Poster Category
3:
Regulation of Gene Expression at the Genome Level
PR3.1
Tianhong Wang,
Li Yan, Guanjun Chen
State Key Laboratory of Microbial Technology,
wangtianhong@sdu.edu.cn,
weifliu@sdu.edu.cn
The interplay between the yeast prototypical
transcriptional activator Gal4p and the inhibitor protein Gal80p determines the
transcriptional status of the genes needed for galactose utilization in
Saccharomyces cerevisiae. In this study, we showed that deletion of
components responsible for mitochondria and cytocytoplasmic synthesis of NADP
including
pos5 and
utr1 impaired the induction of GAL genes. The malfunction of
respiratory oxidative phosphorylation was also found to affect the induced
transcription of
GAL genes. Increase in intracellular level of NADP or ATP in these
mutants restored the normal induction process with the recovered occupancy of
Ser5 phosphorylated RNA polymerase II on the
GAL promoter. On the other hand, deletion of
dsg1 coding for an F box protein impaired the early-onset expression
of a
GAL1-LacZ
reporter. However, the induced transcription of
GAL mRNA was not affected although a decreased occupancy of RNA
polymerase II on the 5’ coding region of
GAL genes occurred. The Dsg1-mediated induction defect was
partly complemented by increase in intracellular level of NADP but not by that
of ATP. Nonetheless all the induction defects were abrogated by the absence of
transcription inhibitor Gal80p.
In vitro analysis revealed that NADP synergizes with ATP to
destabilize the interaction between Gal80 and Gal4. Taken together, these
results suggest that interaction status between Gal4p and Gal80p not only
determines the on-off of transcription of GAL genes, but may also be involved in
the fine tuning of the whole transcription process including the formation of
mature mRNAs.
CARMEN RUGER-HERREROS[1]
Raúl Fernández-Barranco[1] Maria Olmedo[2] Luis M.
Corrochano[1] David Cánovas[1]
1Department
of Genetics,
carmenruger@us.es
The ascomycete
Aspergillus nidulans is a model organism to study fungal
development. Conidiation is controlled by the product of the
brlA gene. Many gene products act upstream of
brlA, probably allowing the synthesis of chemicals or allowing the
transduction of environmental signals to trigger
brlA transcription. The
A. nidulans genome contains genes for a phytochrome (fphA),
two homologs of
N.crassa WC-1 and WC-2 (lreA
and
lreB) and a
veA gene. Red and blue light stimulate conidiation in
A. nidulans, but mutations in the
veA gene allow conidiation in the dark.
Recently, it has been shown that the phytocrome FphA interacts with VeA and LreA
and LreB. However, the mechanism that the photoreceptors employ to activate
conidiation remains unknown. We have found that the expression of several
conidiation genes, including
brlA, fluG flbA, flbB and
flbC, was regulated by light. The photoactivation of these genes
showed a quick reponse with mRNA accumulation increasing after 5 minutes of
illumination.
brlA mRNA accumulation after illumination increased with time
showing maximum values between 30 to 60 minutes and revealed a two-component
activation.
Deletion of the photoreceptor genes
fphA, lreA and
lreB reduced the activation by light of the studied genes. None of
these genes are essential for gene photoactivation since we observed
light-dependent mRNA accumulation in strains with single deletion of
photoreceptor genes. On-going experiments provides a model for the
light-dependent activation of conidiation.
PR3.3
Kinga Krol[1]
Igor Y. Morozov[2] Piotr Weglenski[1] Massimo Reverberi[3]
Mark X. Caddick[2] Agnieszka Dzikowska[1]
1Institute
of Genetics and Biotechnology, University of Warsaw, Poland, 2School
of Biological Sciences, The University of Liverpool, UK, 3Plant
Biology, Universita La Sapienza, Roma, Italy
kinga@igib.uw.edu.pl
RRMA is the RNA binding protein involved in posttranscriptional regulation of
gene expression in
Aspergillus nidulans.
rrmA gene was identified as a suppressor of mutations in
arginine/proline catabolic pathway. Independently RRMA protein was shown to bind
to the 3’UTR of
areA transcript (nitrogen positive regulator).
ΔrrmA mutation results in slow growth phenotype and higher
sensitivity to oxidative stress. Analysis of main antioxidant enzymes revealed
different activity pattern during early development stages in
ΔrrmA strain comparing to the control strain. Transcriptional
analysis has shown that
ΔrrmA mutation results in higher stability of specific transcripts
under conditions of oxidative stress and nitrogen starvation. Our results
indicate that RRMA plays important role in metabolism of
A. nidulans and can be involved in the mechanism of regulated
degradation of specific mRNAs in response to environmental signals.
Marion E. Pucher,
Astrid R. Mach-Aigner, Robert L. Mach
mpucher@mail.zserv.tuwien.ac.at
The saprophytic fungus
Trichoderma reesei (teleomorph
Hypocrea jecorina) is able to degrade a huge variety of biopolymers
such as xylan and cellulose, the predominant compound of plant materials. The
produced hydrolytic enzymes have received great industrially importance (e.g.
food and feed industry, pulp and paper industry). Before we started our
investigations, it was already known that D-xylose is an inducer for
hydrolytic-enzymes encoding genes. In this study we show that the degree of
induction is dependent on the applied D-xylose concentration. We could
demonstrate that high induction of hydrolytic-enzymes encoding genes can be
observed using 0.5 or 1 mM D-xylose for 3 hours of cultivation. D-xylose causes
never glucose-like repression of transcription of xylanase-encoding genes, even
not at high concentrations (66 mM). The investigations show that the
transcription factor Carbon catabolite repressor 1 (Cre1) reduces the expression
of the Xylanase regulator 1 (Xyr1), the main activator of many hydrolytic
enzymes encoding genes, and as a consequence, lower amounts of hydrolytic
enzymes are expressed. In this study we can demonstrate that D-xylose has to be
metabolized via the Xylose reductase (Xyl1) to achieve induction of xylanase
expression. Finally, we show that a strain bearing a constitutively expressed xyr1
could partly overcome the negative influence of Cre1.
Jens Kamerewerd[1]
Birgit Hoff[1] Tim Dahlmann[1] Ivo Zadra[2]
Thomas Specht[2] Hubert Kürnsteiner[2] Ulrich Kück[1]
1Christian
Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl für Allgemeine und
Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780
Bochum, Germany
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria
jens.kamerewerd@rub.de
The filamentous fungus
Penicillium chrysogenum is the main industrial producer of the
β-lactam antibiotic penicillin. Recently the genome sequence of
P. chrysogenum was published, thus strain improvement by reverse
genetics became feasible. For efficient gene replacements, a
P. chrysogenum strain lacking non-homologous end joining (NHEJ) was
constructed by deletion of the
Pcku70 gene, a homologue of human Ku70 gene essential for NHEJ.
Although ΔPcku70 strain lacks any obvious phenotype under native conditions, we
were able to identify a stress-related molecular phenotype by conducting a whole
genome microarray time course analysis. Strain ΔPcku70 was used to generate the
mutants ΔPcvelA and ΔPclaeA. The proteins PcvelA and PclaeA, homologues of the
VeA and LaeA proteins from
Aspergillus nidulans, are part of a multiprotein complex and
positively regulate penicillin biosynthesis in
P. chrysogenum. In order to identify common target genes of PcvelA
and PclaeA, a comparative microarray analysis was conducted with transcripts
from the deletion mutants ΔPcvelA and ΔPclaeA isolated after 48, 60 and 96 hours
of growth. For comparison, we used the data from the time course analysis of the
ΔPcku70 strain. Our results clearly demonstrate common as well as distinct
functions of PcvelA and PclaeA as regulators of secondary metabolism and fungal
development in
P. chrysogenum.
Philipp Wiemann[5]
Daren W. Brown[1] Karin Kleigrewe[2] Jin Woo Bok[3]
Nancy P. Keller[4] Hans-Ulrich Humpf[2] Bettina Tudzynski[5]
1Mycotoxin
Research Unit, USDA, ARS, NCAUR, 2Institut für Lebensmittelchemie,
WWU Münster
3Department of Medical Microbiology and Immunology, University of
Wisconsin
4Department of Medical Microbiology and Immunology / Department of
Bacteriology, University of Wisconsin, 5Institut für Botanik, WWU
Münster
philippwiemann@aol.com
Besides industrially produced gibberellins (GAs),
Fusarium fujikuroi is able to produce additional secondary
metabolites such as the pigment bikaverin and the mycotoxins fumonisins and
fusarins. The global regulation of these biosynthetic pathways is only poorly
understood. Recently, the Velvet complex containing VeA and several other
regulatory proteins was shown to be involved in global regulation of secondary
metabolism and differentiation in
Aspergillus nidulans. Here we report on the characterization of two
components of the
F.
fujikuroi Velvet-like complex, FfVel1 and FfLae1. The gene encoding
this first reported LaeA ortholog outside the class of
Eurotiomycetidae is upregulated in the ΔFfvel1
mutant (shown by microarray-studies) and FfLae1 interacts with FfVel1 in the
nucleus (shown by BiFC). Deletion of
Ffvel1 and
Fflae1 revealed for the first time that Velvet can simultaneously
act as positive (GAs, fumonisins and fusarins) and negative (bikaverin)
regulator of secondary metabolism, most likely by interconnecting with the
nitrogen regulation network. Furthermore, we show that both components affect
conidiation and virulence of
F.
fujikuroi on rice plants. Cross genus complementation studies of
Velvet complex component mutants between
Fusarium and
Aspergillus support an ancient origin for this complex which has
undergone a divergence in specific functions mediating development and secondary
metabolism.
PR3.7
Maria Macios, Piotr Weglenski, Agnieszka Dzikowska[1]
Institute of Genetics and Biotechnology,
adzik@ibb.waw.pl
In
A. nidulans arginine is utilised as a nitrogen and carbon source. It
is a good system for investigation the connection between the two global carbon
and nitrogen repression regulatory systems. Utilization of arginine depends on
the presence and inducibility of two arginine catabolic enzymes arginase and
ornithine aminotransferase (OAT) encoded by
agaA and
otaA genes, respectively. Analysis of different single and double
areA and
areB mutants have shown that two GATA factors AREA and AREB
negatively regulate the
expression of arginine catabolism genes under nitrogen repression
conditions. AREA and AREB activities depend on carbon source. AREA regulator is
necessary for the ammonium repression of arginine catabolism genes under carbon
repressing conditions while AREB - under carbon limited, non-repressing
conditions. AREA activity was shown to be modulated by a direct protein –
protein interaction with NMRA protein which is proposed to bind to the C
terminus of AREA and repress its activity in the presence of glutamine (Platt et
al., 1996; Andrianopoulos et al., 1998; Lamb 2003 et al., Lamb et al., 2004;
Wong et al., 2007). We have shown that these interactions are also important in
nitrogen metabolite repression of arginine catabolism genes.
Rodrigo Anselmo Cazzaniga,
Silveira HCS, Marques MMC, Evangelista AF, Sanches PR, Passos GAS, Rossi A,
Martinez-Rossi, NM
cazzaniga@usp.br
Trichophyton rubrum
is a filamentous fungus that infects human skin and nails, being the most
prevalent dermatophyte worldwide. During its growth on keratin a shift on the
extracellular pH from acidic to alkaline occurs, which may be an efficient
strategy for its successful infection and maintenance in the host.
T. rubrum responds to environmental pH by derepressing nonspecific
proteolytic enzymes and keratinases with optimal activity at acidic pH during
the initial stages of infection, probably in response to the acidic pH of human
skin. Although several factors contribute to its pathogenicity, successful
infection depends on the adherence capacity of the infecting dermatophyte and
its ability to sense and overcome the acidic pH of the skin, and usage of the
molecules from the host tissue as nutrient source. In this work
T. rubrum gene expression kinetics during keratin degradation was
evaluated by cDNA microarrays to gain a better comprehension of the adaptive
responses to molecules presented in the host microenvironment. During
T. rubrum growth on keratin medium, we observed that germination of
the conidia and hyphal formation were accompanied by a gradual increase in the
extracellular pH, ranging values from pH 6.5 to pH 8.5. After hybridization of
the microarrays, and SAM analysis, 124 genes were found to be differentially
expressed throughout the cultivation times. In 24h of cultivation, the
transcript enconding a glutamate carboxypeptidase 2 was up-regulated, which must
be important for protease release contributing to the clivage of keratin. At
48h, in which the extracellular pH was 8.0, we observed an up-regulation of the
genes encoding PalA and PalB proteins, showing the activation of the
environmental pH sensing pathway during keratin utilization, suggesting its role
during
T. rubrum pathogenic process. The genes encountered here are
involved in several cellular processes, and their regulation during keratin
degradation and pH sensing may be important in the initial stages of
dermatophyte infection or in its maintenance in the host tissue.
Igor Morozov[1]
Meriel G. Jones[1] Dave G. Spiller[1] Joseph Strauss[2]
Mark X Caddick[1]
1School
of Biological Sciences, The University of Liverpool, Crown Street, Liverpool L69
7ZB, UK
2Fungal Genetics and Genomics Unit, Austrian Institute of Technology
and BOKU University Vienna, Muthgasse 18 A-1190 Vienna, Austria
igorm97@liv.ac.uk
In eukaryotes differential transcript degradation represents an integral
component of gene regulation. Generally the limiting step in mRNA turnover is
deadenylation, which triggers translational repression and 5’ decapping once the
length of poly(A) tail reached about 15 A residues. We have recently shown that
in
Aspergillus nidulans both the Caf1 and Ccr4 orthologues are
functionally distinct deadenylases:
Ccr4 is responsible for basal degradation while Caf1 is required for
the regulated degradation of specific transcripts and the variation in
Processing (P) body formation, which occurs in response to a wide range of
stimuli. Disruption of the Ccr4-Caf1-Not complex leads to premature, poly(A)
independent decapping. We have shown that decapping is correlated with a
novel transcript modification, the addition of a CUCU sequence. This 3’
modification of mRNA occurs precisely at the point decapping is triggered. The
addition of the CUCU based sequence tag requires a nucleotidyltransferase CutA,
the disruption of which significantly stabilises mRNA and blocks the formation
of P-bodies. Intriguingly, the key enzyme complex responsible for deadenylation
and therefore degradation, Ccr4-Caf1-Not, also protects mRNA from
premature modification and deadenylation independent decapping. We propose that
3’ modification of adenylated mRNA, which is likely to represent a common
eukaryotic process, primes the transcript for dissociation from ribosomes,
decapping and efficient degradation (1).
References:
1. Morozov IY, Jones MG, Razak AA, Rigden DJ and Caddick MX. CUCU modification
of mRNA promotes decapping and transcript degradation in
Aspergillus nidulans. Mol Cell Biol, 2010, V.30 (4) in press.
Agaricus bisporus
as a model to study effects of chromosomes on complex traits
Wei Gao,
Johan J.P Baars, Anton S.M. Sonnenberg
Wageningen UR
wei.gao@wur.nl
The button mushroom Agaricus bisporus
has a typical life cycle in that after meiosis-II, most basidia form two spores
and each spore receives non-sister nuclei. Only a small portion of the basidia
form four spores, each receiving one haploid nucleus. There are indications that
most of the present-day commercial hybrids of the button mushroom are derived
from Horst U1 via fertile single spore cultures. We have isolated both parental
nuclei from one of the “new” commercial lines and compared the genetic make-up
with that of the parental lines of Horst U1 using 580 SNPs. Only three
recombinations between homologous chromosomes were found and both were
completely reciprocal. The redistribution of homologues over both nuclei was
also completely reciprocal when compared to Horst U1. Both strains thus have an
identical allelic constitution but show, nevertheless, clear phenotypic
differences. It indicates that the redistribution of homologues has an influence
on phenotypes and this offers an interesting tool for breeding.
SNP markers were also used for segregation analysis using homokaryotic single
spore cultures of Horst U1. This particular set of offspring showed hardly any
recombination (18 recombinants in 143 individuals). Since all homologues are
segregated independently, this offers an opportunity to generate chromosome
substitution lines within a limited number of generations.
Agaricus bisporus
offers the opportunity to study the effect of individual chromosomes on complex
traits either by chromosome substitution or redistribution of homologous
chromosomes over the parental nuclei.
Hoi Shan Kwan;
AU, Chun Hang; WONG, Man Chun; QIN, Jing; KWOK, Iris Sze Wai; CHUM, Winnie Wing
Yan; YIP, Pui Ying; WONG, Kin Sing; CHENG, Chi Keung
The
hskwan@eservices.cuhk.edu.hk
Understanding the genomics and functional genomics of a mushroom allows us to
improve its cultivation and quality. Sequencing the genome provides a
comprehensive understanding of the biology of the mushroom. We can also develop
many molecular genetic markers for breeding and genetic manipulation. We can
identify genes encoding various bioactive proteins and pathways leading to
bioactive compounds. Our laboratory sequenced the genome of
Lentinula edodes (Shiitake, Xianggu). The genome size is about 60
Mbases. We are annotating the genes and analyzing the metabolic pathways. In
addition, we have been using a battery of molecular techniques to study
Lentinula edodes development. We used RNA arbitrarily primed-PCR,
SAGE, LongSAGE, EST sequencing and cDNA microarray to analyze genes
differentially expressed along the developmental stages. We are learning more
about the molecular biology and genetics of this important mushroom. We will
also know more about mushroom fruiting body development through comparative
genomics among the genome-sequenced mushrooms.
PR3.12
Barbara Reithner,
Enrique Ibarra-Laclette,Verenice Ramírez-Rodríguez, Alfredo Herrera-Estrella
Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav, Unidad
Irapuato
barbara.reithner@tuwien.ac.at
Trichoderma
spp. are capable to recognize and attack plant-pathogenic fungi of distinct
phyla like
Rhizoctonia solani (Basidiomycete),
Botrytis cinerea,
Fusarium graminearum (both Ascomycetes),
Phytophthora spp, and
Pythium spp (both Oomycetes).
The scope of action involving recognition, secretion on cell wall lysing
enzymes, secretion of secondary metabolites, formation of penetration
structures, and lysis of the host fungus is commonly summarized as
mycoparasitism. In the present work the transcriptome of different stages of
interaction of
T. atroviride IMI206040 with
P. capsici and
R. solani were sequenced using pyrosequencing by 454 Life Science
Technologies. Due to the distinct cell wall composition of the host-fungi
chosen, the aim was to obtain differences in gene expression depending on the
presence or absence of chitin in the host cell wall and genes which may be
involved in host specificity. A total number of almost 300.000 reads represented
more than 2000 and up to 6500 different
Trichoderma transcripts, depending on interaction stage and
plant-pathogenic fungus. Statistical analysis revealed that out of 353
differentially expressed genes only 6 were common during mycoparasitism of
T. atroviride with
P. capsici or
R. solani. One of them was identified as
epl1, coding for an elucidating plant response-like protein in
Trichoderma. The remaining 347 transcripts are currently analyzed
due to their possible involvement in the host-specific response of
Trichoderma.
Mark Caddick,
Christopher Sibthorp, Huihai Wu, Prudence Wong, Neil Hall
caddick@liv.ac.uk
Next generation sequencing is transforming the way in which we can analyse the
transcriptome. It potentially provides quantitative information which is
superior to microarray data, whilst also providing information about
differential use of promoters, splicing and transcript 3’ ends. We are
applying this approach to
A. nidulans. In the first instance we have specifically
investigated transcript start site location, by sequencing mRNA 5’ ends.
This has provided genome wide data with over ~3500 transcript start sites being
localised to within two nucleotide and over 7000 localised to within four
nucleotides. From this we have been able to investigate DNA motifs and
features associated with transcription start sites. We will present these
data and current work focused on characterising the full transcriptome.
Our aim is to provide a community resource that will significantly improve
genome annotation and greatly increase our understanding of its flexibility and
regulation.
Jiujiang Yu[1] Natalie
Fedorova[2] Thomas E. Cleveland[1] Deepak Bhatnagar[1]
William C. Nierman[2]
1USDA/ARS,
Southern
jiujiang.yu@ars.usda.gov
A. flavus
is the major producer of aflatoxin, which is responsible for millions of dollars
in losses in the world and for significant health issues in developing
countries, and is the second leading cause of aspergillosis in immunocompromised
individuals.
Sequencing of
A. flavus NRRL3357 showed that its 36-Mb genome contains 13,488
genes including the aflatoxin gene cluster. Here we describe our efforts to use
the RNA-Seq technology to characterize the entire transcriptome of the species
under conditions conducive to aflatoxin production. To that end, we
sequenced cDNA fragments obtained from Poly(A)-enriched total RNA samples
extracted from fungal mycelium grown under 3 conditions: (i) PMS medium, 29 C,
24h, no toxin; (ii) GMS medium, 29 C, 24h, make toxin; and (iii) GMS medium, 37
C, 24h, no toxin. Two cDNA libraries from each treatment were sequenced using
the Illumina (SOLEXA) short-read technology. Over 5 Million 100 nt reads
were sequenced for each cDNA prep, which were combined to generate a powerful
high resolution map of the
A. flavus transcriptome. In addition, we used the RPKM analysis to
determine transcript abundance in the 3 mRNA samples. The analysis
detected expression in at least 50 % of the genes for each condition and
contributed to our understanding of the genetic basis of the aflatoxin
regulation.
PR3.15
Jawad Merhej,
Christian Barreau,
INRA-France
jawad.merhej@bordeaux.inra.fr
Fusarium graminearum
infects wheat and maize and produces type B trichothecenes. These mycotoxins
cause serious problems when consumed via contaminated cereals.
Tri genes, located in the “Tri
cluster”, are responsible for the biosynthesis of trichothecenes B.
In vitro,
Tri genes of
F. graminearum strain CBS 185.32 are expressed at day 3 with the
toxin starting to accumulate one day later. Strikingly, the induction of
Tri genes expression always seems concomitant with a sharp pH drop
in the media. Acidic pH seems a determinant factor for induction, as neither the
toxin nor the
Tri genes are detectable at neutral pH. The pH regulation of gene
expression in fungi is mediated by the Pac1 transcription factor involved in
various secondary metabolites regulation. An
Fg∆Pac1 deletion mutant and a strain expressing a constitutively
active form (FgPac1C)
were constructed in
F. graminearum. Expression of this constitutive Pac1C
factor strongly reduces expression of
Tri genes and toxin accumulation at acidic pH. Unexpectedly,
deletion of
Pac1 does not induce toxin production at neutral pH. However, it
causes an earlier
Tri5 induction and toxin accumulation at acidic pH. In order to
determine the interference with other
Tri genes regulatory mechanisms, exploring general transcriptional
response to pH variation for mutants and wild-type strains were also performed
using microarrays. Preliminary results will be presented.
Raffaello Tommaso,
Fred O. Asiegbu
tommaso.raffaello@helsinki.fi
The basidiomycete
Heterobasidion annosum (Fr.) Bref. is a filamentous white rot
fungus, considered to be the most economical important pathogen of conifer
trees. Very little is known about molecular and biochemical aspects related to
this fungus pathogenicity. The role of some signal transduction genes (MAP
kinases) as well as their importance in the stress response and adaptation of
the conifer pathogen was investigated. To date, no MAP kinases have been
characterized in this white rot fungus
H. annosum. The role of the stress related HOG MAP kinase which is
thought to be involved in the fungal osmotic tolerance was studied under
different osmotic stress conditions. In the bioinformatic analysis the HOG gene
shows a typical MAP kinase domain with high level of similarity among
basidiomycetes. Phylogenetic revealed the basidiomycete HOG genes group together
in a clade quite separated from the ascomycetes. To assay for functional
relevance of the gene during osmotic stress, total RNA was extracted and the
expression level of the HOG transcript quantified by qPCR. In a parallel study,
the full-length HOG gene was cloned and used for a functional complementation
assay in the
S. cerevisiae
hog1∆ mutant strain. The result showed that the fungus displayed a
decreased growth when exposed to an increased salt osmolarity conditions.
Increased levels of the MAPK HOG gene transcript was observed in high stress
conditions. Complementation functional study with full length gene in the
yeast
hog1∆ mutant strain is ongoing and the results will be presented.
Taken together these results show the putative role of the HOG gene in the
basidiomycete
Heterobasidion annosum and its importance in the capacity of the
fungus to overcome osmolarity stress conditions in the natural environment.
Oezlem Sarikaya Bayram[1]
Oezguer Bayram[1] Oliver Valerius[1] Jennifer Gerke[1]
Stefan Irniger[1] Kap-Hoon Han[2] Gerhard H. Braus[1]
1Georg-August-University,
2Pharmaceutical Engineering,
osarika@gwdg.de
We have recently discovered the trimerinc
velvet complex which is comprised of the light-dependent regulator
VeA, Velvet-like protein VelB, and master regulator of secondary metabolism
LaeA. The
velvet complex coordinates development and secondary metabolism upon
light signal in
Aspergillus nidulans. VeA protein serves as a light-dependent
bridging function between VelB and LaeA proteins. We are currently analysing the
functional as well as physical relationships between the components of the
velvet complex. First data suggest that there might be some
subcomplexes regulating development. We have new insights into the function of
the complex. The current state of the ongoing studies will be presented.
Ayumi Ohba,
Takahiro Shintani, Katsuya Gomi
Graduate School of Agricultural Science, Tohoku university, Japan
over-walk@biochem.tohoku.ac.jp
During the long-term usage of the azole drug for antifungal therapy in
aspergillosis, emergence of azole resistant isolates of
Aspergillus fumigatus has been recently documented. One of the
possible mechanisms of azole resistance
is the up-regulation of genes encoding drug efflux pumps, mainly
belonging to ABC transporters. However, the mechanism that regulates ABC
transporter gene expression has not been elucidated in filamentous fungi
including
Aspergillus species. In
Aspergillus oryzae, until now we have shown that overexpression of a
transcription regulator gene
atrR positively regulates expression of ABC transporter genes (atrA,
atrF, and
atrG) leading to azole drug resistance. In this study, expression of
atrR and the three ABC transporter genes was induced by azole drug
added in the wild-type strain. Furthermore, addition of azole drug significantly
enhanced the expression level of the ABC transporter genes in an overexpression
strain of
atrR, although equal amount of the
atrR transcript was accumulated in the strain irrespective of
addition of azole drug. In contrast, in an
atrR deletion mutant, no expression of the above mentioned ABC
transporter genes was observed regardless of the presence of azole. In
Saccharomyces cerevisiae and
Candida glabrata, xenobiotic substrates such as azole drugs have
recently been shown to directly bind and activate the transcription regulator
Pdr1p via a nuclear-receptor-like pathway. Also in
A. oryzae, AtrR protein may be activated by azole drugs through a
similar mechanism to accelerate the gene expression of ABC transporter genes to
survive in such an environment.
Divyavaradhi VARADARAJAN,
Ingo Bauer, Martin Tribus, Peter Loidl, Stefan Graessle
Division of Molecular Biology, Biocenter,
divyavaradhi.varadarajan@i-med.ac.at
Modifications on the N-terminal tails of core histones highly impact the
regulation of many genes in eukaryotic organisms. In filamentous fungi, however,
only little is known about the enzymes that modify histones. Nevertheless, it
has become increasingly evident that histone acetyltransferases and histone
deacetylases (HDACs), which are responsible for a balanced acetylation status of
the histone tails, are crucial for the regulation of genes involved in fungal
pathogenicity, stress response, production of antibiotics or mycotoxins, and
resistance against antifungal drugs. Our work focuses on RpdA, a class 1 HDAC of
the model organism
Aspergillus nidulans. We recently could demonstrate that RpdA is
essential for growth and development of the fungus. Now we investigate the
ability of complementation of class 1 HDACs from different filamentous fungi,
yeasts and human in an
Aspergillus strain with depleted RpdA activity. The RpdA orthologs
of filamentous fungi like
A. fumigatus or
Cochliobolus carbonum were able to complement RpdA minus strains,
whereas orthologs of yeasts (RPD3/Clr6) or human (HDAC1/HDAC2) were not. These
results confirm that RpdA-type enzymes of filamentous fungi comprise distinct
motifs that are essential for fungal growth and development. These
characteristic features and their significance turn RpdA-type enzymes into
promising targets for (fungal specific) HDAC inhibitors (HDACi) with impact on
the vitality of these organisms. Several HDACi were recently approved (or are
under evaluation) as therapeutic and chemo-preventive agents against cancer,
neurodegenerative disorders and transplantation intolerance. In the future,
these molecules may also represent attractive canditates for the development of
novel antifungal agents, or may have potential as compounds used in combination
with drugs administered in classical antifungal therapies.
Taylor Schoberle,
Gregory S May
UT MD
Taylor.Schoberle@uth.tmc.edu
Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, is
important to virulence in certain host models. Most genes involved in
gliotoxin production and transport are located on a gene cluster, which is
co-transcribed. Deletion of an essential gene within the gliotoxin
biosynthetic pathway,
gliP, led to a lack of gliotoxin production within
A. fumigatus, as well as a significant reduction in virulence in a
steroid treated host. Reduced virulence was a result of the presence of
neutrophils within the host, as other labs using neutropenic mouse models did
not see this trend. In microarray studies,
gliA, the gliotoxin efflux pump, is induced over 30-fold in the
presence of neutrophils. Growth of
A. fumigatus in medium containing sodium nitrate also significantly
induces expression of
gliA. Although many studies have been done to elucidate the
effects of gliotoxin on host cells, little is known about the expression of the
gliotoxin cluster. To identify cis-acting regulatory elements in the
gliA promoter, we examined expression from promoter deletion mutants
fused to
lacZ. Several positive and negative regulatory elements were
identified that altered expression in a nitrogen source dependent manner.
Using a
gliA promoter
lacZ fusion reporter construct, we screened for activators of
gliA expression and identified candidate plasmids that activate the
reporter. We are conducting additional experiments to investigate this
regulation. Elucidating the genes that are responsible for the regulation
of
gliA will lead to a greater understanding of gliotoxin synthesis and
transport, which is important to the pathogenesis of
A. fumigatus.
PR3.21
Robin A. Ohm,
Jan F. de Jong, Luis G. Lugones, Han A.B. Wösten
Department of Microbiology and Kluyver Centre for Genomics of Industrial
Fermentations
Genome Of
Schizophyllum Commune Yields Insight Into Regulation Of Mushroom
Formation
The wood degrading fungus
Schizophyllum
commune is a model system for mushroom development. The recently
sequenced 38.5 Mb genome contains 13,210 genes. We performed whole genome
expression analysis using the sequencing-based technique MPSS (Massively
Parallel Signature Sequencing) on 4 developmental stages: monokaryon, stage I
aggregates, stage II primordia and mushrooms. These data yield insight into the
process of mushroom formation in high detail. For example, compared to other
stages of development protein production is up-regulated in stage I, gene
regulation is up-regulated in stage II and metabolism is down-regulated in
mushrooms. Interestingly, 43% of the genes were shown to be also expressed in
anti-sense direction, suggesting a role for anti-sense gene regulation during
development.
We identified 471 transcription factor genes in the genome, of which 27 were
evolutionary restricted to mushroom forming fungi. Moreover, about one third of
all transcription factor genes were differentially regulated during development.
Eleven transcription factor genes were deleted using recently developed and
efficient gene deletion techniques. Inactivation of
reg1,
reg2 or
reg3 resulted in colonies with only vegetative mycelium. Development
stopped in stage I after inactivation of
reg4, whereas inactivation of
reg5, reg6 or
reg7 resulted in more but smaller mushrooms than wild-type.
These are the first strains with a targeted gene inactivation that show an
effect on mushroom formation.
Taken together, we conclude that the genome sequence and genetic amenability of
S. commune strongly contribute to our understanding of mushroom
formation.
Ingo Bauer,
Martin Tribus, Johannes Galehr, Birgit Faber, Divyavaradhi Varadarajan, Gerald
Brosch, Stefan Graessle
Biocenter – Division of Molecular Biology,
ingo.bauer@i-med.ac.at
ct" class="mceAdvanced">In eukaryotic organisms DNA is compacted into an
elaborate structure called chromatin, thus enabling regulation of transcription
by controlling the accessibility of the genetic information for transcription
factors. Among the key players involved in the regulation of chromatin structure
are histone acetyltransferases and histone deacetylases (HDACs) —enzymes
establishing distinct acetylation patterns in the N- terminal tails of core
histones. In filamentous fungi only little is known about the biological
functions of these enzymes; nevertheless recent studies have shown that class 2
HDACs affect the regulation of genes involved in stress response and secondary
metabolite production. Here we report that depletion of RpdA, a class 1 HDAC of
Aspergillus nidulans, leads to a drastic reduction of growth and
sporulation. Functional studies revealed that a short C-terminal motif unique
for RpdA-type proteins of filamentous fungi is required for catalytic activity
and consequently cannot be deleted without affecting the viability of
A. nidulans. Thus, the C-terminal extension of RpdA-type proteins
represents a promising target for fungal specific HDAC-inhibitors that might
have potential as new antifungal compounds with clinical and agricultural
applications.
Miguel Corral González[1], David Lara Martínez[1], Arturo
Pérez Eslava[2], Enrique A. Iturriaga[1]
1
Área de Genética, Departamento de Microbiología y Genética, Facultad de
Biología, Universidad de Salamanca.
2
Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de
Salamanca
iturri@usal.es
The presence of modified bases in the DNA of almost every organism analysed is
known for more than fifty years now. While m5C (5-methyl-citosine)
methylation has been shown and broadly studied in bacteria, fungi, other lower
eukaryotes, plants and animals, the investigation on DNA m6A
(6-methyl-adenine) methylation in eukaryotes has been relatively ignored,
probably by the thinking of it being absent, irrelevant, or difficult to
accomplish. The idea that m6A has a modest (or not at all), role in
the regulation of eukaryotic genomic structure, organisation, and regulation
contrasts with what it happens in bacteria, in which more than one important
cellular and “epigenetic” mechanisms of regulation are mediated by m6A
methylation.
Anyhow, several lower and higher (human, rat and plant) eukaryotes have been
investigated in relation to their m6A presence and content, with some
interesting results. A gap in these studies is the fungal kingdom, which
probably could give mixed (and probably relevant), information on what happened
from prokaryotes to eukaryotes in this matter. Our first approach to answer this
question was based on m6A sensitive digestion with restriction
enzymes, and
in silico analyses of DNA from more then ten fully-sequenced fungi.
High-molecular weight DNA was obtained from the fungi to be analysed and
digested with
DpnI, an enzyme that cuts the sequence GA↓TC only when the A in this
sequence is methylated. What we have found is that several groups of fungi have
in fact an m6A DNA-methylation system in their genomes. Why? How?
What for? These are today questions to be resolved.
These preliminary results only show what happens at the GATC sequence. So, we
also analysed
in silico the fungal genomes looking for N6-methyl
adenine transferases. In some of them, which do not show any significant
digestion with
DpnI, we have been able to find sequences more than similar to m6A
DNA-methyl-transferases.
The data obtained in these two kind of experiments give us new ideas to find out
the presence, importance, and possible function of m6A
DNA-methylation in fungi.
PR3.24
Linda Johnson,
Stuart Card, Wayne Simpson, Anar Khan, Anouck de Bonth, Christine Voisey,
Richard Johnson
AgResearch Limited, Grasslands Research Centre, Palmerston North,
linda.johnson@agresearch.co.nz
We are investigating the molecular mechanisms behind how a mutualistic symbiotic
endophyte in its natural host grass,
Elymus spp. (tribe Triticeae) has become pathogenic on
Triticum spp. To unravel the switch from mutualism to pathogenesis,
we have applied a mycological approach along with the latest transcriptomic
technology. We obtained a low (5-10%) systemic infection rate in a
Jochen Schmid1,3
Dirk Müller-Hagen3 Thomas Bekel2 Laura Funk3
Ulf Stahl3 Volker Sieber1 Vera Meyer3, 4
1Chemistry
of Biogenic Resources, Straubing Centre of Science, Technische Universität
München, Straubing, Germany, 2Computational Genomics, Center for
Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany,3Department
of Microbiology and Genetics, Berlin University of Technology, Berlin, Germany,
4Leiden University, Institute of Biology, Molecular Microbiology and
Biotechnology, Leiden, The Netherlands
The plant pathogenic basidiomycete
Sclerotium rolfsii produces the industrially exploited
exopolysaccharide scleroglucan, a polymer that consists of β-(1→3) linked
glucose with a β-(1→6) glycosyl branch on every third unit. Although the
physicochemical properties of scleroglucan are well understood, almost nothing
is known about the genetics of scleroglucan biosynthesis. Similarly, the
biosynthetic pathway of oxalate, the main by-product during scleroglucan
production, has not been elucidated yet. In order to provide a basis for genetic
and metabolic engineering approaches, we studied scleroglucan and oxalate
biosynthesis in
S. rolfsii using different transcriptomic approaches.
Two
S. rolfsii transcriptomes obtained from scleroglucan-producing and
scleroglucan-nonproducing conditions were pooled and sequenced using the 454
pyrosequencing technique yielding ~ 350,000 reads. These could be assembled into
21,937 contigs and 171,833 singletons, for which 6,951 had significant matches
in public protein data bases. Sequence data were used to obtain first insights
into the genomics of scleroglucan and oxalate production and to predict putative
proteins involved in the synthesis of both metabolites. Using comparative
transcriptomics, namely Agilent microarray hybridization and suppression
subtractive hybridization, we identified ~ 800 unigenes which are differently
expressed under scleroglucan-producing and non-producing conditions. From these,
candidate genes were identified which could represent potential leads for
targeted modification of the
S. rolfsii metabolism for increased scleroglucan yields.
The results provide for the first time genomic and transcriptomic data about
S. rolfsii and demonstrate the power and usefulness of combined
transcriptome sequencing and comparative microarray analysis. The data obtained
allowed us to predict the biosynthetic pathways of scleroglucan and oxalate
synthesis and to identify important genes putatively involved in determining
scleroglucan yields. Moreover, our data establish the first sequence database
for
S. rolfsii, which allows research into other biological processes of
S. rolfsii, such as host-pathogen interaction.
Elisa Zampieri[3]
Raffaella Balestrini[1] Annegret Kohler[2] Simona Abbà[3]
Francis Martin[2] Paola Bonfante[4]
1Istituto
per la Protezione delle Piante del CNR, Sezione di Torino,Torino, Italy;
2UMR INRA-UHP ‘Interaction Arbres/ Micro-Organismes’, Centre INRA de
Nancy, Champenoux, France.
3Dipartimento di Biologia Vegetale dell’Università di Torino, Torino,
Italy
4Dipartimento di Biologia Vegetale dell’Università di Torino, Torino,
Italy; Istituto per la Protezione delle Piante del CNR, Sezione di
Torino,Torino, Italy; 2Istituto per la Protezione delle Piante del CNR, Sezione
di Torino,Torino, Italy; 2Istitu
elisa.zampieri@unito.it elisa.zampieri@unito.it
Free-living fungi often encounter different kinds of environmental stresses,
including changes in temperature, osmolarity, pH, humidity, availability of
O2 and nutrients, exposure to toxins, UV or heavy metals, as well as competition
with other organisms. To understand the cell adaptation and the survivor in
non-ideal conditions, a better comprehension of many basic events is required.
Tuber melanosporum can be subjected to different stress conditions,
considering its life cycle. In this work, the genome sequence of the
ectomycorrhizal ascomycete
Tuber melanosporum was analysed with the aim to identify and
characterize genes involved in environmental stress response. As a second step
whole genome arrays were used to verify the transcriptional profiling in the
presence of a cold shock (4°C for 7 days). In a whole genome microarray (7496
genes/probe), 423 genes resulted significantly differentially expressed (> 2.5
fold; p-value < 0.05) in stressed mycelia compared to the control ones. After
4°C exposure for 7 days the number of up-regulated genes was 187; the
down-regulated genes were 236. The 50-60% of the up- or down-regulated
transcripts had no KOG classification and were clustered as unclassified
proteins, which represent the most abundant category both in up- and
down-regulated genes. A gene subset, concerning a range of biological functions,
was chosen to validate the microarray experiment using qRT-PCR. Sixteen out of
22 considered genes confirmed the array data. At our knowledge this is the first
work, which considers the global gene expression profiling in a filamentous
fungus under cold stress condition.
Delfina Popiel,
Grzegorz Koczyk, Lidia Błaszczyk, Jerzy Chełkowski
dpop@igr.poznan.pl
Zearalenones are economically important group of
Fusarium sp. mycotoxins, exhibiting estrogenic activity and chemical
strucure consisting of a resorcinol moiety fused to a 14-membered macrocyclic
lactone. These compounds are converted into a far less estrogenic product by
incubation with
Clonostachys rosea IFO 7063 expressing
zhd101 zearalenone lactonohydrolase gene. In the present study we
described screening of
Trichoderma/Clonostachys combined collection for new strains with
functional lactonohydrolase homologues. In the screened samples, we observed
degradation reactions in 10 of 79 total
Trichoderma sp. and
Clonostachys sp. isolates and have been able to determine new
lactonohydrolase homologue sequences with average sequence identity of 90%.
PR3.28
Julio Rodriguez-Romero,
Maren Hedtke, Reinhard Fischer
Department of Microbiology. Karlsruhe Institute of Technolgy (KIT),
julio.rodriguez@bio.uka.de
Light serves as one important environmental signal to regulate development and
metabolism in many fungi. The light response has been studied to great detail in
Neurospora crassa (1) and
A. nidulans.
A. nidulans develops mainly asexually in the light and mainly
sexually in the dark. We have discovered phytochrome (FphA) as a red-light
sensor (3), and found that the blue-light receptor system proteins LreA (WC-1)
and LreB (WC-2) along with VeA form a
light-regulator complex (LRC)(2). We propose a functional
relationship and a cross-talk between both photoreceptors.
In order to identify light-regulated genes, we used a two-colour microarray
system. After 30 minutes of illumination about 260 genes (approx. 2.5% of the
whole genome) were differentially regulated. 209 genes were up- and 51
down-regulated. Some of those genes display homology to other photo-inducible
genes identified previously in
N. crassa like
ccg-1,
con-10,
con-6, etc. Among the light-inducible genes were also transcription
factors probably implicated in secondary metabolism regulation and genes
encoding enzymes of the secondary metabolism. Many genes encode proteins
involved in stress responses, and a large group represents uncharacterized
genes.
The regulation of
conJcon-10 and
ccgAccg-1 has been studied in detail. Differential
expression of
conJ and
ccgA was confirmed by real time PCR.
Using chromatin IP (ChIP), we found that the LRC binds directly to the promoters
of
conJ and
ccgA. We do have evidence that not only the WC homologues are able
to bind DNA but also phytochrome. Phytochrome-DNA interaction occurs probably
through the response regulator domain at the carboxy terminus of FphA.
References
1. C. H. Chen, et al.,
EMBO J
28, 1029-42 (2009).
2. J. Purschwitz, et al.,
Mol Genet Genomics (2008).
3. A. Blumenstein, et al.,
Curr Biol
15, 1833-8 (2005).
PR3.29
Transcriptional response is a limiting factor in cellulase overproduction by
Trichoderma reesei
Thomas Portnoy1,3,+,
Antoine Margeot1,+, Verena Seidl2, Stéphane Le Crom3,
Fadhel Ben Chaabane1, Bernhard Seiboth2, Christian P.
Kubicek2
1
IFP, Département Biotechnologie, 1-4 Avenue
2Research
Area Gene Technology and Applied Biochemistry, Institute of Chemical
Engineering, Technische Universität Wien, Getreidemarkt 9/166, A-1060
3
Institute of Biology of the École Normale Supérieure, CNRS UMR8197, INSERM
U1024, 46 rue d’Ulm, 75230 Paris Cedex 05, France
+
Both authors equally contributed to this work.
Due to its capacity to produce large amounts of cellulases, the tropical
ascomycete Trichoderma reesei is
increasingly being investigated in second generation biofuel production from
lignocellulosic biomass. The induction mechanisms of
T. reesei cellulases have recently
been described in some details, but the regulation of the genes involved in
their transcription has not been studied thoroughly. Our work reports the
regulation of expression of the two activator genes
xyr1 and
ace2, and the corepressor gene
ace1 during induction of cellulase
biosynthesis by the inducer lactose in a low producing strain
T. reesei QM 9414. We show that all
three genes are induced by lactose. Xyr1
is also induced by D-galactose, but this induction was independent of lactose
metabolism. Moreover, xyr1 and
ace1, but not
ace2, were carbon catabolite
repressed. For xyr1, this repression
operated both on the basal and also the induced expression level, while
ace1 was mostly affected at the basal
level of expression. Significant differences in these regulatory patterns were
observed in the hyperproducer strain T.
reesei CL847. This suggests that a strongly elevated basal transcription
level of xyr1 and reduced
upregulation of ace1 by lactose may
have been important for generating the hyperproducer strain, and are thus major
control elements of cellulase production.
Matheis, S., Thines, E., Scheps, D. Andresen, K. and Foster A.J.
Institut für Biotechnologie und Wirkstoff-Forschung,
The M. oryzae genes FLB3 and FLB4, orthologues of the A.
nidulans regulators of conidiation FlbC and FlbD respectively were deleted.
Analysis of resultant mutants demonstrated that Flb4p is essential for spore
formation and that strains lacking this gene have a ‘fluffy’ colony morphology
due to an inability to complete conidiophore formation. Meanwhile Flb3p is
required for normal levels of aerial mycelium formation. Using microarray
analysis we identified genes dependent on both transcription factors. This
analysis revealed that the transcription of several genes encoding proteins
previously implicated in sporulation in Magnaporthe or in other
filamentous fungi are affected by FLB3 and/or FLB4 deletion.
Additionally the microarray analysis revealed Flb3p may effectively
metabolically reprogramme the cell by repressing transcription of genes encoding
biosynthetic enzymes and inducing transcription of genes encoding catabolic
enzymes. From seven genes whose transcription is controlled by one or both of
these transcription factors we identified the Flb4p dependent gene CON11
as being required for normal levels of sporulation in M. oryzae.
CON11-deleted mutants also exhibited a reduced growth rate and virulence.
PR3.31
Constructed Aspergillus
Robert A. van den Berg1, Machtelt Braaksma2, Douwe van der
Veen3, Mariet J. van der Werf2,
Peter J. Punt2,
John van der Oost3, Leo H. de Graaff3.
1
SymBioSys, Katholieke Universiteit Leuven, Leuven, Belgium
2
TNO Quality of Life,
3
Laboratory of Microbiology,
The fungus
Aspergillus
Comparative analysis revealed the existence of modules, some of which are
present in all three networks. In addition, experimental condition-specific
modules were identified. Module-derived consensus expression profiles enabled
the integration of all protein-coding
A.
PR3.32
Aspergillus
Benjamin M Nitsche1,
Vera Meyer1,
Arthur FJ Ram2
1Leiden
Universitiy,
2Kluyver
Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE
b.m.nitsche@Biology.leidenuniv.nl
Aspergillus
In the current study we, have built gene co-expression networks from a dataset
of about 100 Affymetrix microarrays covering more than 30 different growth
conditions including those which induce stress related to secretion, maintenance
of polarity, cell wall integrity, carbon-source utilization and starvation.
Exemplarily for a wide range of applications, we show mapping of putative TFs
with unknown functions to these networks. Allocation of TFs to functionally
enriched gene clusters can serve as an indication for their regulatory role and
thereby give valuable leads for further experimental studies.
PR3.33
The flbF gene encoding a putative C2H2-type
transcription factor is involved in the expression of sterigmatocystin genes and
asexual development in Aspergillus
nidulans
Yong Jin Kim,
Yeong Man Yu, Sang Eun An, Sun-Ho Kim and Pil Jae Maeng
Department of Microbiology & Molecular Biology, College of Biological Sciences
and Biotechnology, Chungnam National University, Daejeon, Republic of Korea
Asexual reproduction cycle is a common mode of reproduction for variety of
filamentous fungi. The asexual reproductive cycle in
Aspergillus nidulans can be divided
into two distinct phases : vegetative growth and development. In spite of the
extensive efforts and prominent progress in addressing the functions of the
genes involved in the fungal differentiation, large numbers of the genes
responsible for the developmental process in
A. nidulans remain to be elucidated.
Thus we performed transcriptome profiling during the entire process from spore
germination to asexual differentiation using the 70-base-oligomer microarrays
and ANURR. The array revealed stage-dependent expression of distinct genes set.
The most significantly regulated genes (p<0.05) were grouped in five and six
clusters based on their expression profiles during vegetative growth and asexual
differentiation, respectively. More than 51 genes encoding putative
transcription factors were found to be included in the stage-specific clusters.
To figure out the function of the genes for the putative transcription factors,
we are performing construction of knock-out mutants of the genes.
Here, we report the functional analysis of the
flbF gene which was expected to
encode a potential transcription factor with a C2H2 zinc
finger DNA binding motif, bipartite nuclear localization signal, and glutamine
rich region. Deletion of flbF
resulted in delay of asexual reproduction and partial fluffy phenotype, also
accumulation of brown pigment on media. From these observations, we propose that
flbF is a putative regulator for
initiation of asexual reproduction and secondary metabolism.
This work was supported by grant from Korea Research Foundation