493 MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on opposite membrane surfaces in the mitochondrial inner membrane of Neurospora crassa. Holger Prokisch, Carola Klanner and Thomas Langer. Adolf-Butenandt-Institut für Physiologische Chemie, Universität München, Goethestrasse 33, 80336 München, Germany
Eukaryotic AAA proteases form a conserved family of membrane-embedded ATP-dependent proteases but have been analyzed functionally only in the yeast Saccharomyces cerevisiae. Here we have identified two novel members of this protein family in the filamentous fungus Neurospora crassa which were termed MAP-1 and IAP-1. Both proteins are localized to the inner membrane of mitochondria. They are part of two similar-sized high molecular mass complexes, but expose their catalytic sites to opposite membrane surfaces, namely the intermembrane and the matrix space. Disruption of iap-1 by repeat-induced point mutation caused a slow growth phenotype at high temperature and stabilization of a misfolded inner membrane protein against degradation. IAP-1 could partially substitute for functions of its yeast homologue Yme1 demonstrating functional conservation. Deficiencies of Dyme1 yeast cells in mitochondrial morphology as well as in cell growth at low temperature were suppressed upon expression of IAP-1, whereas respiratory growth at 37°C was not restored. Our results identify two components of the quality control system of the mitochondrial inner membrane in N. crassa and suggest that AAA proteases with catalytic sites exposed to opposite membrane surfaces are present in mitochondria of all eukaryotic cells.
Developing Mycosphaerella graminicola as a model fungal plant
pathogen for analysing gene function. Wendy
Skinner, John Keon, John Hargreaves. IACR-Long Ashton Research Station
Mycosphaerella graminicola (anamorph Septoria tritici), the causal agent of septoria leaf blotch disease on wheat, is an economically important pathogen throughout the world. Little is known about the mechanisms by which this fungus invades and destroys its host. Understanding the genetic determinants that influence the pathogenic habit would greatly expand our knowledge of this disease and may provide clues for the development of novel and environmentally benign control measures.
The genetic tools required for unravelling the basis of pathogenicity in a filamentous fungus include large-scale mutagenesis systems, rapid and reliable infection assays conducive to high through-put screening, a sexual crossing cycle to establish the segregation of the mutation during
recombination, and an efficient transformation system for subsequent genetic manipulation (Sweigard et al., 1998). Here we report on our progress towards developing protocols for M. graminicola for each of these requirements.
Reference: Sweigard, J. A., Carroll, A. M., Farrall, L., Chumley, F. G. & Valent, B.
(1998). Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis. Molecular Plant-Microbe Interactions 11:404-412.
495 Chitin synthases of Coccidioides immitis, the valley fever fungus. Alejandra M. Mandel1, Kris I. Orsborn2, John N. Galgiani2 and Marc Orbach1. 1University of Arizona, Plant Pathology, Tucson, AZ USA. 2University of Arizona, Valley Fever Center, Tucson, AZ USA
Coccidioides immitis is the causal agent of Valley Fever, a disease of humans and other mammals. The infection cycle is initiated when arthroconidia (single cells, approximately 5 microns in size, the product of filamentous saprobic soil growth) are inhaled into the lung and the fungus begins parasitic growth as spherules (multicellular structures, 50 microns or larger in diameter). This morphologic switch must involve the formation of new cell wall. NikkomycinZ, an antifungal that variably inhibits some chitin synthases, is exceptionally therapeutic in experimental murine coccidioidal infections. This suggests that chitin in the spherule cell wall is critical to pathogenicity. These observations have led us to analyze the role of different chitin synthases in spherule morphogenesis. We have isolated and sequenced completely four CHS genes from C. immitis, corresponding to classes I to IV (according to Robbins et al, 1992). These CHSes share similarity with other fungi. In some cases, intron number and positions are shared with other fungi and in other cases they are not. We are disrupting each CHS gene using Agrobacterium-mediated transformation to determine their roles in growth. Each mutant strain will be screened for differences from normal growth in both the saprobic and parasitic phases, with particular interest in defects in their ability to properly form spherules. If a mutant is found to be less sensitive to nikkomycin Z than the wild-type, this may identify the drug's therapeutic target. We are also analyzing the transcription patterns of these genes during the infection cycle of C. immitis. By defining which CHS genes are responsible for spherule morphogenesis, this research should provide a basis for future studies of control.
496 Characterization of Constiutively Active RAS and CAAX Box Deletion Mutants of C.trifolli. S.Memmott, Y.Ha and M.Dickman, Department of Plant Pathology, University of Nebraska-Lincoln, NE 68503-0722
Colletotrichum trifolii is the causative organism of alfalfa anthracnose. In order to characterize the signaling pathways involved in the interaction between this pathogen and its host we previously cloned the small prototypical G-protein, RAS of C.trifolii. Transformants expressing constitutively active (oncogenic) forms of RAS express differential phenotypes, when compared to wild type, which were dependent on the growth media. In nutrient rich media, (eg yeast extract, peptone) the phenotype of the transformants was indistinguishable form wild type. However, under conditions of nutrient starvation, the transfomants exhibited a loss of polarity, distended hyphae, failure to sporulate and produce appressoria. Studies have been conducted to identify the component(s) responsible for reversing the aberrant phenotype observed in minimal media. We have found that proline is responsible for the reversal of the constitutively active RAS phenotype. To understand the mechanism underlying this response, proline analogs have been utilized to see if they gave the same phenotypic reversal. Amino acid analyses of cytosolic extracts are being performed to determine differences in amino acid synthesis and or degradation between strains containing the wild type or constitutively active forms of RAS.
Preliminary studies using the farnesyl-protein transferase inhibitor fusidienol A suggested that the localization of RAS is essential to germination and growth. In the case of wild type and constitutively active RAS strains, germination rates were severely curtailed and germlings were identified that exhibited a distended morphology and stopped growing after 48 hours. To better determine that mislocalisation of RAS is responsible for this phenotype, a RAS clone containing a CAAX box deletion under the control of an inducible promoter was made and transformed into wild type C.trifolli. Under inducing conditions transformants were similar in phenotype to those treated with fusidienol A supporting the idea that proper localization of RAS is necessary for normal growth.
497 Colletotrichum trifolii TB3 kinase, a COT-1 homolog, is light-inducible and nuclear-localized. Changbin Chen and Martin B. Dickman; Department of Plant Pathology, University of Nebraska-Lincoln, NE68583-0722
Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. A serine/threonine protein kinase gene TB3, which is a functional homolog of Neurospora crassa COT-1, has been previously isolated in our lab and appears to be associated with hyphal elongation and branching in the same manner as COT-1. Since COT-1 is light regulated, we evaluated the effect of light on hyphal growth and TB3 expression. Results indicated that similar to COT-1, hyphal branching frequency is increased and TB3 expression is rapidly induced following illumination. It has been shown that COT-1 is expressed in the cytoplasm, membrane and nucleus. Western analysis using TB3 antibodies showed expression in both cytoplasm and nucleus, but not in membrane fractions. Moreover, indirect immunofluorescence indicated that TB3 is abundantly found in the nucleus. In order to detect the subcellular distribution of TB3 protein, we inserted a TB3::GFP fusion construct into Colletotrichum trifolii. Results indicated that the cellular distribution of TB3 changes during developmental transitions. Consistent with our previous observations, TB3 was localized in both the cytoplasm and nucleus but was preferentially abundant in the nucleus during hyphal growth. The amino terminus of TB3 contains several polyglutamine repeats which are absent in COT-1; such tracts are associated with protein-protein interactions, particularly with respect to transcriptional activation. We postulate that TB3 may be positioned in a signaling cascade resulting in proper hyphal growth and development by functioning as a transcription factor.
498A plant cutin activate protein kinase from C. trifolii is required in appressorium development. Youngsil Ha, C. Huang, Z. Yahg and Martin B. Dickman. Department of Plant Pathology, University of Nebraska-Lincoln, NE
Colletotrichum trifolii causes anthracnose of alfalfa. Successful infection is predicated on appressorium development. Pharmacological data indicated that a protein kinase C like gene product was necessary for appressrium formation. Using degenerate primers and PCR, we have identified a PKC like gene encoding 72kd protein in Colletotrichum trifolii. The deduced amino acid sequence of this protein revealed that it has a high degree of similarity to protein kinase C-like proteins of other fungi but only in the catalytic domain. However, we were unable to induce enzyme activity in the presence of PKC activators, diacylglycerol or phorbol esters. Interestingly, we found that the gene expression could be rapidly incuced by plant cutin and its fatty acid monomeric constituents. Structurally similar fatty acids, which are not found in cutin, were uanable to induce the expression of this gene. Over-expression of this Lipid Activated Protein Kinase (LAPK) resulted in abberrant formation of multiple appressoria. Gene disurption of LAPK resulted in the inability of C. trifolii to form appressorium and such transformants were incapable of infecting alfalfa. Thus the fungus uses host plant cues for induction of gene expression required for pathogenic development.
499Transcriptional control of the cellulase genes in Trichoderma Sp. strains FB 1231. Rojas T.1, Rosales RA.1,2, Pérez J.1, Villaverde M.1, Ortiz E.1,3, and Nuñez R3. 1Faculty of Biology, La Havana University, 25 Esq. I. Havana. Cuba. 2Faculty of Sciences, State University, Iguá S/N. Montevideo Uruguay. 3Institute of Oceanology. 1ra. No. 18408. Playa. Havana. Cuba
The expression of the cellulase transcripts of Trichoderma sp. strains FB 1231 is controlled by the nature of the energy carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Evidence is presented suggesting that a low constitutive extracellular cellulolytic system catalyses the formation of a soluble inducer from cellulose, and this inducer triggers the expression of the cellulose transcripts. This basal and cellulose-induced expression of the cellobiohydrolase I mRNAs (cbh1), the major member of the cellulose system, is transcriptional controlled by two independent cis-acting DNA regions. In addition, expression of the cbh1 transcript is influenced by the physiological state of the mitochondria and this sensitivity is controlled through the 5´-flanking DNA sequence of this gene.
500 The Ustilago maydis sequencing project
Jörg Kämper1, Gerhard Weinzierl1, Andreas Brachmann1, Michael Feldbrügge1, Christoph Basse1, Gero Steinberg1, Regine Kahmann1, Gabi Friedrich2, Verena Vollenbroich2, Edda Koopmann2, Isolde Häuser-Hahn2, Dirk Nennstiel2, Kai Sievert2, Rüdiger Suelmann2, Martin Vaupel2, Christian Aichinger3, Ronald Ebbert3, Birgitta Leuthner3, Birgit Jaitner3, Volkhart Li3, Peter Schreier3, Thomas Schlüter4, Dagmar Schütte4, Harald Kranz4, Jürgen Henrich4, Günter Kurapkat4, Monika Arenz4, Hartmut Voss4. 1 Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., 35043 Marburg, Germany. 2 Central Research, Bayer AG, 51368 Leverkusen, Germany. 3Agricultural Center Monheim, Bayer AG , 51368 Leverkusen, Germany 4Lion Bioscience AG, Im Neuenheimer Feld 515-517, 69120 Heidelberg, Germany
The phytopathogenic fungus Ustilago maydis is the causal agent of smut disease on corn. We have sequenced the genome of U. maydis strain 521 which was estimated to comprise 20.5 Mb to foster a molecular understanding of pathogenicity and fungal development. In addition, the sequence will allow the development of novel strategies for the identification of anti-fungal compounds.
Using high throughput hybridization technologies, 258 BAC clones were assembled in 28 contigs representative for the 23 U. maydis chromosomes. Prior to sequencing an ordered set of overlapping plasmid subclones was generated for each BAC. A total of 17.425.931 base pairs were sequenced, covering approximately 85.9% of the genome. The systematic combination of this genome analysis with the sequencing of 2626 unique cDNA clones allowed to access more than 90% of all U. maydis genes. For the identification of potential protein coding regions an automated bioinformatic analysis was used. From a total of 6700 regions with coding capacities, more than 60 % of the open reading frames show significant similarities to entries in protein databases. At least 5 distinct groups of retrotransposons were identified, while no DNA transposons were found. In addition, several duplicated sequences up to 50 kb were identified, a finding that can possibly be linked to the genome variability observed between different U. maydis strains.
501 Activity of transposable element Restless in Neurospora crassa Frank Windhofer1, David E.A. Catcheside2, Frank Kempken1. 1Ruhr-Universitat Bochum, Allg & Molekulare Botanik, Bochum, Germany. 2Flinders University, School of Biol Science, Adelaide Australia
In the past years several fungal transposable elements have been identified (Kempken and K ck, 1998, Bioassays 20:652-659). We have isolated and characterized Restless, a new type of fungal class II transposons from Tolypocladium inflatum (synonym: Beauveria nivea) which so far has not been found in any other fungus (Kempken and K ck, 1996, Mol Cell Biol 16:6563-6572). The predicted amino acid sequence deduced from an open reading frame encoded by Restless shows significant homology to transposases of the hAT transposon family, e.g. the maize Activator element. We were able to proof the usefulness of Restless as a molecular tool to tag and identify a new gene in T.inflatum (Kempken and K ck, 2000, Mol Gen Genet 263:302-308). Here we present new data regarding the activity of the transposon Restless in a foreign host, i.e. Neurospora crassa. We show that multiple copies of the element introduced by transformation are subject to methylation, whereas single copies integrated at the his-3 locus are not subject of methylation (Windhofer et al. 2000, Curr Genet 37:194-199). Single copy Restless transformants were selected for transposition of Restless. Numerous excision events have already been identified and characterized. Currently the ability of Restless to reintegrate into new genomic locations is under investigation.
502 Functional Analysis of a Cdc42 Ortholog from Magnaporthe grisea. Zhiying Zhao1, Zonghua Wang2, Peter Albersheim1, Alan G Darvill1, Zhenbiao Yang3, Shengcheng Wu1. 1University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia, USA. 2Fujian Agricultural Univ, Plant Protection, Fuzhou Fujian China. 3University of California, Botany and Plant Sciences, Riverside California USA
Following contact with a plant surface, spores of some phytopathogenic fungi produce by polar growth a germ tube, which differentiates into a highly pressurized dome-shaped appressorium. The appressorium then develops a penetration peg, which pierces the plant surface and starts the infective growth phase. Understanding the mechanism by which such invasive polarized structures are formed may lead to the development of novel methods for fungal disease control. We have cloned from Magnaporthe grisea, a notorious fungal pathogen of rice, mgCdc42, an ortholog of Cdc42 GTPase that regulates polar growth, bud emergence and pseudohyphal growth in yeast. mgCdc42 complements a lethal cdc42 temperature-sensitive mutant of Saccharomyces cerevisiae. However, in contrast to the case in S. cerevisiae, mgcdc42 knockout mutants of M. grisea are viable and do not exhibit obvious deficiency in germination and hyphal polar growth. However, these mgcdc42 mutants are only lightly pigmented on oatmeal agar, and produce gherkin-shaped conidia rather than the bulb-shaped wild-type ones. Under glass coverslip, wild-type conidia germinate and differentiate appressoria at almost 100% after 6 hours of incubation, whereas mgcdc42 mutants do not produce any. Based on these preliminary results, mgCdc42 appears to play a role in M. grisea different from that in yeast. Further analyses of the mgcdc42 mutants will be presented and discussed at the conference. (This work was supported in part by a grant from Natural Science Foundation of Fujian, China, and by U. S. Department of Energy grant DE-FG05- 93ER20114 and the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates.)
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