Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSregulated after treatment with both 133Cs and 137Cs.730. The completion of meiosis in Ustilago maydis requires an Ndt80 ortholog. C. E. Doyle 1 , H.Y. K. Cheung 1 , B. J. Saville 1,2 . 1) Environmental & LifeSciences Graduate Program, DNA Building, Trent University, 2140 East Bank Dr., Peterborough, ON, Canada; 2) Forensic Science Program, DNA building,Trent University, 2140 East Bank Dr. Peterborough, ON, Canada.Meiosis in the model fungal plant pathogen Ustilago maydis requires growth in the plant host; as such, control of meiosis responds to cues receivedduring pathogenic development. To begin investigating this process, an ortholog of the Saccharomyces cerevisiae meiotic control protein, Ndt80 (nondityrosine) was identified in Ustilago maydis. It was hypothesized to control progression through meiosis and has been designated mcg1 (meiosis controlgene 1). To assess its role in meiosis, mcg1 deletion mutants were constructed in compatible U. maydis haploid strains by replacing the gene with differentselectable markers. This allowed the impact of mcg1 deletion on pathogenesis, teliospore development and the completion of meiosis to be determined.Infections with compatible Dmcg1 strains were fully pathogenic, but teliospores produced from these crosses displayed a distinctive, abnormalmorphology and meiotic segregation assays indicated that they germinated without undergoing meiosis. This suggests that Mcg1 is involved in theregulation of meiosis and teliospore formation. To investigate this possibility that Mcg1 accomplishes this function by acting as a transcription factor, theupstream region of U. maydis genes was searched for variants of sites known as middle sporulation elements (MSE, the binding site of S. cerevisiae Ndt80).89 genes with upstream MSEs were screened by RT-PCR, using RNA from dormant teliospores of wild-type and Dmcg1 strains. The results suggested thattranscript levels for 43 of these genes differed in wild-type, relative to Dmcg1 teliospores, which indicated that the expression of these genes was affected,either directly or indirectly, by Mcg1. Further screens using RT-qPCR allowed the confirmation of genes with increased transcript levels, as well as thosewith decreased transcript levels, in the Dmcg1 teliospores relative to the wild-type teliospores. The upstream regions of these genes were screened for thepresence of conserved sequence elements. In parallel, Mcg1 was aligned with putative orthologs to identify conserved regions. Based on these alignments,mcg1 genes containing targeted mutations were synthesized. Together, these analyses begin the determination of how in planta transitions in U. maydisdevelopment are controlled.731. Impact of changes in the target P450 CYP51 enzyme associated with altered triazole-sensitivity in the Wheat pathogen Mycosphaerellagraminicola. Eileen Scott 1 , Regula Frey 2 , Helge Sierotzki 2 , Michael Csukai 1 . 1) Syngenta, Biological Sciences, Jealotts' Hill International Research Centre,Bracknell, United Kingdom; 2) Syngenta Crop Protection Munchwilen AG, Research Biology Centre, Schaffhauserstrasse, Stein, Switzerland.The triazoles are a widely used class of fungicides, targeting the cytochrome P450 sterol 14a-demethylase Cyp51. They are hence also known as the 14a -demethylase inhibitors, the 14-DMIs. Despite heavy use of this chemical class in the field over a considerable period of time, catastrophic resistance hasnot been observed in the economically important plant pathogen M. graminicola. Rather, there has been a slow shift toward reduced sensitivity. A largenumber of mutations in the Cyp51 gene have been previously associated with this shift in sensitivity to DMIs, although other resistance mechanisms suchas alteration in sterol biosynthesis and fungicide uptake and efflux may also play a role. There have been attempts to correlate changes in resistance levelswith specific Cyp51 mutations in field isolates. However, due to the genetic diversity of Mycosphaerella , the possible effect of non-target site mutationsand issues with expression in exogenous fungi making solid conclusions has been problematic.In order to accurately assess the contribution of each of the target site substitution mutations found in the field associated with 14-DMI resistance wehave introduced mutations individually and in combination into the endogenous Cyp51 gene in a uniform genetic background (M. graminicola genomesequenced strain, IPO323). Here, we present the findings of the comparative efficacy of varying triazole structures against this comprehensive collection ofmutants.732. Molecular Evolutionary Analysis and Synteny of Fungal GAL Genes. Julien S Gradnigo 1 , C. L Anderson 2 , R. A Wilson 3 , E. N Moriyama 1,4 . 1) School ofBiological Sciences, University of Nebraska - Lincoln, Lincoln, NE; 2) Department of Computer Science and Engineering, University of Nebraska - Lincoln,Lincoln, NE; 3) Department of Plant Pathology, University of Nebraska - Lincoln, Lincoln, NE; 4) Center for Plant Science Innovation, University of Nebraska- Lincoln, Lincoln, NE.In many fungal species (including Saccharomyces, Candida, Schizosaccharomyces and related genii), genes involved in successive steps of a metabolicpathway are often physically clustered in the genomes. Within genes involved in the Leloir pathway for galactose catabolism, such clustering is consideredto facilitate niche adaptation via rapid gene inactivation. This pathway involves three structural genes - GAL1, a galactokinase, GAL7 a uridylyl transferaseand GAL10, a bifunctional protein with two epimerase domains. The products of the GAL80, GAL4 and GAL3 genes - a co-repressor, activator and coactivator- cooperatively regulate expression of the structural genes. GAL1 and GAL3 are highly similar (>90% identity) and likely arose from an ancientduplication event. GAL1, 7 and 10 are known to cluster in many divergent fungal lineages, including Saccharomyces, Candida, Schizosaccharomyces, andCryptococcus. To further investigate potential syntenic patterns in a wider range of fungal lineages including filamentous species, we identifiedorthologous GAL proteins from over 60 fungi. An initial set of orthologue candidates was generated using a combination of BLAST, reciprocal BLAST andprofile hidden Markov model searches. Sequences meeting the percent identity and coverage thresholds established for each protein were subsequentlyaligned using MAFFT. We then reconstructed maximum likelihood phylogenies and, where necessary, compared predicted secondary structures toproduce the orthologue dataset. Location information was obtained from the respective source database (NCBI, JGI or the BROAD Institute). Weconfirmed that GAL1, GAL7 and GAL10 are not clustered in all 53 species of filamentous fungi we studied. While in 7 species closely related to S. cerevisiaeas well as C. albicans, as previously reported, the two GAL10 domains exist as a single fused protein, they exist as separate proteins in Yarrowia lipolyticaand not at all in Ashbya gossypii. In all filamentous fungi we examined, these domains exist independently. We found widespread duplication of bothdomains, and are examining the evolutionary origins of GAL10 proteins and the timing of domain duplication and acquisition events. As GAL10 proteinsparticipate in both the first and final steps of the Leloir pathway, such duplication may promote catabolic efficiency.733. Meiotic Drive: A Single Gene Conferring Killing and Resistance in Fungal Spore Killer. Pierre Grognet 1,2* , Fabienne Malagnac 1,2 , Hervé Lalucque 1,2 ,Philippe Silar 1,2 . 1) Univ Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain, 75205 Paris CEDEX 13 France; 2) UnivParis Sud, Institut de Génétique et Microbiologie, Bât. 400, 91405 Orsay cedex, France.Meiotic drives (MD) are nuclear genetic loci ubiquitous in eukaryotic genomes that cheat the Mendel laws by distorting segregation in their favor. Allknown MD are composed of at least two linked genes, the distorter that acts as a toxin by disrupting the formation of gametes, and the responder thatacts as an antitoxin and protects from the deleterious distorter effects. In fungi, MDs are known as Spore Killers (SK). In the model ascomycete Podosporaanserina, MD has been associated with deleterious effect during ascospore formation of the Het-s prion and in Neurospora crassa a resistance gene(responder) to the Sk-2 and Sk-3 distorters has been identified. MDs are easily studied in P. anserina thanks to the ascus structure as SKs are identified bythe presence of 2-spored asci in crosses between strains. Here, we identify and characterize by targeted deletion in P. anserina Spok1 and Spok2, two MD300