Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSSignaling pathways are important in coordinating fungal cellular processes required for stress resistance, development and pathogenicity. The Mps1 MAPkinase pathway of Magnaporthe oryzae is involved in cell wall integrity, sporulation and pathogenicity. Dmps1 mutants displayed an abnormal mycelialgrowth (reduced aerial hyphae and melanisation), did not sporulate and were non-pathogenic on plants as reported (a). Sensitivity of M. oryzae to cell walldegrading enzymes (CWDE) and cell wall inhibitors (CWI) was found to be dependent on pH. Indeed, M. oryzae cell walls display a resistance to enzymaticdegradation at pH 5, while they are sensitive at pH 6. Dmps1 loses this pH 5 induced cell wall resistance, while it is as sensitive to CWDE as wild type at pH6. M. oryzae is highly resistant to calcofluor (cell wall disorganizing agent) at pH 5 (10x) compared to pH 6. Dmps1 loses this pH 5 induced calcofluorresistance, while it is as sensitive as wild type at pH 6. M. oryzae is more sensitive (20x) to Nikkomycin Z (chitine synthase inhibitor) at pH 5 than pH 6,while sensitivity to Aculeacin (glucan synthase inhibitor) is independent of the pH. However, Dmps1 is as sensitive as wild type to these inhibitors at bothpH. We conclude that the pH 5 induced resistance of fungal cell walls to CWDE and calcofluor requires the Mps1 pathway. This also suggests that the Mps1pathway is strongly activated at pH 5 compared to pH 6. To test this hypothesis, we are assaying the phosphorylation status of Mps1 at different pH as wellas under several stress conditions and developmental stages to know when this pathway is activated. Additionally we constructed an activated allele ofMkk1, the MAPKK upstream of Mps1, placed under the control of either its own promoter (b) or the repressible pNIA1 promoter. These transformants willbe used to assess the effect of controlled activation of the Mps1 pathway on M. oryzae cellular functions. The different conditions of Mps1 pathwayactivation will be used for a comparative transcriptomic analysis of wild type and Dmps1 mutants.(a) Xu, 2000. Fungal Genet. Biol. 31:137-152.(b) Fujikawa et al., 2009. Mol. Microbiol. 73(4):553-70.241. A RNA-Seq directed functional genomics screen to identify novel cell wall genes in the hyphal tip of Neurospora crassa. Divya Sain, Lorena Rivera,Jason Stajich. Plant Pathology & Microbiology, University of California, Riverside, Riverside, CA.The cell wall is one of the most important organelles of the fungal cell and differentiates pathogenic fungi from the plants and animals they infect. Thismakes cell wall biosynthesis an excellent target for anti-fungal drugs. To identify new targets we employed a functional genomics approach informed bygene expression patterns based on RNA-Seq of the filamentous fungus Neurospora crassa. The growing tips of fungal hyphae are enriched for cell wallbiosynthesis activity proteins and transcripts (1-2). Based on this idea we sequenced RNA from the tip (1 hr growth) and colony interior (20 hr growth) ofvegetative growing culture of N. crassa. 70 genes were up-regulated in the tip (at least 5 fold) and we supplemented this list with 42 tip expressed genesfrom a study of N. crassa colony development using microarrays, where mRNA transcripts in the colony tips were enriched in functional categories relatedto cell wall growth and morphogenesis (2). We used the N. crassa knockout collection (3) to identify developmental phenotypes and under chemical stressconditions to expose sensitivity in cell wall and growth defects. Almost 60 percent of the genes were found to be sensitive to cell wall stress agents,Caspofungin (cell wall integrity inhibition) and SDS (cell wall disruption) suggesting that our gene-set was enriched for genes having a cell wall defect. Wetested these genes for defects in the hyper-osmolar stress (NaCl & Glycerol) and oxidative stress pathways as well as sexual development pathway. Wefound 20 knockout strains having defects in all or nearly all of these pathways suggesting these cell wall genes are involved in multiple pathways of growthand development of filamentous fungi. This set includes Zn-Cys transcription factors (NCU04866 & NCU04663), Glycoside Hydrolase 13 family proteins(NCU08131 & NCU08132) and genes with no annotated function (NCU04826 & NCU01254). All of these genes possess homologs in other Peziomycotinafungi. Hence our approach using gene expression selected a candidate gene-set enriched for growth processes that may be useful as targets for anti-fungaldrug development against filamentous pathogenic fungi. 1) Bartnicki-Garcia & Lippman. Science 1969; 165(3890):302-4. 2) Kasuga & Glass. Euk Cell 2008;7(9):1549-64. 3) Colot et al. PNAS 2006; 103(27):10352-7.242. Identification of centromeres in the plant pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola). Klaas Schotanus 1 , Lanelle R.Connolly 2 , Kristina M. Smith 2 , Michael Freitag 2 , Eva H. Stukenbrock 1 . 1) MPRG Fungal Biodiversity, Max-Planck-Institute for Terrestrial Microbiology,Marburg, Germany; 2) Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.Several plant pathogenic fungi contain small, apparently dispensable chromosomes, and in several cases pathogenicity genes have been identified onthese chromosomes. The ascomycete Zymoseptoria tritici has up to eight dispensable chromosomes in addition to thirteen “core” chromosomes. Duringmeiosis dispensable chromosomes are lost at elevated rates, resulting in progeny with distinct novel chromosome sets. So far little is known about the roleof these chromosomes and their evolutionary dynamics. We hypothesize that loss of dispensable chromosomes during meiosis may be correlated tounstable centromeres. Thus, one goal was to identify and characterize centromeric regions on core and dispensable chromosomes to allow us toinvestigate the underlying genetics of chromosome instability. Both core and dispensable chromosomes in the Z. tritici reference isolate IPO323 have beensequenced from telomere to telomere, yielding a unique opportunity to identify the centromeric regions in the genome. We tagged the Z. triticicentromere-specific histone 3 (CenH3) with GFP and confirmed correct insertion by Southern analyses. We demonstrate expression of GFP-tagged CenH3by western blot and epifluorescence microscopy. ZtCenH3-GFP was localized in discrete foci in interphase nuclei, but in contrast to other fungi (e.g.,Neurospora, Fusarium, Saccharomyces and Schizosaccharomyces) there are several foci per nucleus instead of a single chromocenter. We also performedchromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) on the CenH3-GFP strains. To confirm the CenH3-GFP results, we tagged twoadditional centromere proteins (CEN-B and CEN-S) with GFP. To assess stability of centromeres during mitosis and meiosis, we obtained evolved asexualprogeny of IPO323 after 50 and 100 generations and progeny from a cross. Comparison of centromeric positions in the genome of the founder strain(IPO323) versus sexual and asexual progeny will allow us to infer dynamics of centromeres on core and dispensable chromosomes, and aid in ourunderstanding of the evolutionary dynamics of dispensable chromosomes in fungal plant pathogens.243. Loss of the RNAi pathway in VGII Cryptococcus gattii sheds light on the intact system in Cryptococcus neoformans. R Blake Billmyre, Xuying Wang,Marianna Feretzaki, Joseph Heitman. Duke University, Durham, NC.Loss of RNAi in VGII Cryptococcus gattii sheds light on RNAi roles in Cryptococcus neoformans R. Blake Billmyre, Xuying Wang, Marianna Feretzaki, andJoseph Heitman RNAi is a broadly conserved homology-dependent silencing mechanism which functions to defend the genome by silencing transposonsand viral elements. The opportunistic human pathogen C. neoformans, utilizes an RNAi-dependent process to robustly silence repetitive elements duringthe sexual cycle. Interestingly, RNAi components have been broadly lost from the VGII subtype of the closely related sister species C. gattii. We have takena comparative genomics approach to compare the RNAi deficient genome of VGII C. gattii with the RNAi proficient genomes of VGI C. gattii and serotypesA and D C. neoformans. This approach has identified a total of fourteen gene losses or truncations of otherwise conserved genes in VGII, including three ofthe known canonical RNAi components. Two of the remaining eleven genes have been shown to have a role in the sex-induced silencing pathway in C.neoformans var. grubii, despite a lack of homology with previously identified RNAi components in other organisms. One of these genes CPR2, waspreviously studied in our lab as a constitutively active G-protein coupled pheromone receptor. cpr2D also confers a moderate defect in sex-inducedsilencing, but no defect in silencing during vegetative growth. Similarly, the zinc finger factor Znf3 was previously identified in our lab and here was180