Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS232. Spliceosome twintrons ( “stwintrons”) revealed by fungal nuclear genomes. Michel Flipphi 1 , Erzsébet Fekete 1 , Claudio Scazzocchio 2 , LeventeKaraffa 1 . 1) Department of Biochemical Engineering, University of Debrecen, H-4010, Debrecen, Hungary; 2) Department of Microbiology, Imperial CollegeLondon, London SW7 2AZ, UK.The spliceosome is an RNA/protein complex, responsible for intron excision in eukaryotic genes. In mitochondria and plastids intron excision does notinvolve the spliceosome. For a class of chloroplast introns (II and III) "introns within introns” (twintrons) have been described. The removal of the internalintron is necessary for the excision of the external intron, and thus RNA maturation. Analogous structures have not been described for splicesomal introns.We have predicted four putative instances of “introns within introns” in nuclear genes of fungi. We call these “swtintrons” for “spliceosomal twin introns”.Putative stwitrons show a variable phylogenetic distribution. The presence of the internal intron predicts specific splicing intermediates. We haveexperimentally confirmed the existence of the predicted intermediate for the splicing of an RNA encoding a putative cyclic imidine-hydrolase of Fusariumverticillioides (Sordariomycetes, Hypocreales), where the internal intron interrupts the donor sequence between the first and second nucleotide andpredicted an analogous structure for a gene encoding a sugar transporter in two Magnaportacea. In the bioDA gene (encoding an enzyme catalysing twosteps of biotin biosynthesis of the Sordariales, an internal intron, predicted to interrupt a donor sequence of an intron between the second and thirdnucleotide has been confirmed by isolation of the splicing intermediate. In the fourth instance the putative internal intron disrupts the donor sequencebetween the fourth and fifth nucleotide of the 5’ sequence. In this instance, the presence of the internal intron was disproved, revealing an unsuspectedcase of alternative splicing.233. NGS data revealed that the NSDA sterile mutant contains a mutation in the SCF ubiquitin ligase subunit gene, culC, in Aspergillus nidulans. Dong-Soon Oh 1 , Dong-Min Han 2 , Masayuki Machida 3 , Kap-Hoon Han 1 . 1) Dept Pharmaceutical Engineering, Woosuk Univ, Wanju, Korea; 2) Division of LifeScience, Wonkwang University, Iksan, Korea; 3) Bioproduction Research Institute, Hokkaido Center, National Institute of Advanced Industrial Science andTechnology (AIST), Sapporo, Japan.Sexual development and fruiting body production of fungi play pivotal roles in production of ascospores by meiosis as well as adaptation of variousenvironmental changes. In a homothallic fungus Aspergillus nidulans, many environmental factors and genes affecting sexual development have beenelucidated. One of the first and important attempts for understanding the sexual development of A. nidulans was isolation of NSD, BSD and ASD mutants,which are defective in the process. Among them, NSD mutants are divided into four different complementation groups, NSDA-D, and two of the mutants,NSDC and NSDD, have already been characterized about the responsible genes, nsdC and nsdD and their functions. However, nsdA and nsdB mutations areremained to be unveiled. Since classical complementation experiments were not successful, we analyzed the whole genome sequence of NSDA mutantobtained from Next Generation Sequencing (NGS) to identify the nsdA4 mutation. As a result, we found three NSDA mutant-specific mutations andconfirmed the mutations by PCR followed by sequencing analysis. One of the mutations was found in AN3939 locus which encodes SCF ubiquitin ligasesubunit CulC. The mutation was G to T transversion, making D468Y amino acid residue change. Since the COP9 signalosome and ubiquitin ligase playimportant roles in fungal development, this mutation could be the correct nsdA4 mutation responsible for the sterile NSDA mutant phenotype. However,since two more mutant-specific mutations were also found in NSDA, detailed genetic characterization and mutation analyses will have to be performed.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No.2012R1A1A4A01012864).234. Whole genome sequencing of two Aspergillus oryzae strains isolated from Meju, a traditional brick of dried fermented soybean, in Korea. Dong-Soon Oh 1 , Seung-Bum Hong 2 , Jong-Hwa Kim 1 , Goro Terai 3 , Hiroko Hagiwara 3 , Masayuki Machida 3 , Kap-Hoon Han 1 . 1) Dept of Pharmaceutical Engineering,Woosuk Univ, Wanju, Korea; 2) Korean Agricultural Culture Collection, NIAB, Korea; 3) Bioproduction Research Institute, Hokkaido Center, NationalInstitute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan.In Korea, various Aspergillus oryzae-like fungi are generally regarded as one of causal agents of Korean Meju, a soybean brick for soybean paste,fermentation. Since the fungal strain plays important roles in Japanese fermented food, A. oryzae type strain of Japan, RIB40, has been sequenced andanalyzed in detail. Despite the importance of the A. oryzae strains in Korean fermented food, not many fungal strains have been isolated from fermentedfoods as well as Meju and the characteristics of the fungi isolated from Meju have not been elucidated so far, especially in molecular genetics andgenomics level. In this study, we tried to reveal the differences between Japanese and Korean A. oryzae strains by characterizing the whole genomestructure and their features. The whole genome sequence of two A. oryzae-like fungi, which were isolated from Korean Meju by Korean AgriculturalCulture Collection (KACC), were obtained by Next Generation Sequencing. Comparison of the genome sequences between RIB40 and Korean isolates byusing ortholog and homolog analyses revealed that, in one of the Korean isolates, about 50 kb subtelomeric region of chromosome III, where the aflatoxingene cluster located, was deleted, suggesting that chromosome deletion have been occurred inside the genome of the same species. Not only theaflatoxin gene cluster but also the other regions were modified in the Korean isolates. Gene annotation analysis and characteristics including those inrelation to closely related species Aspergillus flavus will be discussed.235. Systematic analysis of the uncharacterized genes, which widely conserved among filamentous fungi, in Aspergillus oryzae. N. Imaru 1,2 , F. Senoo 1,2 ,Y. Ikeda 1 , S. Terado 1,2 , K. Iwashita 1,2 . 1) National Research Institute of Brewing, Higashihiroshima, Hiroshima, Japan; 2) AdSM, Hiroshima Univ.,Higashihiroshima, Hiroshima, Japan.The genome sequences of Aspergillus oryzae revealed huge number of uncharacterized genes, which were occupied about 50% of A. oryzae genes. Mostof these genes were widely conserved among other Aspergillus species and filamentous fungi, but not found in other organisms. Moreover, severalgenome array analysis revealed that some of these genes were highly expressed in various conditions, such as liquid or solid-state cultivations. In thiswork, we designated these gene as cff (Conserved among Filamentous fungi and Function unknown genes) genes. The analysis of the functions of thesecffgenes will be important to reveal the novel molecular mechanisms which conserved among filamentous fungi. In this context, we constructed cff genesdisruptants library and analyzed the phenotype of these cff disruptants to examine the function of the genes and to identified new drug or breeding targetgenes. First of all, we isolated function unknown genes according to KOG category of A. oryzae genome database and further selected the genes that areconserved at least 7 species among 14 filamentous fungi as the cff candidate genes. Then we further examined several database, such as Swiss plot, AspGDetc., to verify the function-unknown then decided cff genes. From these cff genes, we performed the disruption of the highly expressed cff 147 genes andobtained 130 cff genes disruptants including 9 heterokaryon type disruptants. We observed the morphological phenotype of these cff genes disruptantson the minimal medium and natural medium using three serial powder plates, as a model assay of industrial conditions. As the result, some disruptantsshowed characteristic phenotypes in the hyphae growth and the conidiation. Furthermore, we examine the drug sensitivity of these disruptants usinghydroxyurea, camptothecin, micafungin et. al.. As the results, significant growth inhibition was observed in some disruptants, while some disruptantshown slight drug resistant. Now we are going to examine stress responses and second metabolite productions. We will further analyses the detail178