461. Possibilities of the maintenance of Shaggy Mane (Coprinus comatus), spawn production and growing. Tamás Szabó, József Szarvas, VHajdú Csaba and Nikolett Tóth. Korona Spawn Plant and Strain Researching Laboratory, 3395 Demjén, POB. 1., Hungary. VPh.D. students at Szent István University, School of Horticulture, Budapest, Hungary
The growing of Coprinus comatus not so widespread as that of Agaricus bisporus and the hybrids of Pleurotus ostreatus, Lentinus edodes. In Hungary few of the growers is engaged in the growing of mushrooms with curative power. We made collections from more sites of the country from which we created a stock culture (culture in test tubes, cryopreservation, maintenance of the stock on compost). We examined the growth of isolates on 10 different substrate types among laboratory circumstances. We used four types of grain substrate for the creation of a mother spawn, on which we examined the intensity of growth and the possible anomalous mycelium improvement. Arising from the mother spawn we used different substrate mixtures for our inoculatum creational experimentations. Next we created spawn producing then growing experimentations on five substrates for each stock. We examined their morphologies, their changes because of environmental factors, perishability.
462. Differentiation of newly breeded Agaricus hybrids with RAPD method. József Szarvas, Tamás Szabó and Csaba Hajdú, Korona Spawn Plant and Strain Researching Laboratory, 3395 Demjén, POB. 1., Hungary, Ph.D. students at Szent István University, School of Horticulture, Budapest, Hungary
On a world scale, but first of all in Europe and North America a white button mushroom, Agaricus bisporus is grown at the highest quantity. Differentiation of champignon species on the basis of traditional morphology and physiology is extremely hard. The appearance of the fruiting body is determined not only by own genetic substance, but by numerous environmental factors as well. Such factors are CO2 level, humidity, temperature and the temperature grade during the yielding period. A strain, which can be marked as a medium hybrid reacts to changes of the environmental factors besides general growing conditions with great versatility, so a medium hybrid can grow as a fresh market hybrid with huge fruiting body or as mushroom to be canned with small fruiting body, depending on the growing circumstances. Starting from the previous facts we would like to differentiate our species – instead of the morphological, perhaps physiological markers, which make the differentiation uncertain – with the help of molecular markers. We used RAPD method for our work, with which - with the use of adequate primers – as a result of the sublimation work the item of difference between our potential strains and primary materials for breeding (431 cultures) can be realized. Through the sequencing of the differentiating bands we synthetized own primers. Our further aims for the future are to be able to detect clones deriving from the cross – inoculation of our own breaded species, so that we can make fingerprints from every of our species with the determined primers. Furthermore we would like to make a selection of homocaryon monospore cultures for the facilitation of breeding work.
463. Characterisation of endopolygalacturonases from Botrytis cinerea. Geja Krooshof1, Harry Kester1, Ilona Kars2, Jan van Kan2, and Jac Benen1. 1Microbiology/Fungal genomics,2 Phytopathology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands
Botrytis cinerea is a plant pathogenic fungus causing serious diseases in a range of economically important crops, such as grapevine, strawberry, kiwi, rose, and flower bulbs. An essential step in the infection process of the fungus is the degradation of the plant cell wall. Therefore, Botrytis cinerea secretes a considerable number of cell wall-degrading enzymes, among which are six endopolygalacturonases (BcPGs). The aim of our research is to characterise all six endoPGs in more detail. The methylotrophic yeast Pichia pastoriswas used to produce the individual BcPGs. Secretion of active recombinant BcPG1, 2, 3, 4, and 6 has been confirmed. Results on the purification and characterization of the BcPG isozymes will be presented.
464. The effect of mismatch repair knockouts on recombination in Neurospora. L. Y. Koh, P. J. Yeadon and D. E. A. Catcheside. School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia.
DNA mismatch repair genes, msh-2 and pms-1, the eukaryotic homologues of bacterial MutS and MutL, function in the long patch mismatch repair pathway. These two genes are involved in the correction of errors from DNA replication and the repair of mismatches in heteroduplex DNA formed during genetic recombination. Knockouts of msh-2 and pms-1 were generated in Neurospora using RIP. Recombination at his-3 was measured in three types of cross; 1) homozygous wild-type, 2) heterozygous msh-2 and 3) homozygous msh-2. Additionally, sequence heterology in each of the diploids allowed identification of the parental origin of each section of a conversion tract. In the msh-2 homozygotes, there was a 2 to 3-fold increase in recombination and no decrease in the frequency of interruptions to conversion tracts.
465. The Functions of a G-alpha and a G-beta Protein in the Life Cycle of the Oomycete Phytophthora Infestans. Maita Latijnhouwers, Wubei Dong and Francine Govers Laboratory of Phytopathology, Wageningen University, The Netherlands
The G-protein mediated signaling pathway is a ubiquitous eukaryotic signaling pathway involved in translation of extracellular signals into intracellular ones. Previous work has shown that G-protein mediated signal transduction in plant-pathogenic fungi is indispensable for effcient adaptation to the plant environment and for the development of structures that are required for virulence. Our aim is to study the role of G-protein signaling in pathogenesis of the oomycete Phytophthora infestans, the causal agent of potato late blight. We isolated and characterized P. infestans genes coding for Galpha (Pigpa1) and Gbeta (Pigpb1) subunits, with up to 42% and 62% identity with known G-protein subunits, respectively. Expression studies showed that both genes are differentially expressed in various stages of the life cycle. By means of homology-dependent gene silencing we obtained P. infestans transformants deficient in either of the two subunits. Those in which Pigpb1 is silenced show a defect in asexual sporulation and produce a dense mat of aerial mycelium. Silencing of Pigpa1 has a major effect on zoospore behavior. Zoospores of Pigpa1-silenced mutants show aberrant swimming patterns and fail to autoaggregate. The virulence of these mutants is severely impaired. To isolate downstream targets of PiGPA1, a comparison of gene expression in sporangia of wildtype and of Pigpa1-silenced mutants using cDNA-AFLP is in progress. In addition, to elucidate targets of PiGPA1 and PiGPB1, large scale expression profiling of wildtype silenced mutants will be performed in the near future.
466. Investigations into the fungal-fungal interaction between Verticillium fungicola andAgaricus bisporus. C. Burns1, R. Amey1, A. Bailey1, J. Bowers1, M.P. Challen2, T.J. Elliott2, P.R. Mills2 and G.D. Foster1. 1School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK 2 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Agaricus bisporus (the white button mushroom) accounts for 10% of total horticultural production in the UK. Retail value is £300M in the UK, with a global value of £3,000M p.a. Pathogenic threats to Agaricus are varied, with the most significant to the commercial industry being Verticillium fungicola. Verticillium infection can occur throughout mushroom development, drastically reducing crop yields. We have used Agrobacterium and T-DNA to introduce disruption constructs into V. fungicolaas part of a molecular investigation into the fungal-fungal interaction between Verticillium and A. bisporus. We have developed an efficient transformation system for Verticillium which we have now adapted to give high levels of targeted mutagenesis. We have used this technology to investigate genes involved in Verticillium pathogenicity. We have also developed T-DNA tagging technology in a mycological context for random mutagenesis in V. fungicola. We will also report our results in using Agrobacterium to transform Agaricus, and the range of promoters tested and developed as part of our Agaricus Molecular Toolkit which will also allow us to investigate gene expression within Agaricus.
467. Transformation of enzyme-treated Rhizopus oryzae germlings by square-wave electroporation. Gyan Rai, John N. Tonukari, John P. Rayapati and Christopher Skory. Fermentation Research, Archer Daniels Midland Company and USDA.
Transformation involves the introduction of foreign DNA molecule into a cell for investigating gene structure and function. While bacterial transformations are relatively simple, eukaryotic transformations are always associated with problems that differ greatly between the transforming organisms. This necessitates optimization of complex variables, thereby resulting in a wide array of published transformation protocols available for eukaryotes. These transformation protocols can vary significantly from one species to another, although they can be usually adaptable to organisms within a species. In fungi, the spheroplasts generated from mycelia or spores are routinely transformed by chemical methods or by electroporation. Protoplasts have also been successfully transformed, but this procedure requires tedious optimization and is quite laborious. In some fungal species, intact spores have also been transformed with foreign DNA molecules, albeit with limited success and efficiencies. The biolistics transformation method has proved very helpful in species, including fungi, that have been recalcitrant to the convetional means. The use of this microprojectile bombartment method has been limited because of its cost. We report here a transformation procedure for the partially digested germlings of the filamentous fungus, Rhizopus oryzae. This protocol is relatively simple and time efficient. An overnight culture of spores was washed and further incubated for 2-4 hours with a mixture of lysing enzymes containing chitinase, chitosanase and zymolyase. The mixture of partially digested germlings and DNA was electroporated in a BTX square-wave electroporator. The transforming DNA consisted of a circular plasmid with a functional lactate dehydrogenase and the pyrG genes under control of their respective promoter and terminator sequences. The transformed cells lacked the functional pyrG gene thus allowing selection by functional complementation on minimal plates lacking uracil.
468. Cytological karyotyping of somatic chromosomes from Phytophthora infestans, Mycosphaerella graminicola, and Fusarium spp. Masatoki Taga1, Cees Waalwijk2, Wilbert G. Flier2, and Gert H. J. Kema2. 1Department of Biology, Okayama University, Okayama, Japan. 2Plant Research International B.V., Wageningen, The Netherlands.
Cytological karyotypes of somatic cells in the ascomycetes, M. graminicola and Fusarium spp. (F. graminearum, F. pseudograminearum, F. culmorum, F. cerealis, and F. lunulosporum in the section Discolor) as well as an oomycete, P. infestans, were analyzed with the germ tube burst method. We found that M. graminicola and Fusarium spp. represent the two extremes of cytological karyotypes in filamentous fungi; M.graminicola had a large chromosome number (ca. 20) with many minute chromosomes (12 chromosomes in a standard strain, < 1 micrometer in length or < 2 Mb in DNA size), whereas only four large chromosomes constituted the genome in the Fusarium spp. In P. infestans, chromosomes had a previously unknown coiled structure. Most chromosomes were very long, heavily twisted and coiled before their condensation reached the peak, and had a segmented appearance in the highly condensed state. Somatic chromosome counts in P. infestans are under way. The results of this study show that cytological karyotyping with somatic nuclei can be practiced in various fungi as well as oomycetes, providing basic information on the genome of each species.
469. Development of Real-time PCR assays for the detection of stem and leaf rust pathogens of wheat. Les J. Szabo. U.S. Department of Agriculture, Agricultural Research Service, Cereal Disease Laboratory, St. Paul, Minnesota, U.S.A.
Historically, stem and leaf rusts have been the one of the most devastating pathogens of wheat, with written accounts of epidemics dating back to ancient Greek and Romans. In North America, major epidemics have occurred from the late 1800's through the 1970's. During the cold war, the United States and the Soviet Union studied Puccinia graminis (wheat stem rust pathogen) as a biological warfare agent. Based on the current concern for biosecurity of agricultural crops we are developing Real-time PCR assays for the rapid detection of P. graminis, P. recondita, P. triticina and P. striiformis using a TaqManTM assay system. Current results on the development of Real-time PCR assays for these wheat pathogens will be presented.
470. Somatic cytogenetis in Neurospora crassa. Yuya Kato1, Chizu Ishii2, and Masatoki Taga1. 1Department of Biology, Okayama University, Okayama, Japan. 2Department of Regulation Biology, Saitama University, Saitama, Japan.
In N. crassa, cytogenetic analysis has been mostly done on meiosis in ascus. Although mitosis in somatic cell or hyphae is a potential target for cytogenetics, the lack of appropriate method to make preparations has hampered the development of somatic cytogenetics of this fungus. In this study, we developed the cytological methods for karyotyping as well as fluorescence in situ hybridization (FISH) with germ tube cells of macroconidia. Chromosome specimens were prepared by the germ tube burst method (GTBM; Taga et al., 1998) combining with thiabendazole treatment to accumulate metaphase cells. In the DAPI-stained samples, seven condensed chromosomes were easily distinguished based on their size and constrictions. In addition, difference in chromosome size was detected between a wild type strain and reciprocal translocation mutants. FISH using cosmid clones located near the centromere and telomere of LGI, rDNA, and beta-tubulin gene as probes was performed onto the interphase and metaphase specimens prepared by GTBM. Fluorescent signals were detected at the expected position for each probe. Thus, somatic cytogentics was shown to be feasible for N. crassa in this study.
471. Recombination in Neurospora declines exponentially with distance from cog, even across heterologous sequences. P. J. Yeadon, L. Y. Koh, F. J. Bowring, J. P. Rasmussen and D. E. A. Catcheside. School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia
We have constructed a set of Neurospora strains in which the distance between the recombination initiation site, cog, and his-3, at which recombination is selected, varies from 1.7 to nearly 6 kb. To maximise recombination, each of the manipulated strains has the more active recombination hotspot allele, cogL, and the recessive allele of rec-2, the unlinked gene controlling local meiotic recombination. In addition each is a histidine mutant, either K26 or K480. We have used these strains to show that the logarithm of the recombination frequency is inversely proportional to the distance fromhis-3 to cog, a relationship that holds even when the sequences between his-3 and cog are non-homologous.
472. Scanning electron microscopy of fungal mitotic chromosomes and nuclei. Dai Tsuchiya1 , Hironori Koga 2 , and Masatoki Taga 1. 1 Department of Biology, Okayama University, Okayama, Japan. 2 Researach Institute of Agricultural Resources, Ishikawa Agricultural College, Ishikawa, Japan.
Fungal chromosomes and nuclei have never been observed by scanning electron microscopy (SEM). In this study, we established the techniques for SEM observation of mitotic chromosomes and nuclei of the ascomycetes of Cochliobolus heterostrophus and Neurospora crassa , and revealed their ultrastructure at the resolution level of chromatin fiber. The nuclei of various mitotic stages were spread on slide glasses by the germ tube burst method. They were impregnated with osmium and tannin for conductive staining, and freeze-dried using t -butyl alcohol. The specimens coated with platinum were observed by field-emission SEM at 20 kV. Metaphase chromosomes were composed of highly condensed chromatin and their overall shape was cylindrical. Apparent constriction indicative of centromere region was found in most chromosomes. Interphase nuclei were more or less spherical, in which 30- nm chromatin fibers were discernible. Each nucleus had a large cavity probably representing the territory of nucleolus. The results from further observation will be presented.
473. Isolation and characterization of DNA polymerase mutants, which are involved in DNA repair and mutagenesis of Neurospora crassa. W. Sakai1, Y. Matsushita2, Y. Wada1, Y. Naoi1, C. Ishii1, H. Inoue1. 1Saitama Univ., 2Toyo Univ., JAPAN
In previous meeting, we reported that the N. crassa upr-1 gene is homolog of the yeast REV3. To understand the mechanisms of mutagenic DNA repair in the N. crassamore extensively, we identified N. crassa homologs of the yeast REV1 and REV7 and obtained mutants ncrev1 and ncrev7, which have similar phenotypes to the upr-1 mutant. ncrev7 mutant was more sensitive to UV and 4NQO than the wild type. The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild type, but the sensitivity to 4NQO of the mutant was similar to that of the wild type. In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, all ncrev mutants showed lower induced-mutability than the wild type. Expression of ncrevs was found to be UV-inducible. All ncrevs mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype. Furthermore, we characterized UV-induced ad-3A mutation-spectrum analysis at the upr-1 mutant with/without photoreactivation (PR). Most of ad-3A mutations at the upr-1 mutant with PR after UV irradiation are large deletion. These results imply that UPR1 product has an important role for translesion synthesis of non-photoreactable UV lesions.
474. Metabolic activity in filamentous fungi can be analysed by flow cytometry. J. Ron Bradner1, Paul V. Attfield2, and K. M. Helena Nevalainen1. 1 Department of Biological Sciences, Macquarie University, Sydney NSW 2109, Australia. 2 MicroBiogen Pty Ltd., c/-Department of Biological Sciences, Macquarie University, Sydney NSW 2109, Australia
The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and isolate bacterial and yeast cells is well established. We have shown that through the judicial choice of stains the non-destructive screening and sorting of filamentous fungal material is possible. The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma have been followed using forward and side angle scatter on a Becton Dickenson FACSCalibur flow cytometer. By staining these isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured. We have been able to demonstrate that there is a 12 -13 hour window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry. An important feature of flow cytometry is its ability to analyse individually thousands of cells per second. This ability to examine and select from such a vast number of events has the potential to significantly improve the chances of identifying particular fungal mutants and transformants within a much shortened time frame by obviating the need to undergo exhaustive screening on selection medium which is the current practice. The incorporation of genes carrying fluorogenic selection markers, such as green fluorescent protein (GFP) or its analogues, with the target gene construct could provide a convenient avenue for identifying genetically engineered strains.
475. Exploring the potential of HEX1 as a vehicle for foreign protein fusion expression in Trichoderma reesei. V. S. Junior Te'o 1, Natalie Curach 1, Peter L. Bergquist1,2 and K. M. Helena Nevalainen1. 1 Department of Biological Sciences, Macquarie University, Sydney NSW 2109, Australia. 2Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland, New Zealand
The search for strong promoters that function under specific conditions, are required for effective gene expression of industrially important gene products. We are currently investigating the use of the catabolite repression insensitive hex1 gene promoter as an alternative to inducible genes promoters in T. reesei (1). A 4376 bp DNA fragment that contains thehex1 gene open reading frame (ORF) as well as promoter and terminator was isolated from T. reesei using Genomic Walking PCR (2). When translated, the 784 bp hex1 gene ORF gives a peptide sequence of 225 amino acids with an expected molecular mass of 25,207. HEX1 isolated from cell envelope fraction of T. reesei, is expressed in different iso-forms also seen on 2-D gels as described in Lim et al., (3). In this report, we discuss the different iso-forms and demonstrate the potential of HEX1 protein as a carrier in foreign protein expression in filamentous fungi such as T. reesei. (1) Curach N, Te'o VJS, Bergquist PL, and Nevalainen KMH (2002). Hex1, a new promoter for gene expression in Trichoderma reesei. Abstracts of the 6th European Conference on Fungal Genetics. Abstract IIo5. (2) Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KK, Allison RW, Nelson PJ and Bergquist PL (1998). Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Appl Environ Microbiol 64:1759-1765. (3) Lim D, Hains P, Walsh B, Bergquist P, and Nevalainen H (2001). Proteins associated with the cell envelope of Trichoderma reesei: A proteomic approach. Proteomics 1:899-910.
476. Characterization of the Neurospora homologue of the human gene nbs1. Yukiko Kikuchi, Akihiro Kato, Hirokazu Inoue. Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama University, Saitama City 338-8570, Japan.
Double-strand break is induced by ionizing radiation and also occur during DNA replication. There are at least two pathways which can repair such breaks. They are non-homologous end joining (NHEJ) and homologous recombination (HR). nbs1 gene in human, which is a responsible gene of Nijmegen breakage syndrome, recruits a hMRE11/hRAD50 complex to sites of DSBs. NBS1/hMRE11/hRAD50 complex plays roles in processing DNA ends, selecting either NHEJ or HR and in activation of cell cycle checkpoint. A newly isolated mutant defective in the Neurospora homologue of the human NBS1 was found to be highly sensitive to a wide variety of mutagenes, including UV light, methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO),N-methyl-N'-nitro-N-nitrosoguanidine (NG), tert-butyl hydroperoxide (TBHP), hydroxy urea (HU) and ethyl methanesulfonate (EMS). This mutant was sensitive to histidine (HIS) and camptothecin (CPT), too. The ncNBS1 gene is located on the right arm of LGVII between met-7 and arg-10. We investigated epistatic relationships between ncNBS1 and other repair genes. Further characterization of the mutant will be presented.
477. Progress in Transforming the Mushroom Agaricus bisporus: Agrobacterium Methodologies and the Development of Novel Marker Genes. K. A. Leach1, 2; M. P. Challen1, T. J. Elliott1 and J. Henderson2. 1Horticulture Research International, Wellesbourne, Warwick, UK, CV35 9EF. 2Coventry University, School of Science and the Environment, Priory Street, Coventry, UK, CV1 5FB
Successful transformation of the button mushroom Agaricus bisporus has recently become possible due to the application of Agrobacterium-mediated transformation. Factors such as the strain of Agrobacterium tumefaciens, co-cultivation conditions, host strain, type of tissue, and the use of various promoters are known to affect rates of transformation. Using gill tissue from a number of commercial A. bisporus strains we have shown that different Agrobacterium strains and the use of vacuum infiltration and sonication treatments can dramatically alter the recovery of transformants. The effect of the virulence inducing phenolic, acetosyringone, on the number of transgene copies integrated into the A. bisporus genome has also been investigated. The rate of gill tissue transformation to hygromycin resistance was highest using A. tumefaciens strain AGL-1 and a sonication treatment (ca. 70 – 99% efficient). There is a need for alternative selectable markers in mushroom transformation. Homobasidiomycete para-aminobenzoic acid genes can confer sulfonamide resistance and have potential as positively selectable markers. The Coprinus cinereus auxotroph, PG78, was transformed to PAB+ prototrophy using PEG-mediated protoplast transformation and the C. cinereus pabA gene. PAB+ prototrophs were also generated using heterologous C. bilanatus and A. bitorquis pab genes. Most PAB+ transformants have proved resistant to sulphanilamide and sulfamethoxazole. Southern blotting is normally used to confirm transgene copy number. With PAB+ transformants we have demonstrated the use of real-time quantitative PCR to determine copy number.
478. Direct cloning of fungicide resistance genes in Botrytis cinerea. Sabine Fillinger, Catherine Lanen and Pierre Leroux Unite de Phytopharmacie & Mediateurs Chimiques, INRA, Versailles, France
Botrytis cinerea is the causal agent of grey mould on fruits, vegetables, ornamentals... Chemical control remains the principal means to reduce the incidence of grey mould on these crops and especially on grapevine, but rapid development of resistant strains involves development of anti-resistance strategies (Leroux et al., 2002). Theses strategies require the understanding of the resistance mechanisms in B. cinerea mutants. Direct cloning of these mutations involves transformation frequencies higher than spontaneous mutation and tagging of the resistance marker for its identification. The autonomously replicating plasmid pFAC1 harboring human telomeric sequences and the hygromycin resistance marker (Javerzat et al., 1993) transforms B. cinerea with frequencies up to 20000 transformants/ug DNA. Combining linearized pFAC1 and restricted genomic DNA for transformation and selecting for the double resistance, to hygromycin and to the fungicide to be analyzed, makes it possible to clone the fungicide resistance gene in B. cinerea. The pFAC1 derivative remains linear in the transformant in the presence of hygromycin and/or the fungicide. The insert can be characterized after PCR-amplification. We will present results obtained with DNA from field-isolates resistant to anilinopyrimidine fungicides. Javerzat, J. P., Bhattacherjee, V., and Barreau, C. (1993). Nucleic Acids Res 21, 497-504. Leroux, P. et al. (2002). Pest Managment Sci 58(9),876-88.
479. Molecular analysis of the multiallelic B mating locus that encodes pheromone and receptors in Coprinus cinereus. Meritxell Riquelme1, Andrew J. Brown2 and Lorna A. Casselton1. 1Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB, UK. 2Molecular Pharmacology, GlaxoSmithKline, Stevenage, SG1 2NY, UK
Pheromone signaling plays an essential role in fungal mating. In the homobasidiomyceteCoprinus cinereus, the mating pheromones and receptors have been recruited for self-nonself recognition and are encoded by multiallelic genes at the B mating type locus. The pheromone receptors belong to the rhodopsine-like superfamily of 7-transmembrane domain G-protein coupled-receptors (GPCRs) and specifically to the S. cerevisiae MATa cell receptor Ste3p subfamily. The lipopeptidic pheromone precursors are processed and secreted in the same manner as the S. cerevisiae a-factor. Rhodopsin-like GPCRs have evolved different specificities to respond to a variety of ligands. In fungi, and specifically in C. cinereus, a similar evolutionary process has created a family of functionally redundant receptors and corresponding ligands. Sequence variation permits them to display highly specific interactions: a single receptor may be activated by several different pheromones and a single pheromone may activate several different receptors. The different versions of the B locus derive from different allelic combinations of three sets of paralogous genes termed groups 1, 2, and 3. We have isolated genes from 18 strains in our collection with different B mating specificities. Cross-hybridisation in Southern blots and PCR amplification has been used to identify homoalleles of a particular subfamily of genes in these different B loci. Long range PCR has allowed us to amplify other new alleles. Thus far, we have identified 5, 6, and 2 alleles in groups 3, 2 and 1, respectively. We predict the existence of at least 4 more new alleles: one for group 1 and two or three for group 3. The family of C. cinereus receptors and pheromones offers a new dimension in studies of GPCRs, which are currently the largest protein family of validated drug targets. Our analysis of these different allelic versions of pheromone and receptors in wild type strains of C. cinereus is allowing us to predict the structure-function relationship determinants of ligand specificity. In addition, molecular genetic analyses of the B locus in C. cinereus provide clues toward understanding the evolution of so many different specificities of the B-mating type.
480. Molecular analysis of breeding behaviour in Agaricus species. Y. Li*, M. P. Challen++, T. Elliott++ and L. A. Casselton*. *University of Oxford, Department of Plant Sciences, Oxford, OX1 3RB, UK ++Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Agaricus bisporus, the cultivated mushroom, has a single mating type locus that determines mating compatibility. As the name implies, only two spores are produced following meiosis. Partitioning of haploid nuclei of opposite mating type into each basidiospore means that each germinates to give a fertile heterokaryon. This breeding behaviour is known as secondarily homothallic. Wild isolates of this species may have a more conventional heterothallic breeding behaviour in which the meiotic nuclei are segregated into four different spores. These spores are self-sterile and develop as homokaryons that must fuse with a compatible partner in order to generate a fertile heterokaryon. Other wild members of the genus may be truly self fertile and no longer need to mate to produce fruit bodies. Our aim is to clone the mating type genes of the cultivated A. bisporus and its close wild relatives to see if breeding behaviour can be related to mating type gene organisation and function. We have identified a pair of divergently transcribed homeobox genes within the mating type locus of A. bisporus. We are currently using PCR strategies to establish how many allelic variants of these genes exist in 15 different strains that exhibit different mating specificities. This analysis should reveal whether a single pair of genes is sufficient to generate the allelic variation displayed in this collection.
481. Identification and mapping of avirulence genes from Magnaporthe grisea. Claudia Kaye1, Joelle Milazzo1, Yanli Wang2, Ying Shen2, Marc-Henri Lebrun3 and Didier Tharreau1. 1UMR 385, Cirad, Montpellier, France 2CNRRI, Hangzhou, China 3UMR1932, CNRS-Bayer Crop Science, Lyon, France
We have begun a study aimed at mapping different Magnaporthe grisea avirulence (avr) genes. Crosses were made between fertile isolates and pathology tests were performed to uncover segregating avr genes. From the first cross, three different avr genes were uncovered. Pathology tests for two of these have been completed. The third gene shows a more complicated inheritance implicating possibly two genes. Fine mapping using AFLP was achieved to find genetic markers closely linked to the first two genes. Markers were found on either side of each of the genes. A second part of this project involved the development and mapping of microsatellite or SSR (single sequence repeat) markers. These microsatellite markers can be used to link maps from diverse crosses with a reference map. Several of these markers enabled us to locate the two avr genes to a specific chromosome and region. A search of the Magnaporthe grisea database allowed us to find other microsatellite markers closely linked to a gene on chromosome 7 and therefore approach the gene for eventual cloning. A BAC library has been constructed from DNA isolated from the avirluent parent for all three of the above described genes. The library has been screened with probes made from sequences closely linked to the gene on chromosome 7. Several candidate BACs have been selected for transformation into the virulent parent. Pathology tests will determine if any of these BAC clones contain the gene of interest.
482. A System for Studying Genetic Changes in Candida albicans During Infection. A. Forche, G. May, J. Beckerman, S. Kauffman, J. Becker, and P. T. Magee. University of Minnesota. GCD
Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the Candida albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of Candida albicans from a commensal to a pathogenic organism. We have identified 432 SNPs (single nucleotide polymorphisms) in the GAL1heterozygote (http://sequence-www.stanford.edu/group/candida/index.html). To study mitotic recombination at the population level, microarrays will be developed to facilitate high-throughput analyses of populations of Candida albicans. To analyze mitotic recombination in vivo, populations will be analyzed by SNP microarrays prior to and after mouse passage. This work was supported by a NIH grant AI46351 awarded to P. T. Magee and G. May.
483. Evidence for repeat-induced point mutation (RIP) among sequences of a putative transposable element from the wheat pathogen Mycosphaerella graminicola. Stephen B. Goodwin1, Yang Tian2, and Jessica R. Cavaletto1. 1 USDA-ARS, 2 Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054, USA
Repeat-induced point mutation (RIP) is a potential mechanism for inactivating transposons in fungi through introduction of C to T transitions into coding regions. This phenomenon has been analyzed thoroughly in Neurospora, but is less well studied in other fungi. A DNA fingerprint probe from the septoria tritici leaf blotch pathogen of wheat, Mycosphaerella graminicola, was shown previously to contain part of an active transposable element. Transposition of this element appeared to be stimulated by meiosis, but also occurred at a fairly high frequency in one isolate during asexual reproduction. To test for possible RIP in M. graminicola, five copies of the reverse-transcriptase gene from the putative transposable element were cloned and sequenced. A high frequency of G:C to A:T transitions characteristic of RIP was found among the sequences of the five clones. These mutations occurred not only in the reading frame for the reverse-transcriptase gene but also in the presumably non-coding regions of the transposon. All copies except one contained one or more stop codons within the region coding for the reverse transcriptase. Most of these stop-codon mutations were caused by G:C to A:T transitions, as expected if RIP is the causal mechanism. The copy of the transposon without stop codons presumably may be active. By comparison, analysis of the single-copy Sln1 gene from M. graminicola showed no evidence of RIP. This may provide the first evidence for RIP in a Loculoascomycete and indicates that this phenomenon may be important for inactivating transposons in many fungi in the genus Mycosphaerella and possibly the order Dothideales.
484. Analysis of sexually compatible C. lindemuthianum isolates from Mexico. Raul Rodriguez, Maria-Teresa Ramirez and June Simpson, CINVESTAV, Unidad Irapuato, Mexico
The genus Colletotrichum contains 39 species, many of which are pathogens of economically important plants. Eight of these species undergo sexual reproduction under laboratory conditions and both heterothallic and homothallic sexual forms have been observed. Sexual reproduction in C. lindemuthianum the causal agent of anthracnose in common bean is of the heterothallic form, but is rare and difficult to reproduce. Few sexually compatibleC. lindemuthianum isolates have been described previously. Compatible isolates produce few reproductive structures many of which are inviable. Nineteen Mexican C. lindemuthianum isolates were analysed in order to determine their capacity to develop sexually reproductive structures. All isolates were self-sterile, eight confrontations produced only inviable proto-perithecia and one confrontation produced fertile perithecia containing asci and ascospores. All other confrontations were infertile. AFLP analysis of colonies derived from individual ascospores confirmed that they were progeny from a sexual cross. Analysis of 44 individual ascospores mainly from different asci showed 42 distinct genotypes, all of which were combinations of the original parental genotypes. Markers analysed segregated in a proportion of 1:1 confirming that the progeny originated through sexual recombination. Analysis of this segregating population will allow genetic analysis of important traits such as avirulence, pathogenicity and sexual compatibility.
485. Meiotic Silencing in Neurospora is Mediated by Specific DNA Elements. Dong Whan Lee, Kye-Yong Seong, Kevin Baker and Rodolfo Aramayo. Department of Biology. College of Science. Texas A&M University. College Station, TX 77843-3258. USA.
Altering the normal ploidy in Neurospora crassa activates RNA-mediated gene silencing mechanisms, called "quelling" in haploid cells and "meiotic silencing" in diploid cells. Although these two events share equivalent molecular components, they respond to different signals. Repetitive elements have been postulated to trigger quelling, while unpaired DNA has been postulated to trigger meiotic silencing. This work was aimed at identifying signals, if any, that would be required to be present in the unpaired DNA to trigger meiotic silencing. We started by scanning the chromosomal region corresponding to the Ascospore maturation-1 (Asm-1) reporter gene of Neurospora crassa (Aramayo et al. (1996). Genetics 144, 991-1003) and found two such regions that we call Meiotic Silencing Inducing RU/u>egions (MSIRs). The first one, located immediately upstream of the coding region can induce meiotic silencing in cis (i.e., silence itself), but not in trans (i.e., silence paired copies of Asm-1 present elsewhere). The second one, located downstream, is equally capable of inducing silencing both in cis and in trans. Here, we describe some of the properties of these MSIRs. In addition, we demonstrate the size of the unpaired DNA loop determines the strength of meiotic silencing. Finally, we demonstrate that the signal produced by the unpaired DNA loop does not propagate to adjacent paired genes. These findings are discussed in the context of the biology of meiotic chromosomes. This work was supported by U. S. Public Health Service Grant GM58770 to R. A.
486. Identification of Suppressors of Meiotic Silencing. Dong Whan Lee, Malcolm McLaughlin, Robert J. Pratt, Kevin Baker and Rodolfo Aramayo. Department of Biology. College of Science. Texas A&M University. College Station, TX 77843-3258. USA.
During meiosis, chromosomes 'sense' each other through a process called meiotic transvection (Aramayo, R. and Metzenberg, R. L. (1996), Cell 86, 103-113), which was discovered by studying the complex Ascospore maturation-1 (Asm-1) locus of Neurospora crassa (Aramayo et al. (1996), Genetics 144 , 991-1003). The presence of unpaired DNA was recently proposed to activate RNA silencing (Shiu et al. (2001), Cell107 , 905-916), based on the demonstration that mutations in an RNA-dependent RNA polymerase (RdRP) gene called Suppressor of ascus dominance-1 (Sad-1), eliminate the ascus-dominance of unpaired DNA from Asm-1 and other genes (Shiu et al, (2001), Cell 107 , 905-916). Scanning of the Neurospora genome revealed the existence of a paralog for quelling deficient-2 (qde-2) which we call Suppressor of meiotic silencing-2 (Sms-2--NCU009434.1) and of two Dicer-like proteins related to the ribonucleases of the carpel factory, which we call Suppressor of meiotic silencing-3 (Sms-3 --NCU08270.1) and dicer-like-2(dcl-2-- NCU06766.1). In this work; we first demonstrate the involvement of both Sms-2 and Sms-3 in meiotic silencing. We next demonstrate that the SAD-1 RNA-dependent RNA polymerase is only essential for amplification, not production, of the meiotic silencing signal if double-stranded RNA corresponding to the silenced gene is produced by an alternative pathway. Finally, we hypothesize that Sad-1, Sms-2 and Sms-3 are components of the same meiotic silencing pathway. R. J. P. was partially supported by the Program in Microbial Genetics and Genomics (PMGG). This work was supported by U. S. Public Health Service Grant GM58770 to R. A.
487. A novel B-ZIP transcription factor from the rice blast fungus, Magnaporthe grisea, is required for pathogenicity on rice. Andrew Tag 1, Pierre-Henri Clergeot2, Karine Lambou2 , Stéphanie Sibuet3, Christelle Barbisan3, Marie Pascale Latorse3, Philippe Perret3, Roland Beffa3, Josh Munson1, Terry Thomas2, and *Marc-Henri Lebrun2. 1Laboratory for Functional Genomics, Department of Biology, Texas A&M University, College Station, TX USA. 2CNRS-Bayer CropScience, Lyon, France3Bayer CropScience, Lyon, France
Genes required for pathogenicity of M. grisea were identified using non-pathogenic mutants obtained by REMI-insertional mutagenesis. We recovered three tagged non-pathogenic mutants (M421/PLS1, M700/BUF1, and M763). The number of lesions caused by mutant M763 was dramatically reduced compared to wild type (-95%), and its colonization of host tissues was also impaired (-80%). Cytological analysis of the penetration process showed that M763 differentiated appressoria at normal rates, but their penetration efficiency was highly reduced. Colonization of epidermal cells occurred normally but the mutant did not efficiently invade underlying leaf tissues. Genomic regions flanking the plasmid insertion were rescued and used to recover corresponding genomic and cDNA clones. Complementation analysis identified an ORF inactivated in M763 that encodes a novel B-ZIP transcription factor from the GCN4 superfamily. This gene is only expressed in spores and during plant infection and it is likely to control the expression of genes that are required for efficient penetration and colonization of host plant tissues. Cell biology and expression studies are being pursued in order to identify the cellular functions controlled by this gene. In particular, genes that are differentially expressed between wild type and the M763 mutant will be identified using a M. grisea EST-microarray.
488. Assessment of basal levels of expression of defense-related genes in soybean cultivars varying in quantitative (partial) resistance to Phytophthora sojae. Miguel E. Vega-Sanchez1, Margaret G. Redinbaugh2, Anne E. Dorrance1. 1The Ohio State University, Ohio Agricultural Research and Development Center, and 2USDA Agricultural Research Service, Wooster, OH 44691, USA.
Phytophthora sojae is an oomycete pathogen that causes economically important root and stem rot diseases of soybean. Quantitative or partial resistance in this pathosystem is characterized by containment of the pathogen to the lower stem and tap root, but the genetic and/or biochemical mechanisms underlying the expression of partial resistance to P. sojae in soybean are poorly understood. To explore the hypothesis that higher basal expression levels of defense-related genes correlate with quantitative disease resistance, Northern blot analysis of root and cotyledon total RNA from 14 soybean cultivars varying in partial resistance levels to P. sojae was carried out. Ranking of cultivars into low, moderate and high partial resistant groups was done based on a standard lesion growth test. The coding sequences of eight defense-related genes including pathogenesis-related (PR) proteins and enzymes of the phenylpropanoid pathway were used to design primers for RT-PCR. The cDNAs representing a portion of the coding regions were cloned into a plasmid vector and used as probes. Preliminary results show that no clear-cut associations exist between constitutive defense-related gene expression levels and quantitative resistance to P. sojae in soybean. However, differential basal levels of expression of defense genes was observed between roots and cotyledons: most transcripts were at higher levels in roots than in cotyledons, with some mRNAs (PR1 and basic peroxidase) being undetectable in cotyledons. Two exceptions were matrix metalloproteinase (GmMMP2) and beta-1, 3-endoglucanase which were expressed at higher levels in cotyledons than roots.
489. H2O2 resistance of microfungi isolated from regions of various radioactivity. Tatiana A.Belozerskaya1, Yulia V.Blazheevskaya2, Konstantin B. Aslanidi3, Anna E.Ivanova4, Nelly N. Zhdanova2. 1A.N. Bach Inst. Biochem., RAS, Moscow, Russia, 2Inst. Microbiol. Virol., NAS, Kyiv, Ukraine, 3Inst. Theor. Exp. Biophys., RAS, Pushchino, Moscow region, Russia, 4Soil Science Faculty, Moscow State University, Moscow, Russia
High resistance of microfungi to radiation in comparison with animals, plants and bacteria puts forward the question about their tolerance to oxidative stress caused by ionizing radiation. Video image analysis was used to study growth peculiarities and H2O2 resistance of micromycetes isolated from territories of background radiation (K), and from such radioactive substrates as the inner locations of 4th Unit of ChNPP and soils of 10-km limit zone (R). Six strains of microfungi were analyzed: Alternaria alternata (K, R), Cladosporium cladosporoides (K, R), and Paecillomyces lilacinus (K, R). Two different growth strategies were elucidated in the (R) fingi. A. alternata (R) and P. lilacinus (R) strains showed hyphal aggregation and an increase in initial growth rates. On the other hand, C.cladosporoides (R) did not differ significantly from the (K) strain, but its growth rate was two times lower than that of the (K) one. It turned out that in all (R) strains growth was inhibited at H2O2 concentrations of 10-2-10-1 M, while in (K) strains - at the concentrations of 10-3-10-2 M. The unique strain (A. alternata K) demonstrated an increase in growth rate at the H2O2 concentrations of 10-7-10-5 M. Restoration of growth was observed only in the (R) strains upon transfer to the control medium following incubation under high (10-2 M) H2O2.
The work was partially supported by the Russian Foundation for Basic Research Grant # 01-04-48567
490. Analysis of a G protein coupled receptor from Neurospora crassa with similarity to putative carbon sensory receptors from yeasts. Liande Li and Katherine A. Borkovich. Department of Plant Pathology, University of California, Riverside. Riverside, CA 92521
We have identified a gene encoding a G protein coupled receptor in the Neurospora genome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora) that is most similar to Gpr1p from Saccharomyces cerevisiae and Git3 from Schizosaccharomyces pombe. The two yeast receptors are coupled to G alpha subunits that modulate cAMP levels in response to carbon availability in these species. The N. crassa homologue of these yeast G alpha proteins is GNA-3, for which our laboratory has previously demonstrated roles in regulation of adenylyl cyclase protein levels and conidiation. We have identified cosmids containing the N. crassa gene (gpr-4; G protein coupled receptor –4), constructed a gene replacement vector and isolated N. crassa strains lacking the wild-type gpr-4 gene. Experiments are in progress to determine the cellular phenotypes of gpr-4 deletion mutants and to explore coupling to the three N. crassa G alpha proteins.
491. Rice blast secondary metabolism signals pathogen attack. H. U. Böhnert, I. Fudal and M.-H. Lebrun. CNRS/Bayer CropScience, Lyon, France.
Rice resistance to the blast fungus Magnaporthe grisea depends on specific interactions controlled by fungal avirulence genes and corresponding plant resistance genes. Isolate Guy11 is avirulent towards rice varieties carrying the resistance gene Pi33. The corresponding avirulence gene ACE1 was isolated by positional cloning and was found to encode a fungal type I polyketide synthase (PKS) bearing high similarity to LNKS from A. terreus (involved in lovastatin biosynthesis). However, in contrast to LNKS, ACE1 carries a complete NRPS module at the C-terminus. Several lines of evidence, including the molecular analysis of a spontaneous virulent mutant, support the notion that this C-terminal NRPS module is functional.
Using an ACE1-GFP fusion, we demonstrated cytoplasmic localization of the synthase. By introducing a point mutation in the ketosynthase domain we created a nonfunctional allele that is unable to confer avirulence. We conclude that it is the metabolite produced by ACE1 which is recognized by resistant host plants and triggers resistance.
Unlike most fungal PKS-encoding genes studied to date, ACE1 is not expressed in culture. Instead, ACE1 expression is restricted to the penetration phase of pathogenic development and can be detected both on plant and artificial surfaces. No expression was detected in the penetration-deficient mutant buf (impaired in melanin biosynthesis). In M. grisea, melanin is required for the build-up of turgor pressure in the appressorium, an essential prerequisite for penetration. Our results suggest that ACE1 expression requires appressorial turgor and is developmentally regulated.
492. Integrating graduate and undergraduate education. Patricia J. Pukkila. Office of Undergraduate Research, U. North Carolina-Chapel Hill.
On many campuses, the opportunity to carry out an original investigation has become a distinctive feature of the undergraduate experience. The Office of Undergraduate Research at UNC-Chapel Hill seeks to expand these opportunities by eliminating some of the artificial boundaries between graduate and undergraduate education. Elements of our programs that are relevant to teaching fungal biology and genetics include collaborative design and facilitation of undergraduate "inquiry seminars" that focus on the logic of scientific investigation, workshops and course work on mentoring skills, graduate contributions to a database of research opportunities for undergraduates, financial compensation for graduate students to encourage careful design of undergraduate research opportunities and subsequent reflection on the mentoring experience, and graduate involvement in undergraduate presentations at national meetings and for elected officials. In addition to enhancing the undergraduate experience, these programs contribute to graduate professional development and to the public understanding of emerging issues in fungal biology and genetics.
493. Using fungal genomes to teach bioinformatics to undergraduates. Steven W. James. Biology Department & Biochemistry and Molecular Biology Program, Gettysburg College, Gettysburg, PA.
Bioinformatics and functional genomics have, in a few short years, become indispensable tools of the molecular biologist. Putting these powerful tools into the hands of aspiring students provides a rigorous intellectual challenge and fosters original thinking and problem-solving skills necessary for success as an independent investigator. Fungal genomes furnish an ideal training ground for in silico gene discovery and analysis, owing to their high gene density and the relative diminution of introns. In the Molecular Genetics course at Gettysburg College, juniors and seniors undertake a six-week project to discover and analyze genes within a 50-100 kb fragment of the Neurospora crassa genome. Using a combination of desktop software and web-based tools, pairs of students collaborate on a segment of their own choosing to identify and characterize a minimum of six genes that share homology with known genes from other organisms. Students use DNA Strider 1.0 to identify ORFs and convert them to protein sequences. They perform blastp searches using Genbank (NCBI), and learn how to judge the quality of hits, predict orthologs, and navigate auxiliary Genbank resources for further information about the genes. Students make extensive use of the Saccharomyces Genome Database (SGD), examining expression profiles and protein-protein interactions of the yeast homologs of their genes. The students assemble their findings into a comprehensive report, in which they analyze gene functions and demonstrate a detailed understanding of microarray techniques and proteomic approaches. (Supported by Gettysburg College).
494. Physical mapping of the genome of the fungal pathogen Ustilago hordei and characterization of the 500 kb MAT-1 sequence. G. Jiang, G. Bakkeren, R. Warren, Y. Butterfield, R. Chui, J. Schein, H. Shin, N. Lee, D. Kupfer, Y. Tang, B. Roe, S. Jones, M. Marra and J. Kronstad. Biotechnology Laboratory, Department of Microbiology and Immunology, and Faculty of Agricultural Sciences, The University of British Columbia, Vancouver, B.C. V6T 1Z3, Canada. Agriculture and Agri-Food Canada, Summerland B.C., Canada. Genome Sciences Centre, B.C. Cancer Agency, 600 W. 10th Avenue,Vancouver, B.C. V5Z 4E6, Canada. The Advanced Center for Genome Technology, The University of Oklahoma, Norman, Oklahoma 73019-3051
The basidiomycete fungus Ustilago hordei causes covered smut disease of barley and oats. In this pathogen, sexual development and the ability to infect the host plant are controlled by the MAT locus with two alternative types MAT-1 and MAT-2. The MAT locus is unusually large compared with other fungal mating loci (~ 500 kb for MAT-1 and ~ 430 kb for MAT-2) and recombination is suppressed between the a and b gene complexes that define the boundaries of the MAT locus. We constructed a physical map of the U. hordei genome by fingerprinting a bacterial artificial chromosome (BAC) library; the map consists of 2030 BAC clones that have been assembled into 51 contigs containing approximately 23 Mb of DNA. The physical map provided information on the rDNA repeat structure for U. hordei and allowed us to define a 1.76 Mb contig that spans the MAT-1 locus. A set of minimum tiling path clones was identified for this contig and the order of these clones was confirmed by BAC-end sequencing and hybridization. Five BAC clones were identified that provided the minimum tiling path between the a and the b gene complexes. These BACs now have been sequenced and reveal a preponderance of retrotransposon elements as well as several predicted genes with functions not obviously related to mating. The characterization of the MAT-1 locus provides an opportunity to compare the organization of mating-type loci between basidiomycete fungi and explore evolutionary aspects of sex-chromosomes.
495. Disruption of chitin synthases in Coccidioides posadasii, the Valley Fever fungus. M. Alejandra Mandel, Ellen M. Kellner, John N. Galgiani and Marc J. Orbach. University of Arizona
Coocidioides immitis and Coccidioides posadasii are dimorphic fungal pathogens of humans and other mammals that are endemic to the desert Southwestern US. During infection, the fungus switches from polar hyphal growth and conidial production to isotropic spherule growth and subdivision of spherules into endospores. This process involves major synthesis of new cell wall material. To investigate the role chitin synthases (CHSes) may play in the infectious phase of Coccidioides sp., we have isolated six CHS genes corresponding to individual members in classes I-IV and two members of class V. Data will be presented on their expression patterns during hyphal growth and the infection cycle of C. posadasii. Agrobacterium-mediated transformation of C. posadasii has been used to disrupt three of these genes. Phenotypic analyses of the disruptants will be presented.
496. A 3-hydroxyacyl-CoA Dehydrogenase Encoded at the Cochliobolus heterostrophus Tox1A Locus is Required for T-toxin
Production and High Virulence to T-cytoplasm Corn.
Thipa Asvarak1, Scott Baker2 and Gillian Turgeon1. 1Department of Plant Pathology, Cornell University, Ithaca, NY 14853 2Torrey Mesa Research Institute, CA 92121
Cochliobolus heterostrophus race T produces T-toxin, a polyketide required for high virulence of the fungus to Texas male sterile (T)-cytoplasm corn. Ability to produce T-toxin segregates as two unlinked loci, Tox1A on chromosome 12;6 and Tox1B on chromosome 6;12. Previous work has shown that genes at Tox1A (PKS1 and PKS2) and at Tox1B (DEC1) are essential for T-toxin production. A second gene at Tox1B (RED1), is not required. To determine if additional genes are required for T-toxin production, the sequences of the known Tox1 genes were used to query the C. heterostrophus genome sequence database (Torrey Mesa Research Institute). Three genes - LAM1 at Tox1A on a contig carrying PKS2, and RED2 and RED3 at Tox1B on a contig carrying DEC1- were recovered. LAM1 (similar to 3-hydroxyacyl-CoA dehydrogenase) was found upstream of PKS2, and RED2 and RED3 (similar to short chain dehydrogenases) were found downstream of RED1. Targeted disruption of LAM1 greatly reduced both T-toxin production and virulence to T-cytoplasm corn, whereas disruption of all three reductase genes RED1, RED2 and RED3 had no apparent effect on T-toxin production or on virulence.
497. Re-annotation of ORF start and stop sites in S. cerevisiae based on comparisons of orthologous ORFs from five other Saccharomyces species. Christie, K. R.1, Sethuraman, A.1, Balakrishnan, R.1, Dolinski, K.1, Dwight, S. S.1, Fisk, D. G.1, Hong, E. L.1, Theesfeld, C. L.1, Kamvysselis, M.2, Paul Cliften, P.3, Costanzo, M. C.1, Engel, S. R.1, Issel-Tarver, L.1, Dong, S.1, Weng, S.1, Johnston, M.3, Lander, E.2, Botstein, D.1, and Cherry, J. M.1. 1 Department of Genetics, Stanford University, Stanford, CA. 2 Whitehead Institute for Biomedical Research, MIT, Cambridge, MA. 3 Department of Genetics, Washington University School of Medicine, St. Louis, MO.
Since the publication of the genome sequence of S. cerevisiae in 1996, corrections have been ongoing to improve the accuracy of the sequence and the annotations of open reading frames (ORFs) and other sequence features. While most of these changes have been made on a gene by gene basis, the availability of genome sequences for five Saccharomyces species (S. paradoxus, S. mikatae, S. bayanus, S. kluyveri, and S. castelli) from groups at MIT and Washington University allowed the Saccharomyces Genome Database (SGD) to make a large scale comparison of orthologous ORFs in these species and re-evaluate ORF boundaries in Saccharomyces cerevisiae. Using the Fungal Alignment viewer and Synteny Viewer (links under Comparison Resources on SGD locus pages), users may view protein and DNA sequence from the other Saccharomyces species. For 402 of the ORFs considered, there were differences in the length of the one or more ORFs relative to other species at either or both the amino-terminal and carboxyl-terminal ends of the predicted protein sequence. Careful comparison of the DNA sequences, predicted protein sequences, and available literature for these ORFs has allowed us to conclude that changes should be made to the ORF start and/or stop sites for 104 ORFs. A further 161 changes were suggested, but require additional sequencing of S. cerevisiae S288C for confirmation. Any changes made will be documented in Locus History notes and a paper will be published detailing the changes to be made. Thus, comparison of S. cerevisiae with three closely related and two more distantly related Saccharomyces species has allowed us to improve the ORF annotations in SGD.
498. Characterization of the Glucose-6-phosphate Isomerase Gene in Phytophthora infestans
Manuel D. Ospina-Giraldo, Richard W. Jones. USDA/ARS Vegetable Laboratory, Beltsville, MD
Glucose 6 phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis and GPI isozyme pattern is the most widely used approach to characterize isolates of P. infestans. In addition to its well-known catalytic activity, GPI is considered a neurotrophic factor. Recent studies on Xanthomonas campestris pv. citri indicate that GPI also plays a role in bacterial pathogenicity. Despite the crucial role of GPI in the studying of P. infestans, the gene encoding this enzyme has not been characterized yet. Furthermore, it has been suggested that P. infestans contains multiple copies of the GPI coding sequence but this hypothesis remains to be demonstrated. In an attempt to elucidate these questions, we have cloned and characterized the GPI gene in various isolates of P. infestans as well as in several species of the genus Phytophthora. The confirmed cDNA GPI sequence consists of 1,671 base pairs. The gene, which has no introns, encodes a protein of 556 amino acids with a molecular weight of 60.78 kD. Multiple different alleles were identified by cloning and sequencing and Southern analysis indicates certain P. infestans isolates carry several copies of the gene. Phylogenetic studies indicate that P. infestans GPI is most closely related to sequences from plant and protozoan origin.