Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSThe ability of Candida albicans to colonize or invade multiple host environments requires that it rapidly adapts to different conditions. Our group hasbeen exploiting ChIP-chip and transcription profiling technologies, together with computer modeling, to provide a better understanding of selecttranscription factor (TF) networks. We used DNA microarrays to measure the changes in transcriptional profiles that occur immediately following thetransfer of C. albicans to hypoxic growth conditions. The impressive speed of this response is not compatible with current models of fungal adaptation tohypoxia that depend on the inhibition of sterol and heme biosynthesis. Functional interpretation of these profiles was achieved using Gene Set EnrichmentAnalysis, a method that determines whether defined groups of genes exhibit a statistically significant bias in their distribution within a ranked gene list.The Sit4p phosphatase, Ccr4p mRNA deacetylase and Sko1p TF were identified as novel regulators of the early hypoxic response. While cells mutated inthese regulators exhibit a delay in their transcriptional responses to hypoxia their ability to grow in the absence of oxygen is not impeded. Promoteroccupancy data on 26 TFs was combined with the profiles of 375 significantly-modulated target genes in a Network Component Analysis (NCA) to producea model of the dynamic and highly interconnected TF network that controls this process. The NCA also allowed us to observe correlations betweentemporal changes in TF activities and the expression of their respective genes, thus allowing us to identify which TFs are potentially subjected to posttranscriptionalmodifications. The TF network is centered on Tye7p and Upc2p which are associated with many of the genes that exhibit the fastest andstrongest up regulations. While Upc2p only associates with downstream promoters, Tye7p is acting as a hub, its own promoter being bound by itself and 7additional TFs. Rap1 and Ahr1 appear to function as master regulators since they bind to a greater proportion of TF gene promoters, including those ofUpc2p and Tye7p. Finally, Cbf1p, Mrr1p and Rap1p show the greatest numbers of unique gene targets. The high connectivity of these models illustratesthe challenges that lie in determining the individual contributions of specific TFs.379. Gene expression and function during invasive Candida infection. Wenjie Xu 1 , Norma Solis 2 , Carol Woolford 1 , Scott Filler 2 , Aaron Mitchell 1 . 1)Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 2) Departments of Medicine and Pathology, Harbor-UCLA, Los Angeles, CA.What genes does a pathogen express during infection? What regulatory pathways contribute to expression of those genes in vivo? Which pathogen genefunctions evoke specific host responses? These questions are beginning to be addressed for many plant pathogens, in which a lesion that is enriched forinfected tissue can be isolated readily. There have also been pioneering studies with human and animal pathogens, but they have been limited by theability to isolate infected tissue and by background problems from microarray technology. We implemented a recently developed technology, nanoStringprofiling, to investigate these long-standing questions with the fungal pathogen C. albicans in a murine model of hematogenously disseminatedcandidiasis. We used whole kidneys, a major target organ, for profiling the time course of both pathogen and host gene expression. NanoString technologyis not genome-wide, so we have selected high-priority fungal and host genes for investigation. On the pathogen side, we find that hyphal genes andneutral/alkaline pH response genes are induced early in infection, while oxidative and cell wall stress genes are induced later. These results are consistentwith the idea that the influx of neutrophils causes cell wall and oxidative stress. Among the 222 C. albicans genes that specify transcription factors, thosehighly expressed or highly up-regulated during infection are enriched for previously demonstrated virulence regulators, and also include many genes notpreviously known to govern virulence. We have profiled attenuated mutants despite their limited growth during infection, and we have found thattranscription factor target genes in vivo differ considerably from the target genes identified in vitro. Finally, we find that the host displays progressiveinduction of cytokines, pattern recognition receptors, and innate immune signaling pathways, with expression changes detectable at 12 hours postinfection,when fungal burden is extremely low. An attenuated rim101 mutant causes a muted host response, but also alters the kinetic profile to yieldprecocious induction of late host response genes. Our data allow the most detailed sketch to date of the dynamics and functions at the host-pathogeninterface during disseminated candidiasis.380. Effects of histone H3 point mutations on centromere maintenance. Steven Friedman 1 , Eric Selker 2 , Michael Freitag 1 . 1) Biochemistry & Biophysics,Oregon State University, Corvallis, OR; 2) Institute of Molecular Biology, University of Oregon, Eugene, OR.Post-translational modifications (PTM) of histone amino acid residues are known to play important roles in chromatin structure and function. InNeurospora crassa, trimethylation of histone H3 lysine 9 (H3K9me3) is essential for cytosine DNA methylation [1]. There is also evidence for a role ofH3K9me3 in the maintenance of centromeres [2]. An in-depth study of a larger set of histone H3 point mutations revealed additional recessive anddominant mutations involved in DNA methylation, including some mutations that proved lethal [3]. In this study, hH3 alleles were integrated ectopically orat the his-3 locus in the presence of a mutant hH3 allele at the endogenous hH3 locus. Here we describe a gene replacement system that allows mutanthH3 alleles to be integrated at the endogenous loci by homologous recombination, yielding hH3 replacement strains. The general approach is versatile andapplicable to studying the role of specific point mutations in other genes. We will present results on how hH3 mutations affect the deposition ofcentromere proteins (e.g., CenH3, CEN-T and CEN-C).[1] Tamaru, H. and E.U. Selker, 2001, Nature 414: 277. [2] Smith, K.M. et al. 2011, Mol. Cell Biol. 31: 2528. [3] Adhvaryu, K.K. et al. 2011, PLoS Genetics 7:e1002423..381. Analysis of the transcriptional regulation of genes involved in the synthesis and organization of the cell wall of Ustilago maydis during infection ofan alternatiive host. Angélica Mariana Robledo Briones, José Ruiz Herrera. Centro de Investigaciones y de Estudios Avanzados del IPN, Km 9.6 LibramientoNorte Carretera Irapuato-León.The cell wall is the most external structure of the cell. Its function is to protect it against the difference in osmotic pressure with the environment andprovide the morphology. The wall in fungi is made of microfibrils of structural polysaccharides (chitin and b-1,3-glucans) immersed in a matrix ofglycoproteins . Ustilago maydis is a dimorphic basidiomycota, pathogen of maize, but under axenic conditions it may infect other plants includingArabidopsis thaliana. Considering the role of the wall in the pathogenic process, we made a transcriptomic analysis of the genes involved in its structureand synthesis, and encoding secreted proteins during the infection of A. thaliana. For this study, one channel chips with high density oligonucleotide wereused. Plantlets of Arabidopsis were infected with a haploid or a diploid strain, and at intervals RNA was isolated, complementary cDNA was synthesizedand used to hybridize the microarrays. Data of microarrays allowed to identify genes involved in cell wall synthesis and organization, and encodingproteins from the secretome of U.maydis whose expression was regulated during the transition from saprophytic to pathogenic stages. These accountedto about 60 per cent; of the total of 639 genes existing in U. maydis, a proportion being slightly higher in the haploid strain. Some differences wereobserved in the regulation of these genes in the haploid and diploid strains. We observed that genes involved in N- and O- glycosylation of proteins wereup-regulated during infection. In addition, some CHS and CDA genes, and some genes involved in the synthesis of b-1,6-glucans and GPI proteins, weredifferentially regulated. A great number of genes encoding secreted proteins with a degradative function were up-regulated (more in the haploid). Thisincreased transcription may be related with degradation of the plant cell wall necessary to establish the infection. All these results demonstrate theusefulness of the Ustilago maydis-Arabidopsis thaliana pathosysthem for identification of the pathogenic mechanisms of U. maydis, and in the case of this27th Fungal Genetics Conference | 213