Ulrike Binder[2]
Meiling Chu[1] Nick D. Read[1] Florentine Marx[2]
1Institute
of Cell Biology, University of Edinburgh, Edinburgh, EH9 3JH, UK
2Biocenter, Division of Molecular Biology, Innsbruck Medical
University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria
ulrike.binder@i-med.ac.at
The antifungal protein PAF from
Penicillium chrysogenum exhibits growth inhibitory activity against
a broad range of filamentous fungi. Recent evidence has suggested that calcium
(Ca2+) signalling may play an important role in the mechanistic basis
of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca2+
concentrations counteracted PAF toxicity towards sensitive moulds. By using a
transgenic
Neurospora
crassa strain expressing codon optimized aequorin, PAF was found to
cause a significant increase in the [Ca2+]c resting level.
The Ca2+ signatures in response to stimulation by mechanical
perturbation or hypo-osmotic shock were significantly changed in the presence of
PAF. BAPTA, a Ca2+ selective chelator, ameliorated the PAF toxicity
in growth inhibition assays and counteracted the PAF induced perturbation of Ca2+
homeostasis. These results indicate that extracellular Ca2+ was the
major source of these PAF-induced effects. The L-type Ca2+ channel
blocker diltiazem disrupted Ca2+ homeostasis in a
similar manner to PAF. Diltiazem in combination with PAF acted
additively in enhancing growth inhibition and aggravating the change in Ca2+
signatures. Notably, both substances, PAF and diltiazem increased the [Ca2+]c
resting level possibly by blocking Ca2+ channel activity. However,
examination of a
N. crassa
Δcch1 deletion strain excluded the L-type Ca2+ channel
cch1 to be the major target of PAF.
Ana Maria Calcagno[2]
Peñalva Miguel[1] Hervás-Aguilar America[1] Bignell Elaine[2]
Scazzocchio Caludio[2] Arst Herb[2]
1Centro
de investigaciones Biológicas, Madrid, 2Imperial College, London
t.brasilensi@imperial.ac.uk
Involvement of MVB class E components in pH signalling is well characterised in
yeasts. In
S. cerevisiae, all components, of ESCRT-I, -II and Vps32p-Vps20p of
ESCRT-III are required for pH signalling. Regulation of gene expression by
ambient pH in
Aspergillus nidulans is mediated by the transcription factor PacC.
The ESCRT –III components Vps32 and Vps24 interact with the pH signalling
components PalA and PalB respectively, thus participating in pH signalling.
We report that the deletion of genes encoding Vps20, Vps32 and Vps36 in
A. nidulans is nearly lethal and nearly always accompanied by
selection of suppressor mutations greatly improving growth. These
(partial) suppressors occur in two genes,
supA and
supB. SupA is a transcription factor and SupB is a putative
protein kinase. The suppressor mutations do not affect pH regulation or
trafficking but they do alter the volume and quantity of vacuoles, even in
vps+ strains.
vps20, -32 and
-36 deletions prevent pH signalling, consistent with results in
yeast. These deletions also impair trafficking of FM4-64 and of the dicarboxylic
amino acid transporter to the vacuolar membrane. We conclude that at least
ESCRT-II and -III components are required for pH signalling.
PR6.3
FANGFANG LIU,
Yanfen Lu, Tajaswini Dhavale, Gregory Jedd
Temasek Life Sciences Laboratory
The differentiation of organelles is fundamental to the growth and development
of eukaryotic cells. Woronin bodies are low -copy fungal organelles produced
from a high-copy number organelle – the peroxisome. Here, we show that Woronin
body producing peroxisomes differentiate
de novo and are hyper-competent for matrix protein import.
This produces a few dominant organelles that receive the majority of nascent
matrix protein import. Differentiation depends on a key oligomeric contact
in the Woronin body core protein HEX. Mutational disruption of this
oligomer abolishes the differentiation of peroxisomes and interaction with two
proteins that are enriched in the membrane of WB producing peroxisomes. Our
results are consistent with a model where the HEX oligomer promotes peroxisome
differentiation through positive feedback to the targeting of key membrane
proteins.
Lukasz Opalinski[1]
Jan A.K.W. Kiel[1] Tim Homan[2] Marten Veenhuis[1]
Ida J. van der Klei[1]
1Groningen
Biomolecular Sciences and Biotechnology Institute (GBB), University of
Groningen, Kluyver Centre for Genomics of Industrial Fermentation
2Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
University of Groningen
L.Opalinski@rug.nl
By genome analysis, we identified Pex14/17p as a putative novel peroxin of
Penicillium chrysogenum. Here we show that Pex14/17p is a component
of the peroxisomal membrane that is essential for efficient PTS1 and PTS2 matrix
protein import, implying that the protein is indeed a
bona fide peroxin. Additionally, a
PEX14/17 deletion strain is affected in conidiospore formation.
Pex14/17p has properties of both Pex14p and Pex17p in that the N-terminus of
this protein is similar to the highly conserved Pex5p binding region present in
the N-termini of Pex14p’s, whereas its C-terminus shows weak similarity to yeast
Pex17p’s. We have identified a novel motif in both Pex17p and Pex14/17p that is
absent in Pex14p.
We show that an N-terminally truncated, but not a C-terminally truncated
Pex14/17 protein is able to complement both the matrix protein import and
sporulation defects of a
Δpex14/17 strain, implying that it is the Pex17p-related portion of
the protein that is crucial for its functioning as a peroxin. Possibly, this
compensates for the fact that
P. chrysogenum lacks a Pex17 protein. Finally, we show that in P.
chrysogenum Pex14/17p plays a role in the efficiency of the penicillin
biosynthesis process.
PR6.5
Stefan Gesing,
Minou Nowrousian
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität
stefan.gesing@rub.de
Many filamentous ascomycetes form fruiting bodies during a highly complex
differentiation process. Four major morphological types (apothecia, perithecia,
pseudothecia and cleistothecia) are differentiated that derive from an ancestral
fruiting body. Thus, fruiting body differentiation most likely is controlled by
a set of common core genes. One way to identify such genes is to search for
genes with evolutionary conserved expression patterns, which is a powerful
criterion for functional importance.
Using “Suppression Subtractive Hybridization”, we selected differentially
expressed transcripts during fruiting body development in
Pyronema confluens (Pezizales). By real time PCR, expression
patterns were shown to be conserved in members of the Sordariales (Sordaria
macrospora and
Neurospora crassa), a derived group of ascomycetes. Knockout studies
with correlated
N. crassa orthologues,
revealed a functional role during fruiting body development for NCU05079, a
putative MFS peptide transporter.
Additionally we verified a conserved expression pattern for the homologues of
yeast
asf1 (anti-silencing function protein 1) during fruiting body
development of four distantly related ascomycetes (N.
crassa,
S. macrospora,
Fusarium graminearum and
P. confluens) by microarray and real time PCR analysis. Asf1 is a
highly conserved histone chaperone involved in the balance of nucleosome
assembly and disassembly. Knockout and complementation analysis with
S. macrospora Asf1 indicate a functional role of the protein during
sexual development of the fungus.
These data indicate conserved gene expression patterns and a functional role of
the corresponding genes during fruiting body development, which are candidates
of choice for further analysis.
Norio Takeshita,
Daniel Mania, Saturnino Herrero de Vega, Reinhard Fischer
Karlsruhe Institute of Technology
norio.takeshita@kit.edu
In eukaryotic cells microtubule (MT) length is determined by polymerization and
depolymerization phases.
One important parameter for length determination is the contact with
the cell cortex. Here, we show in the model organism
Aspergillus nidulans that the contact of MT plus ends with the
cortex is mediated through interaction between a putative MT polymerase
(XMAP215,
A. nidulans homologue AlpA), and a cortical cell end marker protein,
TeaA. Although both proteins localized to MT plus ends during MT growth,
AlpA-TeaA interaction was observed in a bimolecular fluorescence complementation
assay only after MT plus ends contacted the cortex. In the absence of
Loretta Jackson-Hayes,
Terry W. Hill, Darlene M. Loprete, Britany Chavez, Chassidy Groover, Erinn
Ogburn, Michael Pluta
jacksonhayesl@rhodes.edu
We have shown that the
Aspergillus nidulans orthologue of protein kinase C (PkcA)
participates in regulating cell wall integrity (CWI) and localizes at sites of
cell wall synthesis, including growing hyphal tips and septa. PkcA’s role
in CWI is regulated independently of its capacity to target to sites of wall
growth, as shown by the ability of the
calC2 mutation to inhibit resistance to wall-damaging compounds
without affecting growth or cytokinesis. To better understand the
mechanisms by which PkcA localizes to tips and septa, we have observed the
formation of cortical rings at sites of septation by fluorescently tagged PkcA
in hyphae defective in expression of other proteins necessary for septum
formation, using either temperature-sensitive mutants or regulation under the
AlcA promoter. In addition, we have co-imaged PkcA and other
septation proteins bearing complementary tags. Here we report that
localization of PkcA to septa lies “downstream” of the functions performed by
MobA (Mob1p orthologue), TpmA (tropomyosin), SepA (formin), SepD, SepG, and
proteins encoded by two other not-yet-cloned
Sep loci. In the absence of function of these proteins, PkcA
cortical rings were not observed. PkcA localization lies “upstream” of
MyoB (myosin II orthologue), the
A. nidulans orthologue of Bud4p (in yeast, a bud site selection
marker), and a protein encoded by a third uncloned
Sep locus. PkcA cortical rings still form in the absence of
function of these proteins, though septa do not develop. SepA, TpmA, MyoB,
and MobA all appear to colocalize with PkcA during normal septum formation.
While PkcA localizes to the very apex of hyphal tips and to the leading edge of
growing septa, the protein phosphatase BimG localizes to sites lateral to the
most active sites of growth. Studies with other septation-related proteins
are ongoing.
Mojca Bencina[1]
Tanja Bagar[1] Nada Kraševec[2]
1Department
of Biotechnology, National institute of Chemistry, 2Department for
Biosynthesis and Biotransformations, National
mojca.bencina@ki.si
The well tuned orchestra of enzymes is precondition for any cell to function. A
coordination of enzymes’ activities is manly a responsibility of wide variety of
secondary messengers. Some of them like cAMP, PIPs are locally synthesized.
Others like free calcium ions are constantly present, but stored in organelles
and the concentration is rigorously controlled by homeostatic machinery. And
then there is a pH, which might serve as a mechanism by which cells co-ordinate
the regulation of various processes that lack any other common regulating
factors and may provide a link between metabolic state and physiological
responses. However, not lot is known how calcium and pH homeostatic machinery
are working which is most likely due to lack of easy methods to monitor changes
in intracellular free calcium ions concentration and pH
in vivo. The methods for tracking changes in intracellular calcium
concentration and pH will be explained and evidence for calcium and pH
homeostasis will be given. An examination of cytoplasmic pH in growing cells of
Aspergillus
H
1University
of Minnesota, 2Département Environnement et Agro-biotechnologies
(EVA), Centre de Recherche Public-Gabriel Lippmann,
hckist@umn.edu
Niemann-Pick Type C (NPC) disease is a fatal autosomal recessive lipid storage
disorder in humans. An accumulation of endocytosed cholesterol in
lysosomes/late endosomes of spleen, liver and brain cells causes progressive
dementia, usually resulting in death by the age of twenty. NPC is caused
by defects in two genes which function in cholesterol transport,
npc1 and
npc2, with mutations in the former being responsible for 95% of
documented cases. Filamentous fungi make attractive models to study the
endomembrane system, growing rapidly in a highly polarized manor. Here, we
explore the potential of the ascomycete
Fusarium graminearum as a tool to study NPC. A BLAST search
revealed the presence of a putative
F. graminearum homolog of
npc1, possessing 34% sequence identity (51% positives) at the amino
acid level. Deletion of
F. graminearum
npc1 produced a viable mutant which displayed defects in ergosterol
localization. A combination of fluorescence and differential interference
contrast microscopy revealed an accumulation of ergosterol in vacuoles of mutant
cells. Furthermore, a GFP tagged version of NPC1 was found to localize to
the vacuolar membrane, analogous to lysosomal membrane anchored human NPC1p.
Our results suggest that filamentous fungi may provide good model systems to
study NPC.
Akira Yoshimi[3]
Motoaki sano[1] Tomonori Fujioka[2] Yuko Kokubun[3]
Osamu Mizutani[4] Daisuke Hagiwara[3] Takashi Fujikawa[5]
Marie Nishimura[5] Fumihiko Hasegawa[3] Keietsu Abe[3]
1KIT,
2Kumiai Chemical Industry Co., Ltd.,
a-yoshimi@hsq.niche.tohoku.ac.jp
The cell wall of filamentous fungi is a complex structure that is essential for
the maintenance of cell’s shapes and integrity, for the prevention of cell
lysis, and for protection against adverse environmental conditions. We
previously reported that the transcriptional regulation of a MAP kinase gene
mpkA and of cell wall-related genes (CWGs) in
Aspergillus nidulans differs significantly from that in
Saccharomyces cerevisiae. The transcription of two α-1,3-glucan
syntase genes,
agsA and
agsB, were regulated by MpkA pathway, but most CWGs were not.
Recently, the importance of α-1,3-glucan in host-parasite interactions has been
studied in both mammalian and plant pathogenic fungi. In this study, to
understand the role of α-1,3-glucan in
A. nidulans, functional analyses of the
agsA and
agsB genes were performed. The deletion mutants of
agsA gene did not show any significant phenotypes under normal
growth conditions. In contrast, the disruptants of
agsB gene could not be obtained, suggesting that AgsB seems to play
a crucial role in α-1,3-glucan synthesis of
A. nidulans. To assess this issue, we constructed the conditional
agsB strain whose
agsB expression is conditionally regulated under the control of
alcA promoter. The transcription of many CWGs coding for
β-1,3-glucan synthase and chitin synthase were induced under
agsB repressed conditions. This suggests that the decrease of
α-1,3-glucan content was counterbalanced by an increase in other cell wall
components. The results of the sensitivities to cell wall stress compounds such
as micafungin, CFW and Congo Red and the susceptibility test for cell wall
degrading enzymes will be presented.
Darlene Loprete,
Loretta Jackson-Hayes, Terry Hill, Erinn Ogburn, John Musgrove
Decreased expression of the extragenic suppressor, designated
SccA, affects cell wall integrity in the filamentous fungus
Aspergillus nidulans. Overexpression of
SccA suppresses the phenotype of the
calC2 mutation in the
A. nidulans orthologue of protein kinase C (PkcA), which results in
hypersensitivity to the chitin-binding agent Calcofluor White (CFW). In
addition, we have shown that
SccA rescues 6 wall-sensitive strains. In filamentous fungi,
as in yeasts, hypersensitivity to CFW correlates with defects in cell wall
integrity.
SccA is predicted to have a single transmembrane domain with 42% of
its amino acids residues being serine or threonine, which indicates it is bound
to carbohydrates in the cell wall. Homologues exist in the genomes of
other filamentous fungi, but not in yeasts or other organisms.
A SccA-GFP hybrid localizes to the plasma membrane and septa of
vegetative hyphae. When
SccA is placed under the control of the regulatable
AlcA promoter and grown under low expression conditions (glucose),
we observed a sensitivity to CFW, indicating it plays an important role in cell
wall integrity. Taking into consideration the protein’s cell surface location
and its influence on the function of PkcA, we hypothesize that SccA plays a role
in signal transduction as part of a cell wall integrity pathway.
Mizuki Tanaka,
Takahiro Shintani, Katsuya Gomi
Grad. Sch. Agric. Sci.,
mizu-t@biochem.tohoku.ac.jp
The exosome is a multi-subunit 3’→5’ exonucleolytic complex that is conserved in
eukaryotes. The ring-shaped core structure of the exosome is constituted of nine
subunits. In yeast, all of nine exosome subunits are essential for viability. On
the other hand, in plant, Csl4 is dispensable for growth and development,
whereas Rrp41 and Rrp4 are essential for the development of female gametophytes
and embryogenesis. These results suggest that the function of individual subunit
of the exosome is different in each eukaryotic cell. Since there has been no
report on the exosome itself in filamentous fungi, we attempted to construct the
disruptants of genes encoding exosome subunits in
Aspergillus oryzae.
We have chosen two orthologous genes for
csl4 and
rrp4 as targets for disruption, and successfully obtained a
csl4 disruptant but not an
rrp4 disruptant. The disruption of
csl4 gene had no apparent defect on growth in
A. oryzae. Since the
rrp4 disruptant could not been obtained, this gene would be
essential for cell viability. Thus, we generated the conditional
rrp4 expression mutant strain by using the promoter of
nmtA, expression level of which is regulated by riboswitch existed
within its 5’UTR and is repressed considerably in the presence of thiamine. The
resultant conditional
rrp4 expression strain displayed a remarkable growth defect when
thiamine was added to the medium. These results suggested that Rrp4 is essential
but Csl4 is not for cell growth in
A. oryzae and that function of individual exosome components in
A. oryzae is similar to that in plant.
Timo Schürg,
Ulrike Brandt, André Fleißner
tschuerg@tu-bs.de
We are using
Neurospora crassa as a model system to study the molecular
mechanisms of chemotropic growth and cell fusion. When spores of
N. crassa germinate, they attract each other to undergo cell fusion
and to form the mycelial colony. Earlier studies have shown that the MAP kinase
MAK-2 is an essential part of a signaling cascade involved in chemotropic growth
during germling fusion (Pandey
et al. 2004; Fleissner
et al. 2009). To further characterize this signaling pathway we
analyzed the role of BEM-1. The homologous protein in
Saccharomyces cerevisiae is interacting with several upstream
factors of the Fus3 cascade, which is homologous to the MAK-2 pathway in
N. crassa. In contrast to yeast, where BEM-1 is involved in polarity
establishment, a
N. crassa delta bem-1
knock out mutant is not significantly impaired in spore germination and polar
hyphal growth. However, delta bem-1
germlings do not interact chemotropically. Complementation with a
bem delta PB-1
construct did not complement the knock out mutant phenotype, suggesting that the
PB-1 domain is essential for BEM-1 function. Subcellular localization of BEM-1
using GFP fusion constructs showed that BEM-1-GFP accumulates at every growing
hyphal tip. In germling fusion pairs, BEM‑1‑GFP concentrates at the fusion point
and localizes around the opening fusion pore. We also detected BEM-1-GFP at
septa of germ tubes and mature hyphae. Taken together, our data suggest novel
functions of BEM-1 in chemotropic growth, fusion pore formation and at the
septa. In our further studies we will try to unravel and identify the distinct
molecular functions of BEM-1 during
Neurospora development.
Charissa de Bekker, G. Jerre van Veluw, Arman Vinck, L. Ad Wiebenga, Han
A.B. Wösten
Microbiology and Kluyver Centre for Genomics of Industrial Fermentations,
Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The
Netherlands
g.j.vanveluw@uu.nl
Colonies of the filamentous fungus
Aspergillus
Strains expressing
GFP from the glucoamylase (glaA)
or ferulic acid esterase (faeA)
promoters were grown. The Complex Object Parametric Analyzer and Sorter (COPAS)
was used to analyze the diameter and fluorescence of micro-colonies. At least
two populations were found in these cultures, one population of small (25%) and
one of large micro-colonies (75%). Fluorescence correlated with volume. 27% and
73% of the micro-colonies expressing
GFP from the
faeA promoter were lowly and highly fluorescent, respectively. This
implies that heterogeneity in this strain depends on the volume only. In
contrast, lowly fluorescent micro-colonies of the
glaA::GFP strain comprised about 79% of the culture; this implies an
additional factor besides volume.
To assess heterogeneity within a micro-colony, central and peripheral parts were
isolated by laser microdissection and pressure catapulting (LMPC). QPCR showed
that
glaA and
faeA expression is similar in both zones. However, RNA content per
hypha was 50 times higher in the periphery.
Here, COPAS and LMPC were used for the first time to assess gene expression in a
microbial system. Our results show heterogeneity between micro-colonies from a
submerged culture. Moreover, it is shown that zones within a pellet are also
heterogenic. Peripheral hyphae have about 50 times more RNA than central hyphae.
Min Jin Kwon,
Mark Arentshorst, Cees A.M.J.J. van den Hondel, Vera Meyer,
Arthur F.J. Ram
m.j.kwon@biology.leidenuniv.nl
Tip growth in filamentous fungus requires coordination of basic cellular
processes in the cell such as exocytosis, polarity maintenance, endocytosis and
cell wall biosynthesis. The small GTPase RacA, the fungal orthologue of the
human Rac1 protein is important for fungal growth. In the absence of RacA,
apical dominance of tip growth is lost resulting in a hyperbranching phenotype.
Actin patches in the
racA deletion mutant were found to be hyperpolarized at the extreme
apex, while in the wild type strain a smoother gradient of actin patches towards
the tip was observed. GFP-RacA localizes to the plasma membrane at the extreme
apex of growing hyphae probably, marking at the site of exocytosis. To
understand the function of RacA in relation to exocytosis, secretory vesicles
were visualized by tagging the V-SNARE (SynA) with GFP (GFP-SynA). In both
ΔracA and wild type cells, GFP-SynA is present on intracellular
structures representing secretory vesicles and/or endocytic vesicles. High
levels of GFP-SynA are also present in the Spitzenkörper, a filamentous fungal
specific structures that is thought to act as a vesicle supplying center. In
ΔracA the intensity of signal was less. The tips of wild type hyphae
display a ~10-15 mm gradient of GFP-SynA protein, whereas tips in
ΔracA show a much shorter (<5 mm) gradient of GFP-SynA. We conclude
that the
A.
Department of Agricultural Biotechnology, Center for Fungal Genetic Resources,
and Center for Fungal Pathogenesis,
asdfppp2@snu.ac.kr
The most straightforward strategy of reverse genetic approach includes targeted
gene deletion process. It requires homologous recombination (HR) events and
efficiency of gene deletion is subject to frequency of HR. In many filamentous
fungi, however, HR occurs at low frequency due to the dominance of
non-homologous end joining (NHEJ) and in case of the rice blast fungus
Magnaporthe oryzae, frequency of HR is locus-dependent and very low
that has been reported less than 10%. It causes inefficiency of experimental
progress and generates lots of undesirable ectopic transformants. For the
improved efficiency of genetic study, we designed an add-on system named PBS (Phenotype
Based Screening). This system is added to the targeted gene
deletion process and makes efficient use of ectopic transformants previously
abandoned. As a forward genetic approach, ectopic transformants showing gene
disruptions probably in random manner are screened for various defectives in
several phenotypes such as growth rate, pigmentation, colony morphology, and
conidiation. Location of ectopic integration is achieved by inverse-PCR and
sequencing. With aid of PBS system, two growth-retarded ectopic transformants
were confirmed as related to disruption of two loci MGG_00839.6 and MGG_04395.6.
Consequently, coupling of PBS system and targeted gene deletion process can be
regarded as the union of forward and reverse genetic approaches. This
bidirectional approach will advance the functional genomic studies of
filamentous fungi with high efficiency.
PR6.17
Jochen Kleemann, Bleddyn Hughes, Tom Colby, Anne Harzen, Jürgen Schmidt,
Richard J. O'Connell
Max Planck Institute for Plant Breeding Research
hughes@mpiz-koeln.mpg.de
The hemibiotrophic ascomycete
Colletotrichum higginsianum causes anthracnose disease of crucifers,
including the model plant
Arabidopsis. Following conidial germination, the pathogen
differentiates a specialised infection structure called an appressorium that is
essential for initial host invasion. We used a comparative proteomics approach
to uncover changes in the total and secreted proteome during germination and
appressorium formation, taking advantage of the ability to mass-produce
Colletotrichum appressoria
in vitro on polystyrene substrata. Proteins harvested at 5h,
corresponding to early appressorium formation, and 22h, representing mature
melanized appressoria, were compared to the proteome of undifferentiated
mycelia. A total of 677 protein spots were identified using MALDI-TOF-MS and
MS/MS by reference to the
C. higginsianum genome sequence. 27 secreted proteins were
identified in the germination liquid surrounding mature appressoria. Of
the proteins that were co-regulated during appressorium formation, enzymes
involved in fungal wall modification and melanin biosynthesis were represented
during early morphogenesis, while enzymes potentially involved in plant cell
wall degradation and toxin biosynthesis were represented in mature appressoria.
The identified peptides provide experimental support for annotation of the
C. higginsianum genome sequence.
Sara Gremillion[2]
Darlene Loprete[1] Terry Hill[1]
sara.gremillion@armstrong.edu
The
swoP1 (swollen cell) and
podB1 (polarity defective) mutations in
Aspergillus nidulans interfere with establishment and maintenance of
polarity. At restrictive temperatures, conidia of both mutants swell to
approximately 1.5 times the normal diameter. Conidia of
swoP1 also produce abnormally wide hyphae and establish multiple
points of polarity, which grow isotropically before arrest. Genes complementing
the mutations of
swoP1 and
podB1 have strong sequence homology to COG4 (ANID7462.1) and COG2
(ANID8226.1), respectively. In mammals and yeast, COG2 and COG4 are part
of a multi-protein structure called the COG (conserved oligomeric
Golgi) complex associated with retrograde transport within the Golgi
apparatus. A GFP-tagged COG2 displayed a punctuate distribution within fungal
hyphae, a pattern consistent with other Golgi protein localization. COG4 was not
successfully GFP tagged. Protein overexpression studies provided evidence of
intra-complex interactions between COG2 and COG4 as well as between COG2 and
COG3. To study the role of these proteins in growth of filamentous fungi, an
AlcA promoter replacement strategy was performed. When grown on AlcA-suppressive
media, the COG4 AlcA-replaced promoter strain displayed normal growth, while the
COG2 AlcA-replaced promoter strain displayed abnormally wide hyphae. A
lectin blot using concanavalin A revealed significant differences in protein
glycosylation patterns between the
swoP1 and
podB1 mutants when compared to wild type when grown at restrictive
temperatures. The glycosylation patterns of the two mutants were
indistinguishable under these conditions.
PR6.19
Marc Maas,
Martha Merrow
m.f.p.m.maas@rug.nl
The circadian clock is a complex trait, involving a network of interlocked
molecular loops that depend on oscillatory feedback. There are many indications
that also in the fungus
Neurospora crassa the clock consists of multiple oscillators. One
molecular oscillator system that has been described in great detail is the
frequency-white collar (FRQ-WC) transcription-translation feedback loop
(FWC-TTFL). In the absence of a functional FWC loop, strains are still able to
entrain systematically to temperature, they show oscillations in nitrate
reductase activity and, under particular conditions, rhythmic conidiation is
within the circadian range. There thus is ample evidence for one or more
FRQ-less oscillators (FLOs). Microarrays have revealed several candidate
components of these FLOs. We have studied the circadian behaviour of one of
these:
cpc-1 (for cross pathway control), an orthologue of the
gcn4 gene from yeast.
Cpc-1 encodes a transcription factor involved in the coordination of
many amino acid biosynthetic pathways. It is therefore a candidate node in
several metabolic feedback loops. We show that coupling oscillations of FWC and
CPC-1-dependent feedback loops control fundamental clock attributes like
amplitude, precision and robustness.
Regulation of the SNARE complex formation in
Trichoderma reesei
Mari Valkonen1, Markku Saloheimo1, Merja Penttilä1,
and Rory R. Duncan2
1 VTT Biotechnology, P.O. Box 1000, FIN02044 VTT, Finland
2Membrane Biology Group, University of Edinburgh, George Square, EH8
9XD, UK
mari.valkonen@vtt.fi
Eukaryotic cells contain membrane-bound compartments that are connected by
trafficking of vesicular intermediates. Merging of the donor and acceptor
membranes accomplishes three tasks: 1) Surface of the plasma membrane increases
(by the surface of the fused vesicle), which is important for cell growth. 2)
The substances within the vesicle are released into the exterior. 3) Proteins
embedded in the vesicle membrane become part of the plasma membrane.
To maintain compartmental organization, proper targeting of transport vesicles
is required. In the process of exocytosis, where Golgi derived secretory
vesicles fuse to the plasma membrane, three major classes of proteins are needed
SNARE, Sec1 and Rab proteins. Although t- and v-SNAREs are sufficient to drive
the fusion of membranes
in vitro, it is clear that other proteins are important regulators
of this event in the cell.
It has been shown that the formation of SNARE complex is very strictly regulated
in mammalian and yeast cells. The NH2-terminal domain from isolated
mammalian t-SNARE, syntaxin, has been shown to fold back onto the SNARE motif,
and this closed conformation is stabilized by the binding of members
of the Sec1-munc (SM) family, apparently preventing SNARE complex formation. Two
reports have demonstrated that key neuronal mammalian SM protein, munc18-1, can
interact with the assembled SNARE complex, via an N-terminal peptide in
syntaxin. This motif is conserved in some fungal SSO homologs and in mammalian
syntaxin1. The function of the SM family of proteins in membrane fusion is still
controversial, but it has been shown to be essential regulator of the membrane
fusion and in yeast has been shown to concentrate on the sites of secretion.
To address the question of the control of membrane fusion, we have cloned the
Sec1 homologue of
T. reesei and expressed it as a fusion with a yellow fluorescent
marker protein (Venus). The localisation of the fusion protein is cytoplasmic.
Interactions of the SECI protein with two plasma membrane t-SNARE proteins SSOI
and SSOII has been studied using multi-dimensional time-correlated single photon
counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET.
It was shown that there is FRET occurring between mCer::SSOI and the SECI::Venus
fusion proteins indicating that there’s a protein-protein interaction between
these two proteins. The major site of interaction is at the plasma membrane. The
finding was further verified by immunoprecipitation (IP) studies showing that
SSOI and SECI can be isolated as a complex. Both SSOI and SECI proteins seem to
be essential for growth as deletion of either is lethal to the cells. The
FLIM/FRET results from the mCer::SSOII and SECI::Venus interaction were
inconclusive and in IP studies, no interaction has been seen between the SECI
and SSOII proteins.
PR6.21
Daniel H. Scharf[1]
Peter Hortschansky[1] Hubertus Haas[2] Axel A. Brakhage[1]
1Department
of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product
Research and Infection Biology (HKI) and Friedrich-Schiller-University Jena,
Beutenbergstrasse 11a, D-07745 Jena
2Division of Molecular Biology, Innsbruck Medical University,
Fritz-Pregl-Str. 3, A-6020 Innsbruck
daniel.scharf@hki-jena.de
Recently, a putative fourth CCAAT-binding complex (CBC) subunit with an unknown
function was identified in
A. nidulans and designated HapX.
hapX expression is repressed by iron via the GATA-factor SreA.
Various iron-dependent pathways (e.g., heme biosynthesis) are repressed during
iron starvation by the interaction of HapX with the CBC. These data suggest a
model, in which HapX/CBC interaction is regulated at both transcriptional and
post-translational levels. Iron starvation causes expression of
hapX. Subsequent binding of HapX to the CBC results in
transcriptional repression of iron-dependent pathways. During iron-replete
conditions,
hapX is repressed and, therefore, iron-dependent pathways are
derepressed. Moreover, HapX/CBC interaction is inhibited by increased iron
concentrations. This post-translational mechanism allows rapid adjustment to
iron availability by disruption of the HapX/CBC complex. Mutual transcriptional
control of
hapX and
sreA coordinates iron acquisition and iron-dependent pathways,
thereby serving for both iron supply and prevention of iron toxicity. These data
indicate that the CBC has a general role and that HapX function is confined to
iron depleted conditions.
Here, we describe the domain architecture of the HapX protein. Phylogenetic
analysis revealed the conservation of certain domains, which were characterised
further with the help of surface-plasmon-resonance and complementation
experiments.
Colin P. DeSouza,
Stephen A. Osmani
de-souza.1@osu.edu
The spindle assembly checkpoint (SAC) is a universal mechanism which arrests
mitosis if a bipolar spindle cannot be formed. Although higher eukaryotic cells
that are unable to satisfy the SAC eventually die, such a mechanism would not
confer an obvious advantage to filamentous fungi encountering environmental
conditions that interfere with spindle formation. Following the status of
nuclear pore complex and SAC proteins we show that
Aspergillus nidulans cells treated with the spindle poison benomyl
activate the SAC and arrest in mitosis. However after a defined period of time,
the SAC is actively turned off and cells exit mitosis without chromosomal
segregation. Most remarkably we find that cells which have undergone one such
failed mitosis surprisingly transit interphase and enter a second mitosis in
which the SAC is re-activated before again being inactivated. This cyclic
activation then inactivation of the SAC can occur for at least three cell cycles
in which the nucleus completes all aspects of mitosis except those depending on
spindle function. Further, following one or more cell cycles without spindle
function, if cells are allowed to reform microtubules they assemble spindles
upon mitotic entry and can undergo successful mitosis. Therefore, we propose
that inactivation of the SAC allows filamentous fungi to continue growth under
environmental conditions which prevent spindle formation and then periodically
test the environment for conditions which are compatible with mitosis. We
conclude that the SAC can be silenced in a cyclic regulated manner independent
of spindle formation.
Alexander Lichius,
Nick D. Read
A.Lichius@sms.ed.ac.uk
The ability to establish and maintain cell polarity is a prerequisite for spore
germination and cell fusion in
Neurospora crassa. Without it spores are limited to isotropic growth
and unable to protrude germ tubes and conidial anstomosis tubes (CATs). Germ
tubes avoid each other, whilst CATs attract each other. During CAT homing, tip
orientation follows a chemoattractant gradient to establish cell-cell contact
and fusion. Using strains co-expressing Lifeact-TagRFP and β-tubulin-GFP we
recently analyzed the interrelated localization pattern and dynamic
rearrangement of F-actin and microtubules during conidial germination and cell
fusion. We found that recruitment of both cytoskeletal elements occurs in a
distinct but coordinated manner that might influence which protrusion is being
formed and maintained at any point in time. Molecular mechanisms involving
cortical markers whichlocally and specifically recruit the cytoskeleton in order
to achieve re-orientation during germ tube avoidance and CAT homing, are
virtually unknown in
Neurospora. Focused activation
of Rho GTPases, such as CDC42 and RAC-1, at the cell cortex is a
key determinant of polarized F-actin organization, and thought to occur in
sterol-rich microdomains within the plasma membrane. Displacement of these
microdomains in response to external stimuli might regulate directional growth.
Interestingly, ∆rac-1
mutants of
Neurospora still show germ tube avoidance but are unable to undergo
CAT-mediated cell fusion. To study the spatial and temporal relationship between
GTPase activity and F-actin rearrangement during tip orientation in more detail,
we engineered a green fluorescent Cdc42-Rac-interactive binding (CRIB) biosensor
for activated GTPases and co-expressed it with Lifeact-TagRFP-T.
PR6.24
Yasuyuki Kubo[1]
Ayumu Sakaguchi[1] Naoki Fujihara[1] Gento Tsuji[1]
Ulla Neumann[2] Richard O’Connell[2]
1Laboratory
of Plant Pathology, Graduate School of Life and Environmental Sciences, Kyoto
Prefectural University, Kyoto 606-8522, Japan
2Department of Plant Microbe Interactions, Max Planck Institute for
Plant Breeding Research, Carl von Linné Weg 10 D-50829 Köln, Germany
y_kubo@kpu.ac.jp
The cucumber anthracnose fungus
Colletotrichum orbiculare forms an infection structure called an
appressorium. Melanization of appressoria is essential for host penetration and
requires the β-oxidation of fatty acids in peroxisomes. Here, we identified and
characterized a protein specific to filamentous ascomycete fungi, Fam1, that is
essential for peroxisome function and which associates with Woronin bodies. The
FAM1 gene was isolated by screening random insertional mutants for
deficiency in fatty acid metabolism. The
fam1 disrupted mutants were unable to grow on medium containing
oleic acids as the sole carbon source. Green fluorescent protein carrying the
peroxisomal targeting signal 1 (PTS1) or PTS2 were not imported into peroxisomes
of
fam1 mutants, suggesting that
FAM1 is a novel peroxisomal biogenesis gene (peroxin). Accordingly,
fam1 mutants were defective in both appressorium melanization and
host penetration. Microscopy showed that the Fam1p-GFP fusion protein localized
to small punctate structures in the apical region of hyphae, near septa and
adjacent to peroxisomes. This resembled the distribution of Woronin bodies,
which are peroxisome-derived organelles involved in septal pore plugging. After
cloning
CoHEX1, a homolog of the
HEX1 Woronin body structural gene, we generated a Cohex1p-mRFP1
fusion protein, which co-localised with Fam1p-GFP in Woronin bodies.
Furthermore, the apical and septum localization of Fam1p was impaired in
cohex1 mutants. Our results indicate that Fam1p is a novel Woronin
body-associated protein and raise the possibility that filamentous ascomycete
fungi coordinate peroxisome function
via Woronin bodies.
Mordechai Ronen,
Benjamin A. Horwitz
Department of Biology, Technion,
motiron@tx.technion.ac.il
Reactive oxygen species (ROS) are synthesized by specific NADPH oxidases (NOX),
enzymes inserted in the plasma membrane. NOX enzymes use cytoplasmic NADPH to
produce superoxide. ROS can provide both defense and differentiation signaling
roles in animals and plants. Fungal NADPH oxidases have a structure very similar
to the human gp91phox. Specific isoforms of fungal NOX have been reported to be
required for various physiological processes and cellular differentiation
events, including development of sexual fruiting bodies, ascospore germination,
and hyphal growth in both mutualistic and antagonistic plant–fungal
interactions. We identified three NADPH oxidase homologues in the necrotrophic
filamentous fungus
Cochliobolus heterostrophus, and we set out to investigate their
function and importance in the fungal life cycle through study of
loss-of-function mutants in genes encoding catalytic and regulatory subunits of
NADPH oxidases. Mutants in noxA, noxC and the predicted regulatory subunit gene
noxR have decreased pigmentation and delayed conidiation. noxA and noxR show
decreased virulence on the host plant, maize. All nox mutants produce superoxide
as detected by nitro blue tetrazolium (NBT) staining. We are investigating ROS
production in single and multiple
nox mutants under different conditions.
PR6.26
Vera Meyer,
Susann Minkwitz, Tabea Schütze, Cees A.M.J.J van den Hondel, Arthur F.J. Ram,
Leiden University, Institute of Biology Leiden, Department Molecular
Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands &
Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA
Delft, The Netherland
v.meyer@biology.leidenuniv.nl
Many cells and organisms go through polarized growth phases during their life.
Cell polarization is achieved by local accumulation of signaling molecules which
guide the cytoskeleton and vesicular trafficking to specific parts of the cell
and thus ensure polarity establishment and maintenance. Polarization of
signaling molecules is also fundamental for the lifestyle of filamentous fungi
such as
Aspergillus
References:
Meyer V, Arentshorst M, Flitter SJ, Nitsche BM, Kwon MJ, Reynaga-Pena CG,
Bartnicki-Garcia S, van den Hondel CA, Ram AF (2009) Reconstruction of
signalling networks regulating fungal morphogenesis by transcriptomics.
Eukaryot Cell 8: 1677-1691
PR6.27
Subcellular localisation of AreA and MeaB is driven by different nitrogen
sources.
2Institut
für Botanik der Westfälischen Wilhelms-Universität Münster, Schloßgarten 3,D-48149
Münster, Germany.
m.mos@liv.ac.uk
In A. nidulans the utilisation of
nitrogen sources other than ammonium or glutamine is dependent on the
transcriptional factor AreA, which enables transcription of a broad range of
genes specifically under nitrogen limitation. A second transcription factor,
MeaB, also plays a role in the cellular response to nitrogen availability.
Although the function of MeaB is less well characterised it appears, at least in
part, to act in opposition to AreA, being most active under nitrogen
sufficiency. Both AreA and MeaB have been tagged with GFP and their
intracellular localisation monitored in response to changes in nitrogen regime
using confocal microscopy. As controls histone H1:RFP was used to identify the
nuclei and mitochondria were stained with MitoTracker. The subcellular
localisation of MeaB and AreA both respond to changes in nitrogen regime. In the
case of AreA, under conditions of nitrogen repression (eg Gln or NH4+)
the protein appears to be distributed throughout the cell. Surprisingly under
nitrogen derepression conditions (eg NO3- or –N) AreA is
primarily localised to the mitochondria and not the nuclei. In the case of MeaB,
growth on Gln or NH4+ results in localisation in and
around the nuclei and within the mitochondria. In the presence of NO3-
or during nitrogen starvation MeaB accumulates exclusively in mitochondria,
although this is not the case with all deprepressing nitrogen sources (eg
proline) where MeaB is evenly distributed throughout the cell.
In Fusarium fujikuori
subcellular localisation of MeaB is also subject to nitrogen regulation and, as
in A. nidulans, it is located in the
nucleus under nitrogen repressing conditions. However, in contrast to the
A. nidulans, there was no obvious
accumulation of MeaB within the mitochondria under the regimes tested.
In summary, as expected AreA and MeaB both show very distinct response to
nitrogen availability. Unexpectedly, in A. nidulans both transcription
factors localise to the mitochondria under nitrogen regimes.
Joohae Park,
Thomas R. Jørgensen, Mark Arentshorst, Patricia A. vanKuyk, Robbert A. Damveld,
C.A.M.J.J. van den Hondel, Arthur F.J. Ram
A characteristic hallmark of Aspergillus
References:
(1)
Walsh C.T.,
Gehring A.M.,
Weinreb P.H.,
Quadri L.E.,
Flugel R.S.,
Curr. Opin. Chem. Biol. 1,
309-315. 1997.
(2) Shuster J.R., Bindel Connelley
M., MGG 262, 27-34. 1999.