Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS628. Illumina-based genetic linkage map for wheat leaf rust. David L. Joly 1,2 , Barbara Mulock 3 , Christina A. Cuomo 4 , Barry J. Saville 2 , Brent D. McCallum 3 ,Guus Bakkeren 2 . 1) Pacific Agri-Food Research Centre, Agriculutre and Agri-Food Canada, Summerland, British Columbia, Canada; 2) Forensic ScienceProgram and Environmental & Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada; 3) Cereal Research Centre, Agriculture andAgri-Food Canada, Winnipeg, MB, Canada; 4) Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142.Few genetic maps have been made for rust fungi; yet they are useful in identifying candidate loci for phenotypic traits or in unravelling chromosomalarrangements. This lack of maps is, in part, due to the obligate biotrophic nature of rusts and the difficulties in manipulating their life cycle in a way thatenables controlled crosses. Recently, the genome sequence of a wheat leaf rust (Puccinia triticina) isolate was determined and this prompted thesequencing of additional isolates using next-generation sequencing technologies. This has dramatically increased the amount of sequence informationavailable at a substantially decreased per base cost. Fifty-seven F2 progeny of a wheat leaf rust sexual cross between race 9 (SBDG) and race 161 (FBDJ)were sequenced using Illumina. In order to generate a high-resolution genetic linkage map, genome-wide single-nucleotide polymorphisms (SNPs) wereidentified. Employing the genome sequence information from the two parents and the F1 isolate, more than 25,000 SNPs were selected and used togenerate a genetic linkage map. Although they were obtained from different isolates, the genetic map and the reference genome were integrated,allowing the creation of pseudomolecules. Those represent a strong improvement over the currently fragmented status of the reference genome.Moreover, at least 9 seedling and 2 adult-plant avirulence genes were shown to segregate in this F2 population and candidate genes identified using thegenetic map are currently being investigated.629. The deletion of the Histoplasma capsulatum RYP1 homolog in Coccidioides posadasii is avirulent. M Alejandra Mandel 1,3,4 , Hien Trien 2,3,4 , AmrithaWickramage 1 , Lisa Shubitz 2,3,4 , Marc Orbach 1,3,4 . 1) School of Plant Sciences, University of Arizona, Tucson, AZ; 2) Department of Veterinary Sciences andMicrobiology, University of Arizona, Tucson, AZ; 3) The Bio5 Institute, University of Arizona, Tucson, AZ; 4) Valley Fever Center for Excellence, Tucson, AZ.Coccidioides spp. are mammalian fungal pathogens endemic to the desert southwestern US, parts of México and Central and South America that causethe respiratory disease coccidioidomycosis, or valley fever. These dimorphic fungi grow as filamentous saprotrophs in soil, but when a spore is inhaled bythe host and localizes to the lung, it switches from polar to isotropic growth resulting in the development of a spherule. In Histoplasma capsulatum, Ryp1is a master switch required for the transition from the filamentous to the infectious yeast phase, and thus is essential for virulence. We have performed awhole-gene deletion of the RYP1 homolog in Coccididoides posadasii strain Silveira to determine whether it plays a similar role in virulence in thispathogen. Phenotypic effects were observed in both the filamentous and the parasitic phases of C. posadasii. During filamentous growth, there is areduction in colony size, and defects in sporulation. The mutant is avirulent in our susceptible mouse model. Our results indicate that Ryp1 is a masterswitch in different fungal models. Although avirulent, the ryp1 mutant is not able to induce a protective response when used to vaccinate mice prior towild type infection.630. Cpkk2, a MEK from Cryphonectria parasitica is necessary for maintenance of CHV1 virus infection. M. Moretti, M. Rossi, M. Ciuffo, S. Abba', M.Turina. IVV, CNR, Torino, Italy.We have recently obtained and characterized the knock out strains of the three MEKs present in the Cryphonectria parasitica genome, Cpkk1, Cpkk2 andCpkk3, homologues of yeast Mkk1p/Mkk2p, Ste7p and Pbs2p, respectively. We tried to infect each of the knock-out strain with Cryphonectria hypovirus 1(CHV1), a mycovirus causing hypovirulence: Dcpkk1 and Dcpkk3 were easily infected by CHV1 through anastomosis, but we failed to infect Dcpkk2. Wethen showed that hyphal fusion was prevented in such knock-out strain: for this reason we attempted at infecting the Dcpkk2 strain with two alternativeprotocols that overcome the hyphal phusion impairment: stable transformation of protoplasts with a cDNA infectious clone and transfection of protoplastswith viral RNA transcripts obtained in vitro from a cDNA infectious clone. We originated infected strains with both protocols using wild-type C. parasiticaprotoplasts, whereas no stable infected strain was obtained starting from Dcpkk2 protoplasts, which, on the contrary, could be transformed with theempty vector carrying only the resistance gene for selection. Given the uniqueness of such result, we are now trying to show what is the specific molecularimpairment that prevents CHV1 maintenance in Dcpkk2 strain. A proteomic approach was undertaken using 2-DE MALDI-TOF MS/MS and shotgun coupledto LC-MS/MS to compare the WT and Dcpkk2 strains. A number of metabolic pathways are heavily impacted in the mutant. Of interest, proteins involvedin folding, transport and trafficking, are up-regulated suggesting an altered protein turnover. Defence machinery is also up-regulated, indicating that thefungus perceives a stress situation. Moreover, a strong down-regulation of proteins involved in energy production and conversion was detected, indicatinga possible reduction of the energetic metabolism. Among them are some GAPDH isoforms. Given the recent discovery of the role of GAPDH in viralreplication complexes of RNA viruses, we obtained anti-GAPDH antibodies in order to study its possible role in CHV1 viral replication.631. Deep RNAseq of wheat leaf infection by M. graminicola identifies phase-specific in planta expressed genes and varying transcriptionalcontributions of fungal chromosomes. Jason J Rudd 1 , Juliet Motteram 1 , Mark Derbyshire 1 , Keywan Hassani-Pak 2 , Bob Dietrich 3 , Arvind K Bharti 4 , Andrew DFarmer 4 , Ambrose Andongabo 2 , Mansoor Saqi 2 , Mikaël S Courbot 5 . 1) Rothamsted Research, Department of Plant Biology and Crop Science, Harpenden,Hertfordshire, AL5 2JQ, UK; 2) Rothamsted Research, Department of Computational and Systems Biology, Harpenden, Hertfordshire, AL5 2JQ, UK; 3)Syngenta Biotechnology, Inc., 3054 East Cornwallis Road, Durham, NC 27709, USA; 4) National Center for Genome Resources (NCGR), Santa Fe, NM 87505,USA; 5) Syngenta Crop Protection Münchwilen, Schaffhauserstrasse, 4332 Stein, CH.Mycosphaerella graminicola is the causal agent of Septoria tritici blotch disease of wheat. Infection of leaves by M. graminicola involves a characteristiclong period of symptomless intercellular growth of at least 8-10 days prior to the formation of necrotic leaf lesions. The genome sequence of the modelisolate of M. graminicola, IPO323, was recently published by the research community in conjunction with the JGI and has been shown to contain 21chromosomes. We have performed a deep RNAseq analysis to investigate fungal gene expression in vitro (in Czapek-Dox (CDB) and Potato Dextrose broth)and throughout phases of plant infection: day 1 (d1) germination on the leaf surface, day 4 (d4) slow growth in the absence of symptoms within the leaf,day 9 (d9) symptoms of disease become visible, day 14 fungal growth rate increases and finally day 21 when the fungus is sporulating asexually in fullynecrotic plant tissue. Sequencing was performed on the Illumina Hiseq platform. The RNA-seq data was analysed using the Tuxedo tools (Trapnell et al.,2012). Tophat2 was used to map the reads against the M. graminicola genome. Transcript abundance (in FPKM) was determined using Cufflinks. Significantchanges in transcript expression across all 21 pairwise comparisons were determined using cuffdiff (FDR