Heather/Toyon/Acacia
Hyphal Growth and Polarity
1. The CHK Genes in C. albicans: OS1 Homologs important for Cell-Wall Assembly and Osmotic Tolerance.
J.Agnan, C. Korch, M. Schumacher, L. Alex1, M. Simmons1, C. Selitrennikoff. University of Colorado Health Sciences Center, Denver CO. 1California Institute of Technology, Pasadena CA.
The aim of this research is to identify genes involved in cell-wall synthesis for the development of novel antifungal targets. We previously reported the cloning and characterization of the osmotic-1 (os- 1) gene, which is required for normal growth, cell wall synthesis, and osmotic tolerance in Neurospora crassa. Mutants of os-1 have altered cell-wall compositions, are unable to grow on medium supplemented with 4% NaCl, and are resistant to dicarboximide fungicides, such as vinclozolin, iprodione and procymidone. os-1 encodes an osmosensing histidine-kinase, believed to participate in a signal transduction cascade similar to the high-osmolarity glycerol (HOG) pathway of yeast.
We report the isolation, cloning, and sequencing of the os-1 homologs from C. albicans,
an opportunistic fungal pathogen responsible for a variety of diseases in humans. Three different
genes were isolated and are believed to be members of a gene family. The os-1 homologs in C.
albicans have been designated Candida histidine kinase (chk) and the sequence obtained shows
homology to the N. crassa os-1 gene and to a sensor-regulator protein of Saccharomyces
cerevisiae, SLN 1. These data indicate that the chk genes encode an osmosensing protein that is
essential for normal growth and cellwall biosynthesis. Construction of C. albicans chk deletion
mutants is currently underway.
2.The Aspergillus nidulans sodVIC gene is involved in hyphal growth and encodes a subunit of the coatomer complex. Susan J. Assinder, Susan L. Whittaker and John H. Doonan* School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, Wales, LTK; *John Innes Research Centre, Norwich, NR4 7LTH, UK.
The A. nidulans mutation sodVIC (for stabilisation of disomy) causes a high frequency of aneuploid progeny (1). The mutation is lethal at 42 C but allows normal growth at 30 C. Upon upshift from 30 C to the sub-restrictive temperature of 37 C, disomic sectors are produced which carry an extra copy of chrVI. The disomic state is stable at 37 C (unlike the usual case for A. nidulans aneuploids) but downshift to 30 C causes reversion to haploidy. Based on these properties, sodVIC was originally presumed to be mutant in a function required for chromosome partitioning. However, cytological and molecular characterisation have shown that mutant strains are defective in hyphal extension. At 42 C, conidia arrest with 1-2 nuclei and fail to produce a germ tube. The chromosome mitotic index is similar to that of the wild-type, indicating that there is no cell cycle lesion.Temperature-shifts of pre-germinated conidia have shown the defect to be in hyphal growth rather than in a function specific to germination. The mutation has been complemented using a cosmid from a chromosome VI-specific library which has homology to a subunit of the human coatomer complex involved in the early secretary pathway.
(1)Upshall et al. (1979) In: Sebek (ed): Procs. 3rd Int. GIM Symp. pp 197-204.
3. Morphological changes during growth of Ustilago maydis.
Flora Banuett. Department of Biochemistry & Biophysics, University of California, San Francisco, CA 94143-0448.
Ustilago maydis is a Basidiomycete fungus that induces tumors in maize. It can grow as a unicellular haploid yeast-like form that is non-pathogenic and as a filamentous dikaryon that requires the plant for its growth. Growth of the hyphae in the plant leads to formation of tumors within which the fungus produces round diploid spores, the teliospores. In order to determine how the hyphae, which exhibit highly polarized growth, lead to formation of these round teliospores, a study of the infectious process was undertaken. The course of infection was followed from the time of inoculation of the fungus into the plant to the formation of mature telliospores. These studies and analysis of the fuz1- mutant led to the conclusion that a discrete developmental pathway is involved in teliospore formation (Banuett and Herskowitz, 1996 Development 1 22: 2965-2976). Progression of this pathway is arrested in the fuz1- mutant at a discrete step. This work also demonstrates that although different a alleles are necessary for filamentous growth in culture, they are not required for filamentous growth in the plant, suggesting that the a pathway may be activated by a plant signal.
In addition to this work, I will also describe the characterization of mutants with altered
cell morphology as well as the changes in the actin cytoskeleton during the cell cycle and the life
cycle.
4. Neurospora crassa chitin synthase 2 is expressed during aerial structure formation.
Adi Beth-Din, Vered Ziv and Oded Yarden, Department of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
In Neurospora crassa five chitin synthase (chs) genes has been identified so far. Gene inactivation experiments provided evidence for differential chs gene function. Null chs gene phenotypes range from no observed morphological changes (when compared to wild type) to extreme abnormalities in hyphal growth. Under various laboratory growth conditions no change in phenotype was observed in strains in which chs-2 had been inactivated. However, a substantial reduction in chs activity (as determined in vitro) could be attributed to the loss of chs-2 expression. In order to advance our understanding of the possible role of chs-2 in vivo, we examined this gene and it's product during N. crassa growth and development. Aerial structures are richer in chitin than liquid-grown mycelium. In in-vitro assays, total chs activity was found to be much higher during aerial hypha formation than in mycelium growing in liquid culture. Polyclonal antibodies raised against an overexpressed fraoment of chs-2, identified a ~80Kd polypeptide in protein extracts of aerial hypha that
was absent from the mycelial extract. acon-2, acon-3 and fl, mutants blocked at various staores of
conidiation, were used to analyze chs-2 transcription during aerial structure formation. chs-2
transcript levels were found to be dramatically increased in stages of aerial hypha formation and
major constriction development; We suggest that chitin synthase 2 expression is both temporally
and spatially regulated.
5.Characterization of double disruptants of chsA and either chsC or chsD of Aspergillus nidulans.
Hiroyuki Horiuchi, Makoto Fujiwara and Masamichi Takagi. Department of Biotechnology, The University of Tokyo.
We have isolated four chitin synthase genes (chsA, chsB, chsC, and chsD) of A. nidulans
and disrupted them. The disruptant of chsB showed severe defect of hyphal growth but the
phenotypes of the other three disruptants were similar to that of the parental strain. We have
constructed disruptants of two of three genes, chsA, chsC and chsD. Double disruptants of chsC
and chsD ( CD disruptants ) showed no phenotypical change. While, efficiency of conidia
formation of AD disruptants was about 10% of that of the parental strain and that of AC
disruptants was less than 0.1% of that of the parental strain. AC disruptants showed pleiotropic
defects, such as sensitiveness to high concentration of salts, SDS, Calcofluor white and Congo
Red. Calcofluor white and Congo Red are known as chitin binding dyes. AC disruptants were also
sensitive to a chitin synthase inhibitor, Nikkomycin Z. Moreover, abnormal structures of
conidiophores of AC disruptants were observed by a scanning electron microscopy. These
structures are similar to that of the medA mutant. These results suggested that chsA and chsC
have important but redundant functions for tip growth of hyphae and formation of conidiophores.
6. Effect of water activity on the rate of hyphal growth and pectinase production by 2 deoxy-glucose resistant mutants of Aspergillus niger.
O. Loera-Corral, C.P. Larralde-Corona and G. Viniegra-González. Universidad Autonoma Metropolitana- Iztapalapa, México D.F. México.
Water activity (aw) is a major environmental factor affecting the specific growth rate (m) and also
the yields of enzyme production of molds such as A. niger, in terms of substrate or biomass
changes (Yp/s or Yp/x). Therefore, aw depressants such as ethylene glycol (EG), can be used to
select and screen for A. niger strains adapted to produce pectinases in solid substrates with low aw
values, i.e. coffee pulp. Antier et al. (1993) found that deoxy glucose resistant mutants from wild
strain A. niger C28B25 were adapted to such purpose when selected at low aw values (15% EG).
Estimation of m can be done, according to Larralde (1996) by formula m= ur ln2 / [Lav
ln(Lav/Dh)], where ur=maximal rate of apical elongation, Lav= average length of distal branches,
Dh= hyphal diameter. The patterns of specialized mutants AW96-4 and AW99-iii with respect to
hyphal growth (m) and enzyme production (Yp/s or Yp/x) using aw =0.96 to 0.99 in a medium with
pectin as sole carbon source, were compared to the patterns of wild and dikaryon strains,
suggesting that growth and pectinase production are regulated by different sets of genes in a
complex and non-coordinated fashion.
7. Evidence of Cryphonectria hypovirus replication in putative fungal transport vesicles. Patricia M. McCabe and Neal K. Van Alfen. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas.
The Chestnut blight fungus, Cryphonectria parasitica, contains a dsRNA virus, CHV1, which causes a reduced virulence of the fungus and changes in expression of specific fungal genes. Previously a host vesicle fraction was isolated and was shown to contain dsRNA. A similar fraction can also be isolated from non-viral infected strains, but in viral infected strains there is at least a 6 fold increase in the number of these vesicles. Viral replication has also been correlated with this vesicle fraction and the CHV I polymerase was also identified with these vesicles. Some of the genes which are down-regulated by the virus have been cloned and have been shown to be secreted proteins, some of which contain a signal peptide cleaved by a Kex2p-like serine protease. One of these proteins, cryparin, which is down-regulated in virus infected strains, has been found in this vesicle fraction. Treatment of the vesicles with proteinase K suggested that most of the cryparin is protected from the protease by the vesicles. When isolated with the vesicles, cryparin was not only present in its regular form, but a higher molecular weight glycosylated form was also isolated. Often on transport through a secretary pathway proteins are glycosylated, suggesting the vesicle fraction utilized by the virus for replication is a normal host secretary vesicle whose normal function is to transport, among others, cryparin.
8. Regulation of glucan synthase activity in Aspergillus fumigatus.
Pieternella C. Mol, Laboratoire des Aspergillus, Institut Pasteur, Paris, France.
The filamentous fungus Aspergillus fumigatus may cause pulmonary invasive aspergillosis
which is a life-threatening infection in immunocompromised patients. To gain insights into the
morphogenesis of this fungus and possible targets for chemotherapy, we study the structure and
formation of the cell wall. Synthesis of (1,3)--linked glucan in the fungal cell wall is catalysed by
the enzyme complex 1,3--D-glucan synthase (GS). Activity is stimulated by micromolar amounts
of GTP, and as in S. cerevisiae, the enzyme complex contains a plasma membrane bound catalytic
subunit that is activated by a GTP-binding protein. The latter can be substituted by the
homologous yeast protein, Rho1p, in an in vitro glucan synthase assay. Upon incubation of crude
and detergent-solubilized membrane fractions with C3 exoenzyme from C. botulinum a single 21
kDa protein was ADP-ribosylated. A genomic library was screened using PCR fragments
amplified from A. fumigatus DNA with oligonucleotide primers raised against the highly
conserved GTP-binding and interaction regions of Rho-family genes. Two genes were cloned.
Sequence analysis of RHO1 revealed a 950 nt region interrupted by 4 introns coding for a protein
of 21.400 MW. RHO2 is 1022 nt long, interrupted by 6 introns, and encodes a peptide of 21.900
MW. Rho1p and Rho2p show highest identity with Rho1p of S. pombe and with Rho3p of S.
cerevisiae, respectively. To identify all components of the GS enzyme-complex recombinant
Rho1p has been isolated which will be used in the analysis of protein-protein interactions.
Furthermore, we are studying the cellular localization of Rho1p and Rho2p.
9. Hyperbranching mutants of Aspergillus nidulans.
S. Pollerman and G. Turner, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, U.K.
Early studies on mycelial growth in Aspergillus nidulans led to a model for the duplication cycle of this organism which related hyphal elongation, mitosis, septation and branch development. More recently, commercial production of the edible mycoprotein QuornTM (Fusarium graminearum) by continuous culture has led to the undesired selection of colonial mutants in fermenters, some of which exhibit an increased frequency of branching, and a number of genetic loci appear to be implicated. Hyperbranching mutants have also been isolated in Neurospora crassa, and the cot-1 gene, encoding a protein resembling cAMP dependent protein kinase, has been cloned.
In order to understand better the control of branching frequency, and its relation to the
duplication cycle in fungi, we have isolated and characterised a number of mutants exhibiting
hyberbranching (Hbr) in Aspergillus nidulans. Most of these were identified by screening
microscopically a collection of 1200 Ts mutants for the desired phenotype, and 25 have been
selected for further study following backcrossing to the wild-type. The hyperbranching
phenotypes have been confirmed by measurement of the hyphal growth unit G. Complementation
tests and assignment of mutations to linkage groups should facilitate the isolation and molecular
characterisation of some of these genes with the aid of the chromosome specific cosmid library.
10. Impairing calcineurin of Neurospora crassa reveal its essential role for hyphal Growth, morphology and maintenance of the apical Ca 2+-gradient.
Holger Prokisch1, Oded Yarden2, Margit Mischler3, Maximilian Tropschug3, Ilse Babette Barthelmess1, +Institut fur Angewandte Genetik, Universitat Hannover, D-30419 Hannover, Germany; 2Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and 3Institut fur Biochemie und Molekularbiologie, Universitat Freiburg, D-79104 Freiburg, Germany.
The function of Neurospora crassa calcineurin was investigated in N. crassa strains
transformed with a construct for the inducible expression of antisense-RNA for the catalytic
subunit of calcineurin (cna-1). Antisense-RNA expression, reduced levels of cna1 mRNA and of
immunodetectable CNA1 protein and decreased calcineurin specific enzyme activity, on induction
medium only, were evidence that a conditional reduction of the target function had been achieved
in antisense-transformants with multiple construct integrations. Induction conditions procured a
growth arrest which indicated an essential function for the cna- 1 gene of N. crassa. Growth
arrest was preceded by increased hyphal branching, changes of hyphal morphology and
concomitant loss of the distinctive tip-high Ca2+-gradient typical for growing wild-type hyphae.
This demonstrates, for the first time, a specific role of calcineurin in the precise regulation of
apical growth, a common form of cellular proliferation. In vitro inhibiton of N. crassa calcineurin
by the complex of cyclosporin A (CsA) and cyclophilin2O and increased sensitivity of the induced
transformants to the calcineurin specific drugs, CsA and FK506, were evidence that the drugs act
in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin.
Consistently, exposure of growing wild-type mycelium to the drugs led to a phenotype very
similar to that of the cna-1 antisense-mutants.
11. Directionality of growth in fungal hyphae.
M. Riquelme, C.G. Reynaga-Pena, G. Gierz and S. Bartnicki-García. University of California, Riverside.
Hyphal tips of higher fungi contain a characteristic phase-dark body: the Spitzenkörper
(Spk). Former studies suggested a correlation between the position of the Spk and the direction of
hyphal growth. We have studied Spk behavior in growing hyphae of Neurospora crassa. Hyphae
of this fungus have a tendency to meander, yet they maintain an overall straight direction of
growth. We used video-enhanced phase contrast microscopy to study in fine detail positional
changes of the Spk in living hyphae of N. crassa. We found that Spk position and growth
directionality were closely correlated. A change in growth direction, i.e. the establishment of a
new axis, was correlated with a sustained shift of the Spk position away from the existing cell
axis. By using a computer program based on the hyphoid model for fungal growth and
morphogenesis, it was possible to duplicate the meandering behavior observed in living hyphal
cells. This supports the idea that hyphal morphology is controlled by the Spk functioning as a
vesicle supply center (VSC). Although external factors are known to affect directionality of
hyphal growth (tropisms), we have no evidence to believe that they are the primary determinants
of growth direction. Hyphal tips of N. crassa exposed to microtubule inhibitors became highly
branched and lost their growth directionality, suggesting a role of the microtubules in maintaining
growth direction. We propose that an intrinsic, cytoskeletal-based, mechanism keeps hyphae
growing in a fixed direction while allowing minor reversible departures of the Spk away from its
original trajectory.
12. Biochemical analysis and immunolocalization of ApsA, a protein involved in nuclear positioning in Aspergillus nidulans.
Nicole Sievers and Reinhard Fischer, Universitat Marburg and MPI fur terrestrische Mikrobiologie, Marburg, Germany
Nuclear migration is very important in many eucaryotic cells and absolutely crucial for apical extension of fungal hyphae. Microtubules and the microtubule dependent motor protein dynein are essential components for the translocation process. The regulation of the basic machinery and its coordination to other cellular functions like cell cycle, cell differentiation and morphogenesis are still unknown. In A. nidulans two genes were identified which seem to be involved in nuclear positioning, apsA and apsB (anucleate primary sterigmata). In the mutants nuclei are clustered in hyphae in contrast to evenly distributed nuclei in the wild type. In addition, the nuclear positioning defect leads to the formation of anucleate metulae and thus to a block of development.
The two genes apsA and apsB were cloned and sequenced. apsA encodes a 180 kD coiled coil
protein with similiarity to the yeast nuclear migration protein NUM1. Amino acid sequence
motifs suggest a role in signal transduction or an association to the cytoskeleton. The ApsA
protein and a hemeagglutinine (HA) epitope tagged version of the protein were detected in
Aspergillus protein extracts with polyclonal and monoclonal antibodies, respectively. The protein
could further be detected in germlings, hyphae and conidiophores by immunofluorescence using
HA-tagged ApsA and monoclonal anti-HA antibodies. ApsA was localized at the cell membrane
with a spot like distribution. Similiar results could be obtained with ApsA tagged with the green
fluorescent protein (GFP) of the marine yellyfish Aquorea victoria. Analysis of a cell cycle
dependent regulation of ApsA are under way.
13. Investigating the nuclear cycle in Ustilago maydis vegetative cells and mating hyphae.
Karen M. Snetselaar, Michael P. McCann. St. Joseph's University, Philadelphia PA 19131.
Nuclear cycle events are traditionally studied using 3H-thymidine, which cells take up from
culture medium and incorporate into DNA, but fungi apparently do not incorporate exogenously
supplied thymidine. Some methods used to study fungal nuclear cycles involve DNA extracted
from synchronous cell cultures, but because we wish to correlate nuclear condition in U. maydis
with nonsynchronous events such as bud formation, development of mating tubes and infection
hyphae, we are using microdensitometry. Cells from liquid cultures were attached to microscope
slides, stained with DAPI, and viewed using epifluorescence microscopy. Digital images of stained
nuclei were captured, and densitometry software was used to quantitate their relative
fluorescence. When nuclei were grouped by relative density, two peaks presumably corresponding
to 1C and 2C amounts of DNA were observed. The methods used also allowed correlating
measurements of nuclear density with cell morphology. Ustilago maydis sporidia completed DNA
synthesis prior to visible evidence of bud formation, and cytokinesis was completed before DNA
synthesis begins. Mid log-phase cultures had approximately equal numbers of cells with 1C and
2C nuclei, but as doubling times for cultures increased, the number of cells in G1 increased
dramatically, indicating that the length of G1 varies with growth conditions. Nuclei in cells
induced by pheromone to form mating tubes are apparently arrested in G2 because they have the
2C amount of DNA.
14. Conventional kinesin from the plant pathogen Ustilago maydis is involved in vacuole formation and cytoplasmic migration.
Gero Steinberg1,3, Christiane Lehmler2, Michael Bolker2, Manfred Schliwa3, J. Richard McIntosh1, and Regine Kahmann2. 1MCDB, Univ. of Colorado, Boulder, CO 80302, USA; 2Inst. for Genetics, LMU, Maria-Ward-Str. la, 80638 Munich, FRG; 3Inst. for Cell Biology, LMU, Schillerstr. 42, 803 3 9 Munich, FRG.
Using PCR a gene encoding a kinesin motor (kin2) was isolated from the dimorphic
basidiomycete Ustilago maydis. The predicted amino acid sequence displayed a high similarity
with the conventional kinesin from Neurospora crassa (Nkin). Purified Kin2 and Nkin proteins
share some unique features as a high in vitro motility and the absence of light chains. Haploid
strains carrying a deletion of the kin2 gene were viable and showed no obvious defects. Upon
fusion of compatible haploid wild type or kin2 mutant cells a filamentous dikaryon is produced
but kinesin-deficient mutant hyphae were significantly shorter, curved, often branched and failed
to create empty compartments that are normally left behind the growing tip cell. Moreover,
kin2 mutant cells showed dramatically reduced pathogenity. A biologically functional kin2-GFP
fusion protein displayed a diffuse distribution within the hyphal cells, suggesting that this kinesin
transports small vesicles. Although kin2 sporidia grew apparently normal, these cells were
defective in the accumulation of lucifer yellow in the vacuole. Moreover, kin2 mutant sporidia
contained more but smaller and often misplaced vacuoles. In hyphae produced by kin2 mutants
subapical vacuoles could not be detected. In wild type hyphae such vacuoles expand and
approach a size similar to the empty compartments behind the tip cell. We suggest that Kin2 is
involved in membrane traffic of vacuole precursors promoting the migration of cytoplasm during
growth of the filamentous dikaryon.
15. Nuclear migration and positioning in Aspergillus nidulans.
Suelmann R.*; Galetzka D.*, Robertson L1. Timberlake W. E1. and Fischer R*. *Universitat Marburg
and MPI for Terrestrische Mikrobiologie, Marburg, Germany, 1University of Georgia, Athens, GA.
Nuclear migration is important in many eucaryotic cells and crucial for apical growth of fungal hyphae. We have visualized this organelle movement by time-lapse video microscopy in vivo in A. nidulans using a GFP fusion protein localized in nuclei. First results will be presented.
Besides the microscopic characterization of the process the nuclear basis of the movement
is of great importance. Essential components are e.g. microtubules and the microtule associated
motor protein dynein. In addition two genes were isolated in A. nidulans which are involved in
nuclear positioning and thus in regulation of this process. In contrast to wildtype hyphae where
nuclei are evenly distributed, nuclei are clustered in mutant hyphae. Here, one of the genes apsB
(anucleate primary sterigmata), was analyzed at a molecular level. The gene contains one 48 bp
intron close to the 5' end of the transcript and encodes an open reading frame of 1052 amino
acids. Secondary structure predictions suggest coiled-coil domain at the N-terminus of the
hydrophilic protein. A HA:ApsB fusion protein was detected in crude cell extracts. In vivo
localization experiment (HA:ApsB; GFP:ApsB) are under way. Potential interactions with the
coiled-coil ApsA protein will be discussed.
16. Generation and Characterization of new mutations affecting tip growth and branching in N. crassa via insertional mutagenesis.
Michael Watters and Tony Griffiths, University of British Columbia, Vancouver B.C. CANADA.
Neurospora normally grows forming characteristically branched, spreading colonies. Many genes are known to affect this morphology altering either the branching pattern direction of growth or overall colony size. Reletively few of these genes however have been clones and their function in the cell determined. We have begun a study to induce new (or at least new alleles of) morphological mutations, using gene disruption by a plasmid transformed into cells. These mutants should have the advantage over existing alleles of being tagged with the inserted sequences. We plan to make use of these tags to aid in recovery of the affected genes. We have used this approach thus far to generate several mutants with a range of phenotypes and these have been partially characterized. The poster will report on a work in progress.