From the
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
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,
Materials and Bacterial Strains-E. coli DH5
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.
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 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.
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank
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.
(
)
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.
(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.
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) .
(
)
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.
/EMBL Data Bank with accession number(s) U23722.
-D-thiogalactopyranoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.