Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSFusarium head blight, an important disease of wheat and barley is primarily caused by Fusarium graminearum in many parts of the world. In thishomothallic ascomycete, sexual reproduction and the mating type locus play a critical role in its infection cycle because ascospores are the primaryinoculum. In this study we identified and characterized the FgMCM1 gene in F. graminearum that is orthologous to yeast MCM1 MADS-box transcriptionfactor. Deletion of FgMCM1 resulted in the loss of perithecium production and pathogenicity. The Fgmcm1 mutant rarely produced conidia with abnormalmorphology and germination and was defective in response to various stresses. The FgMcm1-GFP fusion proteins localized to the nucleus and fullycomplemented the Fgmcm1 mutant. Interestingly, approximately half of the sub-cultures of the Fgmcm1 mutant often were significantly reduced ingrowth rate. These spontaneously occurred stunted subcultures had similar or more severe defects than the original Fgmcm1 mutant in most of thephenotypes. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1, Fst12, and Tup1 but not with Mat1-2-1. The Fgmcm1 mat1-1-1 double mutantwas stable, suggesting that defects of the Fgmcm1 mutant may be related to the interaction of FgMCM1 with the other MAT TF genes. The Fvmcm1mutants of F. verticillioides had similar defects but were not unstable. To further understand the instability of the Fgmcm1 mutant and slow growthsubcultures, RNA samples isolated from the wild type, original Fgmcm1 mutant, and a stunted subculture were sequenced. RNA-seq data and analyses willbe presented. Overall, our data indicate that FgMcm1 may interact with MAT locus and other transcription factor genes to regulate cell identity and fungaldevelopment and pathogenesis in F. graminearum.269. Genome sequencing of the Fusarium graminearum species complex in Korea. Haeyoung Jeong 1 , Ulrich Güldener 2 , Hee-Kyoung Kim 3 , SeunghoonLee 3 , Theresa Lee 4 , Sung-Hwan Yun 3 . 1) Systems & Synthetic Biology Research Center, KRIBB, Daejeon 305-806, South Korea; 2) Institute of Bioinformaticsand Systems Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Ingolstädter Landstrabe 1, 85764 Neuherberg,Germany; 3) Dept Med Biotech, Soonchunhyang Univ, Asan, Chungnam 336-745, South Korea; 4) Microbial Safety Team, National Academy of AgriculturalScience, RDA, Suwon 441-707, South Korea.The Fusarium graminearum (Fg) species complex, the causal agent of Fusarium head blight of small grain cereals, comprises at least 15 lineages, orphylogenetically distinct species. Among these, lineages 6 (F. asiaticum) and 7 (F. graminearum sensu stricto) are major populations of the Fg complexrecovered from rice and corn, respectively in Korea; lineages 3 (F. boothii) and 2 (F. meridonale) were also recovered from corn. The F. asiaticumpopulation is clearly different from the F. graminearum population in terms of self-fertility, trichothecenes production, and host preference. We havesequenced the genomes of 19 Fg complex isolates belonging to the 4 clades or species found in Korea using 454 pyrosequencing or Illumina Hiseqtechnologies. As a representative genome sequence for F. asiaticum, we reconstructed five linear replicons from the F. asiaticum SCKO4 strain, whichconsist of four chromosomes, each corresponding to those of the previously sequenced F. graminearum strain PH-1 along with a separated small segment(451kb). By integrating multiple gene models that included the results obtained by ab initio gene prediction tools which incorporated RNA-seq data wemanually identified a set of 12,448 protein-coding genes in SCKO4. Genome-wise comparison between SCK01 and PH-1 revealed a remarkable level ofgenomic synteny throughout the four chromosomes, but several rearrangements including inversions being located on chromosomes II and III in SCK04.Interestingly, the 451-kb fragment in SCK01 showed little sequence relatedness with the PH-1 genome. Similarly, we were able to assemble into 7 largecontigs from the genome of a representative strain (GWS2-6-3) of F. boothii. Using these three representative genomes, we have intensively analyzed andcompared the genomes of the Fg complex field isolates to provide insights into understating of evolutionary relationship among the Fg complex in Korea.270. Identification and functional analysis of virulence genes in different host-pathogenic forms of Fusarium oxysporum. P. van Dam, S.M. Schmidt, M.Rep. Molecular Phyopathology, University of Amsterdam - SILS, Amsterdam, the Netherlands.The species complex Fusarium oxysporum (Fo) represents one of the most abundant and widespread microbes of the soil microflora, including plantpathogenicstrains that, together, are able to infect a broad host range. In the tomato-pathogen Fusarium oxysporum f. sp. lycopersici (Fol), 11 smallSecreted In Xylem (SIX) effector proteins were identified. These were later shown to be encoded on Fol's mobile ‘pathogenicity-chromosome’ that can betransferred horizontally to non-pathogenic Fo strains, resulting in acquired pathogenicity. The goal of this project is to identify host-specific virulencegenes in other formae speciales of Fo.A representative set of isolates from different formae speciales, vegetative-compatibility groups and races will be selected for genome sequencing. Thedevelopment of a comparative genomics bioinformatics pipeline, relying on sequence homology and specific patterns in the promoter regions (e.g.transposable elements) for identification of putative effector genes in newly sequenced F. oxysporum genomes takes a central position in our strategy.Using this approach, several putative effector genes were already identified in a strain of Fo f. sp. melonis, which infects muskmelon.In combination with functional analysis of candidate effector genes, we want to compare the sets of virulence genes of formae speciales that infectmembers of the Cucurbitaceae and Solanaceae families, such as cucumber, muskmelon, watermelon, tobacco, sweet potato and eggplant. Newlydiscovered virulence genes can be used as molecular markers for diagnostic purposes. Additionally, phylogenetic analysis of virulence genes across formaespeciales will help to reconstruct the evolution of host-specific pathogenicity in Fo, and the dynamics of the mobile accessory genome.271. Protocol for generating gene knock-out transformants of the fungal pathogen Verticillium albo-atrum. M. Flajsman, S. Mandelc, B. Javornik. Univ ofLjubljana, Ljubljana, Slovenia.A protocol for generating knock-outs of Verticillium albo-atrum, which is a destructive soilborne fungal pathogen that causes vascular wilt diseases, wassuccessfully established. V. albo-atrum, along with V. Dahliae, is a significant source of crop plant disease, since between them they infect a broadspectrum of host species, from ornamental trees to major crops such as potato, tomato, cotton, tobacco and hop. The genom of V. albo-atrum has alreadybeen sequenced. Translation of genome sequence information into biological functions is therefore possible. One of the most powerful approaches fordissecting the gene function in phytopathogens is the study of the phenotypes of mutants in which a genomic locus has been altered by insertion (genedisruption) or replacement (gene replacement) with heterologous DNA. This is a high throughput reverse genetics approach, which greatly contributes tounderstanding the gene function of fungal pathogens. Our protocol for generating knock-outs of the fungal pathogen V. albo-atrum comprises twomethods: the creation of knock-out plasmids by the USER Friendly cloning technique and transformation of the fungal pathogen by Agrobacteriumtumefaciens-mediated transformation (ATMT). Knock-out strains of V. albo-atrum were made by site directed modifications of the pathogen genome bymeans of homologous recombination and achieved by introducing a DNA fragment containing two homologous recombination sequences flanking aselection marker. pRF-HU2 plasmid, containing a hygromycin resistance gene, was used for USER Friendly cloning of knock-out plasmids. Two PCRamplicons, containing homologous recombination sequences flanking a deletion gene, were inserted into the vector, which was used to transform E. colicells and isolated plasmids were electroporated into A. tumefaciens. V. albo-atrum knock-outs were generated by ATMT. Knock-out strains for two genesfound to be highly expressed at the protein level in the xylem of infected hop plants, were generated. V. albo-atrum knock-out transformants were verifiedby PCR testing and Southern blot analysis, which confirmed that deletion of the target gene had been successful. This is the first report, to our knowledge,of the creation of V. albo-atrum gene knock-outs. It demonstrates that knock-out transformants of this fungal pathogen can be efficiently made.27th Fungal Genetics Conference | 187