Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS716. pH-enotype array: a novel phenomics platform for filamentous fungi. Jaejin Park 1 , Yong-Hwan Lee 1,2 . 1) Department of Agricultural Biotechnology,Seoul National University, Seoul 151-921, Korea; 2) Center for Fungal Pathogenesis, Center for Fungal Genetic Resources, Plant Genomics and BreedingInstitute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.Rapid increase of genome information and application of high-throughput mutagenesis technology allowed large-scale gene characterization infilamentous fungi. However, phenotype screening is a bottleneck as it entails time-consuming and labor-intensive processes. Thus there is a demand for anovel platform for high-throughput phenotype screening. Although several microplate-based assays are currently available, their application on organismsgrowing in filamentous form has been met with considerable difficulty due to uneven distribution of cells. As a solution, we developed pH-enotype array -a new phenomics platform in filamentous fungi. This platform is based on the pH change in the culturing media, reflecting viability and physiological statusof cells. The pH in culturing media is continuously measured by a microplate spectrophotometer using two pH indicators, bromocresol purple and phenolred. The validity of the system was comprehensively evaluated with Magnaporthe oryzae strains and we confirmed that the growth responses to variousstresses or nutrient conditions can be characterized reliably within 24 hours using the optimized medium. This phenomics platform would provide a novel,high-throughput phenotype screening method in filamentous fungi and promote phenotype standardization for comparative functional genomic studies.717. GFP analysis of meiotic recombination in Neurospora Dmsh-2 homozygotes. P Jane Yeadon, Frederick J Bowring, David E A Catcheside. Sch BiologicalSci, Flinders Univ, Adelaide, South Australia, Australia.We have confirmed that, as expected for a mismatch repair gene with both vegetative and meiotic roles, the phenotype of a msh-2 deletion inNeurospora crassa is recessive. Chromatid data indicates that deletion of msh-2 increases allelic recombination at his-3 by a factor of 1.6 with no effect oncrossing over in the lys-4 to ad-3 interval in which lies both his-3 and the recombination hotspot cog. Although analysis of a small number of octads froman msh-2 deletion cross suggested that the only non-Mendelian segregation is post-meiotic, the ease by which octads can be scanned for recombinationevents under a fluorescent microscope when GFP is inserted close to cog allowed us to reveal the wide range of recombination outcomes normally hiddenby the activity of Msh-2. We report that a degree of mismatch repair is retained in the absence of Msh-2 and that recombination initiated by cog exhibits astrong bias for repair in the direction of restoration rather than conversion. In contrast to recombination events in budding yeast, symmetric heteroduplexappears to be frequent in the his-3 region, suggesting variation between recombination pathways utilised in the two species.718. Residual recombination in Neurospora crassa spo11 mutant homozygotes occurs during meiosis. Frederick J Bowring, P Jane Yeadon, David E ACatcheside. Sch Biological Sci, Flinders Univ, Adelaide, South Australia, Australia.We have previously shown that although most genomic regions of Neurospora spo11 RIP mutants lack meiotic recombination, crossing over in the his-3region persists at close to wild-type levels. However, this residual recombination could conceivably occur after meiosis, during a transient period of partialdiploidy. Using crosses heteroallelic for his-3 mutations, we have shown that in spo11 RIP homozygotes, as in wild type, stable His + progeny are generated athigh frequency. We have utilised mutations in either end of a histone H1-GFP fusion gene, inserted between the recombination hotspot cog and his-3, toshow that the frequency at which GFP + spores arise in a cross homozygous for spo11 + is comparable to the frequency of His + spores, and that glowingnuclei first appear during pachytene, as expected for a product of meiotic recombination. In similar spo11 deletion homozygotes, GFP + spores also arise athigh frequency and glowing nuclei are also first seen at pachytene. Thus, spo11 mutant homozygotes experience both crossing over and allelicrecombination during meiosis, suggesting there is a spo11-independent mechanism for initiation of recombination at his-3.719. Controlled synthesis of gold nanoparticles by Neurospora crassa extract and their SERS properties. Katrin Quester 1 , Ernestina Castro-Longoria 1 ,Miguel Avalos-Borja 2,3 , Alfredo Rafael Vilchis-Nestor 4 , Marco Antonio Camacho-López 5 . 1) Departamento de Microbiología, Centro de InvestigaciónCientífica y de Educación Superior de Ensenada. Ensenada, B.C. México; 2) Centro de Nanociencias y Nanotecnologia, UNAM. Ensenada, B.C. México; 3)División de Materiales Avanzados, IPICyT. San Luis Potosí, S.L.P. México; 4) Centro Conjunto de Investigación en Química Sustentable, UAEMex. Toluca,Estado de México, México; 5) Laboratorio de Investigación y Desarrollo de Materiales Avanzados, Sección de Espectroscopía Raman, Facultad de Química-UAEMex, Unidad Rosedal. Toluca, Estado de México, México.Nanotechnology, the study of the controlling matter of an atomic and molecular scale, has emerged as an interesting and important scientific field andthe controlled synthesis of nanostructures from different chemical composition as well as their shape, size, and dispersity are important areas of research.The so-called ‘green chemistry’ or ‘nanobiotechnology’ employs microorganisms to fabricate nanostructures and has the benefit of improving thebiocompatibility of nanomaterial, however the control over average particle size and uniform particle morphology is required but still a challenge. Thiswork bases on the use of Neurospora crassa, a non-pathogenic filamentous fungus with rapid growth rate, for the biosynthesis of gold nanoparticles ofcontrolled size and shape. Briefly, the fungal extract was incubated with the gold precursor solution at different conditions of temperature, pH and time ofreaction. The best results obtained were from incubations at 60°C; at pH 3, particles of different shapes (e.g. spheres, triangles, hexagons, pentagons,rhombs and bars) were formed while at pH 5.5 and pH 10 small quasi-spherical particles were formed with size ranges of 6 to 21 nm and 3 to 12 nm,respectively. High resolution transmission electron microscopy (HRTEM) using a FEI Tecnai F30 transmission electron microscope confirmed the crystallineand elemental character of the gold nanoparticles. The synthesized gold nanoparticles of different shapes were shown to possess excellent surfaceenhancedRaman scattering (SERS) enhancement ability relative to quasi-spherical gold nanoparticles. Small quasi-spherical nanoparticles of 3 to 12 nmenhances the Raman signals of methylene blue about 2 times, those of 6 to 23 nm enhance the Raman signals about 25 times whereas nanoparticles ofdifferent shapes with a broad size range enhances the Raman signals of methylene blue about 40 times. Results are promising and show that these goldnanoparticles might have potential applications for biological sensing and labeling systems.720. Cellulase production in Neurospora crassa. M. Reilly, L. Glass. Energy Biosciences Institute, University of California, Berkeley, CA.The filamentous fungus Neurospora crassa is a well-studied model organism that is frequently isolated from the environment in association with burnedvegetation. Enzyme activities related to the metabolism of plant cell wall material have been observed in N. crassa, but the overall cellular response of theorganism to such a recalcitrant carbon source remains poorly defined. In order to investigate the elements involved in fungal growth on cellulose, weutilized the near full genome collection of single gene knockout strains that has been generated under the auspices of the Neurospora Genome Project.Strains with deletions in loci thought likely to play a role in cellulase activity or production - including the known secretome of N. crassa when cultured onplant biomass, homologs of the Saccharomyces cerevisiae secretion apparatus, and proteins predicted to traverse the secretory pathway - were singledout for analysis. This subset of the N. crassa deletion collection was cultured on a cellulosic substrate and the levels of secreted protein and cellulaseactivity were compared to wild-type. A number of hyper- and hypo-secretion mutants have been identified. Sequence analysis suggests that while many ofthe loci encode fungal-specific proteins of undetermined functions, some of the deleted genes likely act in transcription, protein synthesis, andintracellular trafficking. Early work with the latter found that their hyper- or hypo-secretion phenotypes were a specific response to cellulose, but not27th Fungal Genetics Conference | 297