant-fungus interactions. With the cloning of PBP, it will be possible now to further dissect the molecular events that lead to the activation of the cutinase gene by the hydroxy fatty acids of plant cutin.


FOOTNOTES

*
This work was supported in part by National Science Foundation Grant IBN-9318544. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U23722.

§
To whom correspondence should be addressed: Dept. of Neurobiotechnology, 206 Rightmire Hall, 1060 Carmack Rd., Ohio State University, Columbus OH 43210. Tel.: 614-292-5682; Fax: 614-292-5379. E-mail: Kolattukudy.2@osu.edu.

The abbreviations used are: bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; PBP, palindrome-binding protein; PCR, polymerase chain reaction; IPTG, -D-thiogalactopyranoside.

U. Kämper and P. E. Kolattukudy, unpublished data.

U. Kämper and P. E. Kolattukudy, unpublished data.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.