Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSturgor pressure and act as a focal point for cell wall degrading enzymes to penetrate the host cell. A highly strengthened cell wall is thus essential for theonset of infection. Here we present the results of our detailed biochemical analyses, using GC-MS and methylation analysis to determine the neutral sugarcomposition and glycosidic linkages of the cell wall structural carbohydrates present at these key points in the lifecycle. Having previously established anessential role for a cellulosic cell wall in appressorium production and infection of potato by P. infestans (Grenville-Briggs et al 2008), we are now workingto elucidate the precise functions of the individual cellulose synthase (CesA) genes. Silencing each CesA using RNAi reveals overlapping functions withsubtle differences in phenotype. These results will be presented. Since the genome of P. infestans also contains a putative chitin synthase, but hyphal cellwalls are devoid of measurable chitin we are also investigating the role of this gene in the P. infestans cell wall and in pathogenicity and here we presentthe latest findings of this work.120. Analysis of the cell wall integrity (CWI) pathway in Ashbya gossypii. Klaus B Lengeler, Lisa Wasserström, Andrea Walther, Jürgen Wendland.Carlsberg Laboratory, Yeast Biology, DK-1799 Copenhagen V, Denmark.Fungal cells are constantly exposed to rapidly changing environmental conditions, in particular considering their osmotic potential. The cell wall takes onan important function in protecting the fungal cell from external stresses and controlling intracellular osmolarity, but it is also required to maintain regularcell shape. At the same time, cells must still be able to remodel the rigid structure of the cell wall to guarantee cell expansion during cell differentiationprocesses. While several signaling pathways contribute to the maintenance of the cell wall, it is the cell wall integrity (CWI) pathway that is most importantin regulating the remodeling of the cell wall structure during vegetative growth, morphogenesis or in response to external stresses. To characterize theCWI pathway in the filamentous ascomycete Ashbya gossypii we generated deletion mutants of several genes encoding for the most importantcomponents of the CWI pathway including potential cell surface sensors (e.g. AgWSC1), the following downstream protein kinases including a MAPKsignaling module (AgPKC1, AgBCK1, AgMKK1 and AgMPK1), and transcription factors known to be involved in CWI signaling (e.g. AgRLM1). An initialcharacterization of the corresponding mutants is presented. While a mutant in Agpkc1 shows a strong general growth defect, mutants in several othercomponents of the CWI pathway, in particular in the MAPK module, show a noticeable colony lysis phenotype. Finally, we show that the colony lysisphenotype may be useful to easily isolate recombinant proteins from A. gossypii.121. Dynamics of exocytic markers and cell wall alterations in an endocytosis mutant of Neurospora crassa. Rosa R. Mouriño-Pérez, Ramón O. Echauri-Espinosa, Arianne Ramírez-del Villar, Salomón Bartnicki-García. Microbiology Department, CICESE, Ensenada, B.C., Mexico.Morphogenesis in filamentous fungi depends principally on the establishment and maintenance of polarized growth. This is accomplished by the orderlymigration and discharge of exocytic vesicles carrying cell wall components. We have been searching for evidence that endocytosis, an opposite process,could also play a role in morphogenesis. Previously, we found that coronin deletion (Neurospora crassa mutant, Dcrn-1) causes a decrease in endocytosis(measured by the rate of uptake of FM4-64) together with marked alterations in normal hyphal growth and morphogenesis accompanied by irregularitiesin cell wall thickness. The absence of coronin destabilizes the cytoskeleton and leads to interspersed periods of polarized and isotropic growth of thehyphae. We used CRIB fused to GFP as an exocytic reporter of activated Cdc-42 and Rac-1. By confocal microscopy, we found that CRIB-GFP was present Inwild-type hyphae as a thin hemispherical cap under the apical dome, i. e. when growing in a polarized fashion and with regular hyphoid morphology. In theDcrn-1 mutant, the location of CRIB-GFP shifted between the periods of polarized and isotropic growth, it migrated to the subapical region and appearedas localized patches. Significantly, cell growth occurred in the places where the CRIB-GFP reporter accumulated, thus the erratic location of the reporter inthe Dcrn-1 mutant correlated with the morphological irregularity of the hyphae. We found that the Dcrn-1 mutant had a higher proportion of chitin thanthe WT strain (14.1% and 9.1% respectively). We also compared the relative cell wall area (TEM images) and we found a different ratio wall/cytoplasmbetween the Dcrn-1 mutant and the WT strain. In conclusion, we have found that the mutant affected in endocytosis has an an altered pattern ofexocytosis as evidenced by its distorted morphology and displaced exocytic markers. A direct cause-effect relationship between endocytosis andexocytosis remains to be established.122. Comprehensive genome-based analysis of cell wall biosynthesis in the filamentous phytopathogen Ashbya gossypii. R. Capaul 1 , M. Finlayson 1 , S.Voegeli 1 , A. I. Martinez 2 , Q. Y. Yin 3 , C. de Koster 3 , F. M. Klis 3 , P. Philippsen 1 , P. W. J. de Groot 2 . 1) Biozentrum, Molecular Microbiology, University of Basel,Klingelbergstr. 50-70, CH 4056 Basel, Switzerland; 2) Regional Center for Biomedical Research, Albacete Science & Technology Park, University of Castilla-La Mancha, Spain; 3) Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.The filamentous ascomycete Ashbya gossypii and the yeast Saccharomyces cerevisiae are phylogenetically closely related. It is not known how A. gossypiihas evolved an exclusively hyphal growth mode with very rapid apical extension requiring cell wall expansion rates that are up to 40-fold faster comparedto S. cerevisiae. The genome of A. gossypii encodes 44 putative cell wall-associated GPI proteins, 10 without a homolog in S. cerevisiae. This analysis alsorevealed amplification of several cell wall protein-encoding genes, notably CWP1. Transcriptome studies showed that one third of the CWP-encodinggenes are expressed at higher levels than ribosomal protein genes. Mass spectrometric analysis of protein extracts from purified walls of rapidly growinghyphae resulted in the identification of 14 covalently bound cell wall proteins (CWPs). Some CWPs that are common in hemiascomycetes are missing in A.gossypii. On the other hand, the chitin deacetylase Cda1/Cda2 was identified in addition to three novel proteins (Agp1, Awp1, and Sod6), all withouthomologs in baker's yeast (NOHBYs). Phenotypic analysis confirmed the importance of these NOHBYs for cell wall integrity. Interestingly, hyphal walls of A.gossypii contain very little chitin and orthologs of genes required for cell wall remodeling and degradation of septa during cell division in S. cerevisiae showlow expression or are absent. Conclusions: Loss of distinct cell wall genes, acquisition of novel genes, and amplification as well as increased expression ofevolutionary conserved fungal cell wall genes led to the evolution of fast polar surface expansion of A. gossypii hyphae.123. cAMP regulation in Neurospora crassa conidiation. Wilhelm Hansberg, Sammy Gutiérrez, Itzel Vargas, Miguel-Ángel Sarabia, Pablo Rangel. Institutode Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., México.In N. crassa, conidiation is started when an aerated liquid culture is filtered and the resulting mycelial mat is exposed to air. Three morphogenetictransitions take place: hyphae adhesion, aerial hyphae growth and conidia development [1]. Each transition is started by an unstable hyperoxidant state(HO) and results in growth arrest, autophagy, antioxidant response and an insulation process from dioxygen [2,3]. These responses stabilize the systemand growth can restart in the differentiated state. We found that ras-1 bd has increased ROS formation during conidiation resulting in increased aerialmycelium growth and increased submerged conidiation. Different ras-1 point mutations were generated that affected growth and conidiation. Only threeproteins have a predicted RAS association domain: NRC-1, the STE50p orthologue (STE50) and adenylate cyclase (AC). The Dncr-1 was more resistantwhereas the Dste50 more sensitive to added H 2O 2. The AC mutant strain cr-1 affects vegetative growth and aerial hyphae formation. Oxidative stress andRAS-1 determined partially cAMP levels during the first two HOs of the conidiation process. Higher cAMP levels than Wt were observed in ras-1 bd . In bothstrains, [cAMP] decreased within minutes at the start of the first two HOs and thereafter, as rapidly, levels recover to initial values. N. crassa has a high27th Fungal Genetics Conference | 151