Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSresistance mutations. Xiaoxiao Sean He, Shengnan Jill Li, Susan Kaminskyj. Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.Systemic fungal infections are estimated to contribute to ~10% of hospital deaths. Systemic fungal infections are most dangerous for the young, the old,and the already sick, since their immune systems are less vigorous. Most antifungal drugs in current clinical use target ergosterol (polyenes) or theergosterol biosynthetic pathway (azoles and allylamines). Drugs against beta-glucan synthesis (echinocandins) are effective against aspergillosis andcandidaisis. The use of compounds that target fungal enzymes inevitably leads to the development and natural selection of drug resistant fungal strains.Not only are the anti-fungal drugs in current clinical use losing efficacy in some situations, but in addition the high level of conservation between animaland fungal physiology leaves relatively few relevant targets to explore. However, it is likely that for any drug-enzyme combination there will be relativelyfew mutations that could increase drug resistance while still maintaining enzyme function. We are using Aspergillus nidulans as an experimental modelsystem to assess the number and identity of mutations that lead to drug resistance. As proof of concept, we grew wild type A. nidulans on replicate platescontaining a sub-lethal concentration of Calcofluor. These developed fast-growing sectors beginning at ~ 5 d (70 rounds of mitosis). Preliminary resultsshow that many of these sectors harboured heritable, single-gene mutations. To date, mutated genes that confer robust, heritable resistance to Calcofluorthat were identified by next generation sequencing have roles in cell wall synthesis, cell wall integrity regulation, or drug detoxification. We suggest thisstrategy will be useful for predicting genetically-mediated anti fungal resistance adaptation and help us to be ahead in the drug-resistance arms race.88. Aspergillus nidulans cell walls lacking galactofuranose are more susceptible to glucan degrading enzymes. Biplab Paul 1 , Tanya Dahms 1 , SusanKaminskyj 2 . 1) Dept Chemistry and Biochemistry, Univ Regina, Regina, SK, Canada; 2) Dept Biology, Univ Saskatchewan, Saskatoon, SK, Canada.The cell wall of Aspergillus is a dynamic organ, consisting of a semi-permeable network of mannoprotein, and alpha- and beta-glucans. Thesecomponents are remodeled as fungal cell grows and responds to its environments. By weight, fungal walls are estimated to be 35-45% alpha-(1,3)-glucan,20-35% beta-(1,3)-glucan, 20-25% galactomannan, 7-15% chitin (beta-1,4-glucan), and 4% beta-(1,6)-glucan. Evidence from literature sources suggest thatthe Aspergillus wall 'core' is chitin and galactomannan linked to beta-1,6- and beta-1,6-glucan. Galactofuranose (Gal-f) appears to play a central role inAspergillus cell wall maturation. Previously, we showed that Gal-f biosynthesis is important for wild type chemical, physical, structural properties of the A.nidulans cell wall. We propose that the lack of Gal-f disrupts the proper packing of cell wall components, giving rise to more disordered surface subunitsand so to greater deformability. Here, we show results from an investigation of the susceptibility of Aspergillus Gal-f biosynthesis deletion strains to glucandegrading enzyme using atomic force microscopy. Topographic images of glucanase- and laminarinase-treated wildtype strains suggest that glucan is atleast one component of the cell surface subunits. Strains that lacked Gal-f were more susceptible to beta-1,3-glucanase.89. The GATA-type transcription factor NsdD is a key regulator of conidiation and secondary metabolism in Aspergillus. Mi-Kyung Lee 1 , Nak-Jung Kwon 1 ,Im-Soon Lee 2 , Jae-Hyuk Yu 1 . 1) Bacteriology, University of Wisconsin Madison, MADISON, WI, USA; 2) Department of Biological Sciences, KonkukUniversity, Seoul, Republic of Korea.Asexual development (conidiation for higher fungi) is the most common reproductive mode of many fungi; yet, its regulatory mechanisms remain to beunderstood. In this study, we carried out a multi-copy based genetic screen in the absence of the repressor of conidiation sfgA, which is designed toidentify a new set of negative regulator(s) of conidiation. Among over 100,000 colonies, 45 transformants showing altered conidiation were isolated, ofwhich 10 defined the nsdD gene (AN3152), a key activator of sexual fruiting. The others have defined AN7507, AN2009, AN1652, AN5833 and AN9141. Aseries of verification, genetic and mycotoxin analyses revealed that only NsdD is a true negative regulator of brlA (an essential activator of conidiation) andconidiation, and that NsdD acts downstream of fluG and flbA~E, but upstream of brlA. The removal of NsdD was sufficient to cause hyper-activeconidiation even in liquid submerged culture, as well as early and prolonged activation of brlA, suggesting that NsdD is indeed a key repressor of brlA andconidiation. Moreover, the deletion of nsdD results in hyper-active conidiation and altered production of mycotoxins in the opportunistic human pathogenAspergillus fumigatus and the aflatoxin-producing human/plant pathogen Aspergillus flavus. Importantly, we have discovered that nsdD encodes twodifferentially expressed mRNAs and polypeptides (b and a). Finally, the subsequent transient promoter analysis using the brlA promoter::luciferase fusionconstructs have revealed that NsdD negatively regulates the brlAb promoter activity. In summary, NsdD is a key negative regulator of conidiation actingdirect upstream of brlA in A. nidulans, and couples conidiation and mycotoxin biosynthesis in Aspergilli.90. THE velvet regulators in Aspergilli. Heesoo Park, JJae-Hyuk Yu. Bacteriology, University of Wisconsin Madison, Madison, WI.The velvet regulators are the key players coordinating fungal growth, differentiation and secondary metabolism in response to various internal andexternal cues. All velvet family proteins contain the conserved velvet homology motif (~190 a.a.), and define a novel class of fungal specific transcriptionfactors with the DNA binding ability. Some velvet regulators form time and/or cell type specific complexes with other velvet regulators or non-velvetproteins. These complexes play differential roles in regulating growth, development, sporogenesis and toxigenesis. Among the velvet complexes, the VelB-VosA hetero-complex acts as a functional unit conferring the completion of sporogenesis (focal trehalose biogenesis and spores wall completion), and thelong-term viability of spore, and the attenuation of conidial germination in the model filamentous fungus Aspergillus nidulans. Both velB and vosA areactivated by AbaA in developing cells, and the VelB-VosA complex plays a dual role in activating genes associated with spore maturation and in exertingnegative feedback regulation of developmental genes. Interestingly, the VelB-VosA complex plays similar yet somewhat distinct roles in spore maturation,dormancy and germination in Aspergillus fumigatus and Aspergillus flavus. A comprehensive model depicting the roles of the velvet regulators in aspergilliis presented.91. Coordinated regulation of asexual development, cell death and autolysis by the C2H2 zinc finger transcription factor BrlA in Aspergillus nidulans .István Pócsi 1 , Jae-Hyuk Yu 2 , Tamás Emri 1 . 1) Department of Microbial Biotechnology and Cell Biology, University of Debrecen, Debrecen, Hungary; 2)Departments of Bacteriology and Genetics, University of Wisconsin, Madison, WI, USA.Carbon starvation elicited in submerged cultures of Aspergillus nidulans triggers all various physiological responses affecting cell wall composition, stresstolerance, protein synthesis and primary and secondary metabolisms. Particularly, function of vacuoles and endoplasmic reticulum is drastically affectedleading to the re-utilization of cellular biopolymers through macroautophagy and the removal of damaged cells by apoptosis. Autolytic cell walldegradation is also an integrant part of this highly complicated and delicate regulatory process. Importantly, although the development of conidiophores isinitiated in carbon-starving submerged cultures, these structures are underdeveloped and only simple conidia are observable. There is an increasing bodyof evidence supporting the idea that the transcription factor BrlA, a well-studied central regulator of conidiation in aspergilli, is one of the most importantmaster controllers orchestrating development and autolysis in submerged culture of aspergilli. Major processes subjected to BrlA-dependent regulationunder these conditions include the production of autolytic enzymes, rodlet proteins and melanins. In fungal biology, the concerted and well-balancedregulation of conidiogenesis, cell death and autolysis is of primary importance because any overproduction of cell wall hydrolases may affect cell vitalityand colony propagation rather disadvantageously. The age-dependent production of autolytic hydrolases coincides with the synthesis of antimicrobial27th Fungal Genetics Conference | 143