Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana
Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana
Chi Song 0 1 2
Almudena Ortiz-Urquiza 0 1 2
Sheng-Hua Ying 0 1 2
Jin-Xia Zhang 0 1 2
Nemat O. Keyhani 0 1 2
0 1 Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences; Key Laboratory of Microbial Resources, Ministry of Agriculture , Beijing 100081, China , 2 Department of Microbiology and Cell Science, Institute of Food and Agricultural Science, University of Florida , Bldg 981, Museum Rd., Gainesville, FL 32611 , United States of America, 3 Institute of Microbiology, College of Life Sciences, Zhejiang University , Hangzhou 310058 , China
1 Funding: Funding provided by National Science Foundation grant (IOS-1121392) to NOK, University of Florida, Institute of Food and Agricultural Science funds to NOK, and a China Scholarship Council Fellowship (No. 201303250074) to CS
2 Editor: Olaf Kniemeyer, Hans-Knoell-Institute (HKI) , GERMANY
TATA-binding protein (TBP) is a ubiquitous component of eukaryotic transcription factors that acts to nucleate assembly and position pre-initiation complexes. Multiprotein bridging factor 1 (MBF1) is thought to interconnect TBP with gene specific transcriptional activators, modulating transcriptional networks in response to specific signal and developmental programs. The insect pathogen, Beauveria bassiana, is a cosmopolitan fungus found in most ecosystems where it acts as an important regulator of insect populations and can form intimate associations with certain plants. In order to gain a better understanding of the function of MBF1 in filamentous fungi, its interaction with TBP was demonstrated. The MBF1 and TBP homologs in B. bassiana were cloned and purified from a heterologous E. coli expression system. Whereas purified BbTBP was shown to be able to bind oligonucleotide sequences containing the TATA-motif (Kd 1.3 nM) including sequences derived from the promoters of the B. bassiana chitinase and protease genes. In contrast, BbMBF1 was unable to bind to these same target sequences. However, the formation of a ternary complex between BbMBF1, BbTBP, and a TATA-containing target DNA sequence was seen in agarose gel electrophoretic mobility shift assays (EMSA). These data indicate that BbMBF1 forms direct interactions with BbTBP, and that the complex is capable of binding to DNA sequences containing TATA-motifs, confirming that BbTBP can link BbMBF1 to target sequences as part of the RNA transcriptional machinery in fungi.
Competing Interests: The authors have declared
that no competing interests exist.
Promoters for the three eukaryotic RNA polymerases contain variable sets of core and
regulatory elements that are the targets for the general and regulatory transcription factors (TFs),
respectively. The RNA polymerases themselves are incapable of recognizing target promoters,
but are recruited by the polymerase specific TFs, i.e. each polymerase interacts with its own set
of general and regulatory TFs. TATA-binding protein (TBP) has been considered a universal
TF due to the fact that it is a subunit constituent of the various general transcription factors
used by each polymerase [1,2]. Thus, even if a promoter target lacks a defined “TATA”
sequence, TBP is often present as part of a subunit complex during transcription mediated by
any of the three RNA polymerases.
Multiprotein bridging factor (MBF) was first identified as a non-DNA binding factor that
participates in transcriptional activation by the nuclear hormone receptor fushi tarazu (ftz)
gene product in the silkworm Bombyx mori and in the fruit fly Drosophila melanogaster [3,4].
Yeast MBF1 is activated by the transcription factor GCN4 , and human MBF1 has been
shown to interact with the mammalian homolog of FTZ-F1, namely, Ad4BP/SF-1 . These
findings suggest that MBF acts as a link between TBP and various transcription factors and
subsequently MBFs have been found ubiquitously distributed in eukaryotes and archaea, but
are absent in prokaryotes . Some organisms contain multiple MBF genes that are thought to
mediate interactions between different transcription factors, but all MBFs appear to share a
core interaction with TBP . Aspects of this protein-protein interaction have been
investigated using yeast MBF1 and TBP . These data have revealed the occurrence of compensatory
changes in the respective binding interface domains of these proteins, suggested the
co-evolution of specific compensatory amino acids, and thus underscoring the importance of the
binding interaction in the functions of these proteins. However, MBF1 is not essential in the
budding yeast and its role in other eukaryotes in transcription remains unclear.
Despite their potential critical roles in transcription, there are few reports examining TBP
and/or MBF1 in filamentous fungi. The solution structure of (the C-terminal domain of) an
MBF1 has been derived from the Trichoderma reesei homolog ; which aside from work on
MBF1 in Beauveria bassiana, further described below, are, to the best of our knowledge, the
only reports dealing with MBFs from filamentous fungi. Similarly, there are surprisingly few
examinations of TBP in filamentous fungi. The TBP gene from Aspergillus nidulans has been
shown to substitute for the yeast TBP in vivo  and a conditional lethal disruption of TBP in
Penicillium marneffei was used to show that TBP in this fungus was essential for filamentous
growth, but played a less significant role in growth and development during the yeast phase, a
finding that suggests unique role for TBP in filamentous fungi . Thus far, however, there
have been no biochemical investigations of the interaction of TBP with cognate
TATAsequences and/or of TBP-MBF1 interactions in filamentous fungi.
The insect pathogenic filamentous fungus, Beauveria bassiana, has emerged as a new model
system with which to examine fungal stress and development and host-pathogen interactions
. The fungus is also of significant interest as a microbial biological control agent capable of
targeted different insect pests, especially as a more environmentally friendly alternative to
chemical pesticides [14,15]. B. bassiana displays a broad arthropod host range with infection
occurring via attachment and penetration of the host cuticle [16–18]. Fungal hyphae grow in a
filamentous form to penetrate the host integument, producing free-floating yeast-like cells
termed hyphal bodies once the fungus reaches the hemocoel [19,20]. The hyphal bodies are
able to evade the insect immune system, a process that parallels several other microbial
pathogens [21,22]. Infected hosts typically die 3–10 d post inoculation, after with the fungus
sporulates on the host cadaver to produce new dispersal and infectious cells.
A genetic characterization of the single MBF1 homolog found in B. bassiana has recently
been reported . BbMBF1 was not essential, but ΔBbMBF1 mutants displayed abnormal
hyphal morphogenesis, were deficient in carbon dependent development, and were reduced in
virulence. In addition, loss of BbMBF1 resulted in a wide range of increased stress
susceptibilities including, increased sensitivity to osmotic, oxidative, and UV-stress. Here, we report on
the heterologous expression and purification of BbMBF1 and TBP and their use in
demonstrating (a) the binding of BbTBP to TATA-sequences, revealing a Kd = 1.3 nM, (b) the interaction
between BbTBP and BbMBF1, and (c) the formation of a ternary complex between BbTBP,
cognate TATA sequences, and BbMBF1.
Materials and Methods
Strains and reagents
E coli strain Rosetta DE3 (Promega, Madison WI, USA) was routinely grown in Luria-Bertani
broth (LB) and/or on LB-agar (1.5%) plates containing 50 μg/ml kanamycin as indicated.
Reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and/or
Thermo Fischer Scientific (Waltham, MA, USA) except as noted. Potassium ferrocyanide
trihydrate (K4[Fe(CN)6] 3H2O) was purchased from EMD chemicals (Billerica, USA). Ni- and
Co-NTA agarose was purchased from Gold Biotech (St. Louis, MO, USA). Thrombin was
purchased from (Amersham-Pharmacia Biotech).
Heterologous expression and purification of BbTBP and BbMBF1
Nucleic acid manipulations. Primers used for cloning of B. bassiana TBP and MBF1
genes are given in Table 1. The open reading frames (ORFs) corresponding to BbTBP and
BbMBF1 were amplified by polymerase chain reaction (PCR) using primer pairs PTBP-F/
PTBP-R and PMBF1-F/PMBF1-R, respectively. Forward and reverse primers were designed to
contain unique EcoRI and HindIII, respectively. PCR products were digested with EcoRI and
HindIII, and cloned into respective sites in the pET28a (Novagen) expression vector, to
produce plasmids pET28a-BbTBP and pET28a-BbMBF1, resulting in 6xHis C-terminal-tagged
fusion proteins containing thrombin cleavage sites between the His-tag and the protein ORF
that could be used to remove the His-tag from the protein. The integrity of the plasmid inserts
was verified by sequencing (UF-ICBR Sequencing CORE).
TBP expression vector cloning
MBF1 expression vector cloning
Protein expression and purification. Expression plasmids were transformed into
competent E. coli Rosetta DE3 cells. For expression and purification, E. coli transformants bearing either
pET28a-BbTBP or pET28a-BbMBF1 were cultured in LB broth containing 50 μg/ml kanamycin
at 37°C with aeration to an optical density of OD600 = 0.8 at 37°C, before induction with
isopropylthiogalactoside (IPTG) as follows; for the BbTBP construct, cultures (2x400 ml) were induced
using 0.3 mM (final concentration) IPTG and incubated at 18°C, with aeration for an additional
12 h before harvesting; for BbMBF1, cultures were induced using 0.5 mM (final) IPTG and
incubated at 30°C with aeration for 4 h. Bacterial cells were harvested by centrifugation, 6000xg, 10
min, washing once with starting buffer (SB, 100 mM NaCl, 100 mM HEPES, 10 mM imidazole,
pH 7.5), and then resuspended in 1:50 vol of the same buffer before lysis by French Press. Cell
debris was removed by centrifugation (10,000xg, 15 min), and the supernatant was loaded onto a
pre-equilibrated (SB buffer) Ni- or Co-NTA agarose column (1 ml). After loading, columns were
washed with SB buffer (5 ml) and step-wise eluted (2x5 ml each) with SB buffer containing 100,
200, 300, and 500 mM imidazole. Fractions were assessed for purity by SDS-polyacrylamide gel
electrophoresis (PAGE) and pooled (100–500 mM imidazole for BbTBP and BbMBF1). Purified
BbTBP was dialyzed against TBP Buffer (20 mM Tris-HC1, pH 7.9, 10% glycerol, 20 mM
2-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 200 mM NaC1, 0.01%
Nonidet P-40, ) and BbMBF1 was dialyzed against Dialysis Buffer (DB, 10 mM HEPES, pH 7.5,
100 mM NaCl). Proteins were concentrated using PEG and BioMax concentrator (Millipore,
Billerica, MA) as needed. Protein concentrations were determined using Pierce™ BCA Protein
Assay Kit (Thermo Scientific). Purified proteins were aliquoted and stored at -80°C until use.
Electrophoretic gel mobility shift assay (EMSA)
Four oligonucleotide templates were prepared for EMSA experiments with primer sequences
given in Table 1. Templates were prepared as follows; primer pairs in 0.2 x SSC buffer (3 mM
sodium citrate, pH 7, 30 mM NaCl) were heated to 95°C in a water bath for 5 min and allowed
to anneal by cooling to room temperature for 30 min. Templates derived from primer pairs
PCTRL-F/PCTRL-R, PPRA-F/PPRA-R and PCHIT-F/PCHIT-R were annealed using 1:1
ratios, and the template derived from primer pair PCHIT-TEX-F/PCHIT-R was prepared
using a 1:5 primer ratio. Templates were designated as TATA0, TATAP, TATAC, and flTATAC,
respectively, to indicate the absence of a TATA sequences and derivation from the protease or
chitinase gene promoter sequences, and whether the template contained a fluorescent (fl) label,
respectively. Incubation mixtures for EMSA reactions (20 μl) contained binding buffer (4 μl, 60
mM KCl, 20 mM Tris (pH 7.9), 5.0 mM MgCl2, 10 mM dithiothreitol, 0.2 mg/ml bovine
serum albumin, and 10% glycerol), oligonucleotide template and purified BbTBP and/or
BbMBF1 as indicated in the results. Samples were incubated for 30 min at 37°C, analyzed by
6% acrylamide-bisacrylamide non-denaturing gels . In the case of non-labeled templates,
gels were stained using the EMSA dual protein-nucleic acid staining kit (Molecular Probes)
and visualized using a Gel Doc EZ System (Bio-Rad, Hercules, CA, USA). Experiments using
the TEX-615 labeled template were visualized using the same system directly. Band intensities
were quantified using the Gel Doc visualization software. All experiments were performed in
triplicate and data were analyzed by Excel and GraphPad Prism 5. Protein pI values were
calculated using the DNA MAN software (Lynnan Corp, Pointe-Claire, QC, Canada)
The gene loci for BbTBP and BbMBF1 were identified in the B. bassiana genome sequence
database as locus tags: BBA_05384 and BBA_01830, respectively . BbTBP and MBF1
encoded for proteins consisting of 253 amino acids with a pI of 13.7, and 153 amino acid and a
pI of 10.9, respectively. The open reading frames were used to design primers for cloning of
each respective gene into the E. coli pET28a expression system adding a C-terminal 6 amino
acid histidine tag. Optimal conditions for expression of BbTBP and BbMBF1 were found to be
induction at 18°C for 12 h and induction at 30°C for 4 h, before harvesting of cell cultures,
respectively. The proteins were purified in a single step by immobilized metal affinity
chromatography using Co2+-agarose as detailed in the Experimental Procedures section (Fig 1).
Kinetics of TBP binding to the TATA box and formation of a ternary
Preliminary sets of experiments were performed examining the binding of BbTBP to
oligonucleotides derived from B. bassiana promoter sequences and well as one in which the TATA-sequence
was removed. A 40 bp sequence containing two potential TATA motifs was derived from the B.
bassiana subtilisin-like protease promoter (gene locus BBA_04617). The first element (cggcA
TATgt), however poorly conformed to the canonical TATAAAAAA/G consensus, whereas the
second elements (TATAAAGAG) was a better match. The second sequence used (40 bp), derived
from the chitinase (GenBank: AY145440.1) promoter contained a single element that only poorly
conformed to the consensus (attTATAtcttc). The constructs were termed TATAP and TATAC,
respectively, and a control 35 bp sequence lacking the TATA sequence in TATAC, termed
TATA0 was also constructed. Two electrophoretic gel mobility shift (EMSA) assay protocols
were used to examine TBP binding to DNA. The first involved nucleotide and protein staining of
the gels in which the target DNA template was not labeled, followed by densitometric
quantification of the visualized bands . Nucleic acids were stained using SYBR Green, and a standard
curve of staining of the TATAP template indicated a linear sensitivity between 3–70 nM (Fig 2).
Gel shifts were observed in reactions containing BbTBP and TATAP or TATAC, but not when
the protein was incubated with the TATA0, template (Figs 3 and 4). Although these data provided
qualitative confirmation of BbTBP binding to TATA-sequences, quantitative analyses and curve
fitting (for Kd determination) of these data were not possible due to the high concentrations of
protein and template needed for detection using this method. In this method, for [BbTBP] >>
Kd, the binding curve saturates at ~ 1:1 resulting in a curve profile for which accurate fitting of
the data was problematic, and the best approximation that could be calulated was a Kd < 500
nM. In order to increase the sensitivity needed for accurate affinity constant determination, a
fluorescent-tagged version of the TATAC template (flTATAC) was synthesized, enabling use of
protein concentrations ranging from 0.2–22 nM. Quantification of the binding kinetics resulted
in a Kd = 1.3 nM for BbTBP binding to the TATA-sequence (Fig 5). Competition experiments
were performed to confirm the specificity of binding. Addition of unlabeled TATAC or TATAP
templates were effective in competing off BbTBP from the flTATAC template, whereas no
competition was seen upon addition of the TATA0 template (data not shown).
In order to test whether BbMBF1 could bind to the DNA templates directly, a series of
EMSA experiments were performed using purified BbMBF1 and the oligonucleotide templates.
No gel shift was seen in reactions containing BbMBF1 and any of the template sequences, i.e.
TATA0, TATAC, or TATAP (Fig 6). However, super gel-shifts were seen in reaction mixtures
containing the purified proteins, BbMBF1 and BbTBP, and the oligonucleotide targets TATAC
(Fig 7A) and TATAP (Fig 7B). No shifts were seen when these proteins were incubated in the
presence of the TATA0 template (data not shown). Reactions containing the individual
proteins, and their combination in the absence of template indicated that BbTBP and BbMBF1 did
not appear to form a stable complex (in the absence of DNA template) under the conditions of
the (non-denaturing) polyacrylamide gel electrophoresis (Fig 6).
Fig 1. Expression and purification of BbTBP and BbMBF1. (A) SDS-PAGE analysis of BbTBP purification, left panel, lane S, molecular weight markers,
lane 1 before induce, lane 2 after induce with 0.3 mM IPTG, 18°C, 12 h, lane 3 flow through, lane 4 wash with starting buffer, lane 5–8 wash with (100, 200,
400, 500) imidazole; right panel, lane 1 final purified, dialyzed product. (B) SDS-PAGE analysis of BbMBF1 purification, left panel, lane S, molecular weight
markers, lane 1 before induce, lane 2 after induce with 0.5 mM IPTG, 30°C, 4 h, lane 3 flow through, lane 4 wash with starting buffer, lane 5–8 wash with (100,
200, 400, 500) imidazole; right panel, lane 1 final purified, dialyzed product. Lane 2 final his-tag cleaved product.
B. bassiana is an insect pathogenic fungus that is ubiquitously distributed in almost all
ecosystems and is currently being exploited as a biological control agents of a wide range of insect
pests, where molecular manipulation hold promise for increasing the effectiveness of the
fungus in various applications[15,28–30]. The fungus and their insect hosts are engaged in a
coevolutionary arms race, in which, for certain targets, the fungus is winning whereas for others
the insect remains ahead . Little is known, however, concerning the underlying
transcriptional machinery involved in mediating B. bassiana gene expression. TBP and MBFs are highly
conserved proteins involved in transcription found in all eukaryotes and archaea examined to
date, but are absent in prokaryotes. TBP nucleate assembly and position of RNA polymerase
pre-initiation complexes. Amongst DNA binding proteins, TBP is unique as it binding in the
minor groove of target sequences, resulting in significant bending of the DNA, although
Fig 2. Sensitivity of nucleic acid stain. (A) Increasing concentrations of TATAC-oligonucleotide (0, 3.35,
6.7, 13.4, 20.1, 26.8, 33.5, 46.9, and 60.3 nM) were resolved on 6% non-denaturing SDS-PAGE and
analyzed as detailed in the Experimental procedures section. (B) Quantification of fluorescent signal from
panel (A). Linear fit: y = 38.94x + 196.3; R² = 0.95782.
Fig 3. EMSA analysis of BbTBP binding to TATA-containing oligonucleotides. (A) Binding of purified
BbTBP to TATAC-template. Increasing concentrations of BbTBP (0, 280, 560, 1120, 1680, and 1960 nM)
were incubated with TATAC-template (16.8 nM) and analyzed on 6% non-denaturing PAGE as detailed in the
Methods section. Lanes labeled with an asterisk; 1680 nM BbTBP with no template. Left panel, gel visualized
with nucleic acid strain (green), right panel, gel visualized with protein stain (red). (B) Binding of purified
BbTBP to TATAP-template. Increasing concentrations of BbTBP (0, 280, 560, 1120, 1680, and 1960 nM)
were incubated with TATAC-template (18.3 nM) and analyzed on 6% non-denaturing PAGE as detailed in the
Methods section. Lanes labeled with an asterisk; 1680 nM BbTBP with no template. Left panel, gel visualized
with nucleic acid strain (green), right panel, gel visualized with protein stain (red).
Fig 4. Control EMSA experiments showing no gel shift in reaction mixtures containing purified
BbTBP and a TATA-less oligonucleotide. Reaction mixtures containing TATA0 template (16 nM) with
increasing concentrations (0, 280, 1120, and 1960 nM) of BbTBP, were resolved on 6% non-denaturing
SDS-PAGE and analyzed as detailed in the Experimental procedures section. Left panel visualized with
nucleic acid stain (green), right panel, protein stain (red).
important differences between eukaryotic and archeal TBPs in mediating DNA bending has
been noted [32–33]. Furthermore, structural analyses have revealed that the saddle-shaped
archaea and eukaryotic TBPs can be in opposite orientations when bound to TATA-sequences
when driving transcription in the same direction [34,35]. MBF proteins have been grouped
into three families; group I that contains the archaea proteins, group II contains the animal,
protist, select fungal, and plant proteins, and group III with yeast and filamentous fungal
proteins [7,8,36]. The B. bassiana MBF1 belongs to the group III family, within which the
filamentous fungal MBF lineages separate from the yeast proteins in a phylogenetic analysis.
Both B. bassiana TBP and MBF1 were purified from a heterologous E. coli expression
system. Although both proteins were expressed from the same expression vector, the conditions
and yield of each protein was different. BbMBF1 was induced to high levels and could be
readily purified from the recombinant host. In contrast, BbTBP1 expression was low and
significantly less protein could be purified/L of growth culture. As the expression vectors were
constructed from cDNA clones, this could be due to differences in transcriptional and/or
translational efficiencies of the constructs. In addition, BbTBP was prone to the formation of
aggregates/precipitation. Purified BbTBP was shown to bind to TATA-containing oligonucleotide
sequences but not to TATA-less sequence by EMSA analysis. Initial experiments using a dual
dye, i.e. nuclei acid and protein staining protocol , while successful in qualitatively showing
Fig 5. EMSA analysis of BbTBP binding to flTATAC oligonucleotide. (A) Gel shift on 6% non-denaturing
PAGE using 0.1 nM fluorescent labeled TATAC-oligonucleotide and indicated concentrations of purified
BbTBP (0–22.4 nM). (B) Quantification and analysis of BbTBP binding to flTATAC oligonucleotide. Data fit to
Y = Bmax X/(Kd + X), resulting in a Kd = 1.3 ± 0.16 nM, Bmax = 1, R2 = 0.97.
TBP-DNA binding, required protein and template concentrations that were >> Kd values,
resulting in data that could not be accurately quantitatively analyzed. Use of a fluorescently
labeled template, however, resulted in experiments within the appropriate range for analysis,
yielding a Kd = 1.3 nM for BbTBP binding to the TATA-sequence. This number is similar to
that reported for yeast TBP, i.e. 4–5 nM  and from 0.44 to 5 nM for TATA-binding protein
interactions to variously modified TATA boxes [38,39]. The oligonucleotide sequences used
were derived from the B. bassiana chitinase and protease genes , enzymes critical for
targeting of insects hosts by the fungus [40,41]. Indeed, biotechnological application of a fusion
chitinase-protease protein has shown promise as a means of increasing the ability of the fungus to
target insect hosts [42,43]. Both enzymes are considered inducible [44–46], and consistent with
the presence of TATA-sequences in the promoter regions of inducible genes, putative
TATAmotifs were identified in the promoter regions of both genes. The protease promoter sequence
contained two TATA-sequences, one of which conformed poorly to the context consensus
sequence, whereas the other motif was a better match. The chitinase TATA-sequence also did
Fig 6. Control EMSA experiments containing indicated proteins in reaction mixtures. Concentrations of TATAC = 18.3 nM, TBP = 1129 nM, and
MBF1 = 855 nM. Left panels visualized with nucleic acid stain (green), right panels, protein stain (red).
not conform to the canonical TATA-sequence consensus. Our data indicate that both template
sequences were capable to mediating TBP binding, and that TBP affinity to the motif found in
the chitinase promoter was comparable to that seen in other systems. These data are consistent
with a recent survey of TBP binding sites in which chromatin immunoprecipitation (ChIP)
was used to identify novel human genome TBP binding sites. Many such sites were located in
introns or lacked gene/mRNA annotation. However, the authors report that these sites could
still direct transcription, suggesting widespread targeting of TBP and associated complexes to
non-canonical sites .
Despite MBF proteins having one (putative) DNA-binding helix-turn-helix (HTH) domain
, these proteins do not bind DNA directly, a finding confirmed by our results. B. bassiana
MBF1 was unable to bind to oligonucleotides irrespective of the presence of a TATA-motif,
whereas BbTBP was dependent upon the presence of a TATA-motif in the target DNA. A
super shift in EMSA experiments was noted in reactions containing TATA-motif DNA targets,
BbTBP, and BbMBF1, thus providing evidence for ternary complex formation between these
partners. Our data are consistent with a model in which TBP bridges MBF1 with target DNA
sequences. As MBF1 is supposed to then bridge transcription factors to the (TBP-containing)
initiation complex future work identifying the partners of BbMBF1 is warranted. Only a
handful of transcription factors have been characterized in B. bassiana. The carbon catabolite
repressor transcription factor, BbcreA has been shown to be important for nutrient
assimilation, development, differentiation, temperature sensitivity, and virulence , and the stress
response transcription factor Bbmsn2, has been linked to production of secondary metabolites,
pH dependent growth, and stress response . Intriguingly, putative binding sites for CreA
and Msn2 have been identified in the promoter sequences of the chitinase and protease genes,
however, whether there are any links between these transcription factors and MBF1 remains to
Fig 7. EMSA analysis of BbMBF1-TBP-TATA-oligo complex formation. (A) Interaction of BbTBP and
BbMBF1 to TATAC-template. BbTBP (1129 nM) was incubated with TATAC-template (16.8 nM) with and
without BbMBF1 (855 nM) and analyzed on 6% non-denaturing PAGE as detailed in the Methods section.
Left panel, gel visualized with nucleic acid strain (green), right panel, gel visualized with protein stain (red).
(B) Interaction of BbTBP and BbMBF1 to TATAP-template. BbTBP (1129 nM) was incubated with
TATACtemplate (18.3 nM) with and without BbMBF1 (855 nM) and analyzed on 6% non-denaturing PAGE as
detailed in the Methods section. Left panel, gel visualized with nucleic acid strain (green), right panel, gel
visualized with protein stain (red).
This work was supported in part by a National Science Foundation grant (IOS-1121392) to
NOK, University of Florida, Institute of Food and Agricultural Science funds to NOK, and a
China Scholarship Council Fellowship (No. 201303250074) to CS. The authors wish to thank
Collin Osborne for assistance with some experiments.
Conceived and designed the experiments: CS AO-U S-HY J-XZ NOK. Performed the
experiments: CS AO-U. Analyzed the data: CS AO-U NOK. Contributed reagents/materials/analysis
tools: S-HY JXZ. Wrote the paper: NOK.
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