1 The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last oxidative reaction in the Neurospora carotenoid pathway.
Alejandro F. Estrada1, Loubna Youssar1, Daniel Scherzinger2, Salim Al- Babili2, and Javier Avalos1 1Departament of Genetics, Faculty of Biology, University of Seville, E-41012 Seville, Spain. 2Faculty of Biology, Albert-Ludwigs University of Freiburg, Schaenzlestr. 1, D-79104 Freiburg, Germany.

The surface cultures of Neurospora are orange because of the accumulation of the acidic apocarotenoid neuroporaxanthin and variable amounts of precursor carotenes. The enzymes identified so far in the Neurospora carotenoid pathway, Al 1, Al 2, Al 3 and Cao 2, lead to the synthesis of beta apo 4´ carotenal, the aldehyde version of neurosporaxanthin. The yellow mutant, ylo 1, lacks this xanthophyll and accumulates a mixture of carotene intermediates, but the biochemical basis of this phenotype has been elusive. Based on former genetic data and unpublished observations, we identified the gene ylo-1 in the Neurospora genome and demonstrated that it is responsible for the yellow phenotype by complementation. Accordingly, two independent yellow strains contained mutated ylo-1alleles. The predicted YLO-1 protein belongs to the aldehyde dehydrogenase family, and contains a presumptive transmembrane domain in its carboxy-end. In contrast to the precedent structural genes of the carotenoid pathway, light does not induce the synthesis of ylo-1 mRNA. To investigate its enzymatic activity, the ylo-1 gene was expressed in E. coliand the YLO-1 protein was purified. In vitro incubation of YLO-1 with beta apo 4´ carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. YLO-1 was also active on shorter aldehyde carotenoids, including acyclic ones, such as 8'-apo-lycopenal, indicating the irrelevance of the cycled end of the molecule for substrate recognition. The identification of YLO-1 closes the set of enzymes required to produce neurosporanthin in Neurospora.

2 Functional characterization of phosphorylation sites in White Collar Complex.
Gencer Sancar, Erik Malzahn and Michael Brunner. University of Heidelberg Biochemistry Center, Im Neuenheimer Feld328, Heidelberg, Germany.

Frequency (FRQ) and the White Collar complex (WCC), consisting of the GATA-type transcription factors White Collar-1 (WC- 1) and WC-2, are essential element of interconnected negative and positive feedback loops of the circadian clock of Neurospora crassa. Posttranslational modification of clock proteins is crucial for the function of the circadian clock. In particular, the activity of the WCC is regulated by FRQ-dependent and FRQ-independent phosphorylation. Using a tandem affinity purification tag (TAP-tag) we purified the WCC to homogeneity and identified in vivo phosphorylation sites in WC-1 and in WC-2 by mass spectrometry. Site directed mutagenesis of newly identified phosphorylation sites in WC-1 alters period and phase of the conidiation rhythm of Neurospora.

3 Genetic and Functional Characterization of Three K+ Transport Systems in Neurospora
Alberto Rivetta, Kenneth Allen, & Clifford Slayman; Yale School of Medicine, New Haven CT

Molecular characterization of the primary active transporter for K in Neurospora requires identification of the established electrophoretic H-K symport process(1) with one product from three possible ORF's: NCU06449 (TRK1), NCU02456 (TRK2), and NCU00790 (HAK1). At our request, the 'knockout team' within the Neurospora Genome Project(2) produced homokaryon knockout strains for all three genes, and we subsequently crossed these strains to obtain all combinations of knock-outs. Combinations were checked by PCR, and growth tests were carried out on Vogel-sorbose-agar medium, plus KCl at concentrations between 0.03 mM and 100 mM. The following unequivocal results were obtained: i) deletion of all three genes (trk1 trk2 hak1) completely abolishes growth below 1 mM KCl; ii) TRK1 alone (TRK1 trk2 hak1) sustains robust growth at KCl concentrations as low as 0.1 mM, essentially identical with the wild-type control (TRK1 TRK2 HAK1); iii) HAK1 alone (trk1 trk2 HAK1) allows weak growth at 0.1 mM KCl; iv) TRK2has no influence on growth. Thus, TRK1 clearly mediates high-affinity transport, but HAK1 could be either a low-affinity system or a high-affinity system of low velocity. The overall results imply that TRK1 supplies the major current and voltage signals observed in K-starved wild-type cells(1), with perhaps a minor contribution from HAK1. Direct electrophysiological experiments are now under way. We are also exploring the use of Neurospora as an expression system for other fungal and plant K-transporters, by means of a strain (trk1 trk2 hak1 mus51) optimized for homologous recombination at the NcTRK1 locus. References: (1) Rodriguez-Navarro, et al., 1986. J. Gen. Physiol. 87: 649. (2) Colot, et al., 2006. Proc. Natl. Acad. Sci. 103:10353. [We are especially indebted for the advice and assistance of Ms. Hildur Colot.]

4 Development of new biochemistry laboratory exercises using knockout mutants in Neurospora crassa
Kelly Keenan, Matt Scarnati, Xiao Shao, and Amber Cardani, Richard Stockton College, Pomona, NJ

The goal of this project is to develop new laboratory exercises for an undergraduate biochemistry laboratory using knockout mutants created inNeurospora crassa. The goal in one lab is to identify various extracts by measuring arginase specific activity in heat treated as well as in the knockout mutant for the gene that encodes arginase. The specific activity results cannot distinguish these extracts and immunoprecipitiation was used and results showed that a commercially antibody detected a protein with the expected molecular weight of arginase in the heat treated but not the knockout mutant. The second project involves the oxidative phosphorylation and alternate oxidase pathway and uses a knockout mutant in an enzyme of the oxidative phosphorylation pathway. Enzyme assays have been developed that allow measurement of sum of oxidative phosphorylation and alternate oxidase pathway as well as each component individually. Immunoprecipitation experiments with a commercially available antibody have been used to show a protein that appears in wild type but not the knockout mutant and has the expected molecular weight. These experiments are being repeated using the Western blot method.



5 A Screen for mutations displaying rate-dependent branching in Neurospora crassa.
Michael Watters and Erik Lindamood., Valparaiso University, Valparaiso IN USA.

In a previous study of branch frequency in Neurospora crassa focused on the wild-type, no relationship between growth rate and the frequency of hyphal branching was observed. In subsequent experiments, it became clear that while this independence is valid for the wild-type and most mutant strains, it fails to hold for a subset of morphological mutants. This study distinguishes a subset of Neurospora morphological mutants for their morphological response to altered growth rate. The observed effect provides an additional method for characterizing morphological mutants. It also provides support for models of branching in which control of branching is tightly linked to mechanisms of tip growth. It further seems likely that the mutants showing a morphological response to growth rate are more likely to be involved in the homeostatic system previously proposed to govern branch initiation.


6 Calcium sequestration and transport within cells: using green and red fusion proteins to identify organelles in N. crassa.
Barry Bowman, Marija Draskovic, Emma Jean Bowman. Department of Molecular, Cell and Developmental Biology. University of California, Santa Cruz CA 95060

Calcium is an important signaling molecule in all cells. In fungi calcium has been proposed to play a key role in polar growth, although we know little about the mechanisms by which calcium is sequestered and released. Analysis of the Neurospora crassa genome led to the identification of at least 5 genes that encode putative calcium transporters (nca-1, nca-2, nca-3, pmr, and cax). We have characterized the phenotypes of strains with mutations in these genes. To investigate the cellular locations of the transporters we made use of both GFP and dsRED tags. Marker enzymes for the vacuolar network, Golgi, ER and nuclei have also been tagged with GFP and dsRED. Analysis of the fluorescent-tagged proteins in living cells shows that the organelles are part of a dynamic system of small vesicles and tubules. The data also suggest that the nuclear envelope may be a significant part of the ER in N. crassa. Different calcium transporters appear to be located in the ER, the vacuolar network and the Golgi.

7 Identification and partial characterization of Neurospora anastomosis mutants.
Angela Stout, Mash’el Al Dabbous, Julia Abdullah and Stephen J. Free; Department of Biological Sciences, University at Buffalo, Buffalo, NY;

We have been able to identify seventeen genes that are required for the process of cell fusion (anastomosis) in the filamentous fungus Neurospora crassa. Some of these genes were identified by a map and sequence strategy while others were identified from among the mutants found in the KO gene library available from the Neurospora genome project. Many of these genes encoded transcription factors and elements of a MAP kinase signal transduction pathway. There are also cell wall proteins, transmembrane proteins, and cytosolic proteins that have been identified as being required for cell fusion.

8 Expression of the Red Fluorescent Protein mCherry During the Circadian Rhythm of Neurospora crassa
E. Castro-Longoria1, M. Ferry2, S. Brody3, and S. Bartnicki-García1. 1Department of Microbiology, CICESE, Ensenada, B. C., México. 2 Department of Bioengineering, UCSD, USA. 3Division of Biology/Molecular Biology, UCSD, USA.

The circadian rhythm in Neurospora crassa is expressed by the production of conidiation bands alternated by interbands with low or null conidiation. The rhythm is regulated by a transcription/translation clock that revolves around a feedback loop involving the central frequency (frq) gene. In N. crassa, a number of clock-controlled genes (ccgs) have been identified. In this work, we describe the behavior of a band (bd) strain of N. crassa containing the ccg- 2promoter driving the production of the red fluorescent protein (RFP) mCherry (Abs/Em 587/610 nm). Colonies were grown in special chambers and scanned from the edge inward by confocal microscopy. In fully grown colonies scanned at low magnification (10x), we noticed a strong accumulation of mCherry in the conidiation bands, but not in the interbands. To trace the expression or accumulation of mCherry we scanned growing colonies at higher magnification (100x) every hour for an entire circadian cycle. During development of the interband, mCherry was detected in vegetative hyphae at the growing edge of the colony. The level of expression increased gradually and reached maximal intensity during the first 3h. The intensity remained high for the following 7h and then decreased rapidly during the next 2 h. During development of the conidiation band, the expression of mCherry in vegetative peripheral hyphae was very low during the entire phase (11h), but a strong fluorescence developed in aerial hyphae and conidia. It remains to be seen whether the observed differences in mCherry accumulation in the colony represent circadian control of gene expression or are due to differential transport of the RFP from vegetative hyphae to aerial hyphae and conidia, or both. If differential transport is excluded, do differences in mCherry expression suggest the existence of spatially different morphogenetic control of the promoter? Clearly, the Pccg-2 - mCherryNC construct is a useful tool for studying gene dynamics in aerial hyphae development and conidiogenesis of fungi and may provide a new tool for labeling proteins involved in the morphogenesis and apical growth of N. crassa.

9 Characterization of the Neurospora cell wall

Angela Stout, Abhiram Maddi, Julia Abdullah, and Stephen J. Free; Department of Biological Sciences, University at Buffalo, Buffalo, NY USA 14260;

We have identified the major cell wall proteins from different stages of the Neurospora life cycle, and demonstrated that the cell wall is a cell type-specific structure. We have focused our analysis of the cell wall on the vegetative hyphal cell wall and havecharacterized KO mutants affected in most of the identified proteins within the vegetative hyphal cell wall. We identified two cell wall proteins that appear to function as upstream elements in the MAP kinase pathway controlling cell development in Neurospora. We also found that Neurospora has gene families encoding cell wall enzymes that function in cross-linking cell wall polymers (glucans and chitin) and demonstrate that such enzymes are expressed in the cell wall. The analysis of KO mutants shows that these cell wall polymer cross-linking enzymes are necessary for the formation of a normal cell wall.

10 Withdrawn

11 Plasmid-induced senescence in Neurospora crassa triggers mitochondrial-nuclear communication
Maze B. N. Ndonwi and John C. Kennell, Department of Biology, Saint Louis University, St. Louis, Missouri 63103

Wild-type strains of Neurospora grow indefinitely and senescence (growth cessation) is generally restricted to strains that harbor certain mitochondrial plasmids. These plasmids can integrate into mitochondrial DNA (mtDNA) or over-replicate, which inhibits the synthesis of vital mitochondrial proteins. Here, we study senescence in the MS4416 strain that harbors a variant form of the Mauriceville mitochondrial retroplasmid (pMS). Senescence in MS4416 strains is associated with plasmid over-replication and pre-senescent cultures express increased levels of nuclear-encoded mitochondrial genes involved in respiratory function and mtDNA synthesis. Induction of these genes suggests that plasmid over-replication triggers a mitochondrial-nuclear communication pathway that leads to the synthesis of genes involved in mitochondrial function and biogenesis. Significantly, spontaneous isolates of MS4416 that harbor high levels of pMS plasmid, yet escape senescence, fail to induce these genes. Genetic studies of these long- lived mutants suggest that longevity is controlled by a single nuclear mutation that interferes with the expression of nuclear- encoded mitochondrial genes that are induced when mitochondrial function is compromised. Confocal microscopy analyses reveal that the abundance of mitochondria is elevated in senescent cultures, although the relative levels of active mitochondria remain the same. Interestingly, the pMS/mtDNA ratio increases in senescent cultures, while the mtDNA/nuclear DNA ratio is not elevated. Our findings support the hypothesis that senescence is associated with a mitochondrial-nuclear communication pathway that leads to the accumulation of dysfunctional mitochondria.


12 SpaNc localizes at the cellular apex of germlings and mature hyphae of Neurospora crassa
Cynthia L. Araujo-Palomares, Meritxell Riquelme and Ernestina Castro-Longoria Department of Microbiology. CICESE. Km. 107 Ctra. Tijuana-Ensenada. 22860 Ensenada, Baja California, México.

In polarized fungal cells multiple protein complexes assemble at sites of apical growth to ensure that cellular components get incorporated in to the plasma membrane and provide precursors and enzymes required for building new cell wall. One of these protein complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p, Aip3p and Bni1p. Analysis of the genome sequence of N. crassaallowed us to identify the homologues of Spa2p, Bud6p and Bni1p. To investigate the spatial and temporal distribution of the polarisome in N. crassawe labeled SpaNc (NCU03115.1) with the green fluorescent protein (GFP) and analyzed growing cells by laser scanning confocal microscopy. The distribution of SpaNc-GFP was monitored from germination phase until mature hyphae were formed. SpaNc-GFP was localized at the cellular apex during germ tube elongation phase and in mature hyphae. SpaNc-GFP was observed in the apical region of all cells as a crescent shape with higher intensity at the center. Also it was found that SpaNc is not required for septum formation, but it may be involved in the development of lateral branches. The vital dye FM4-64 was used to determine the colocalization of SpaNc with the Spitzenkörper (Spk). As the germ tube elongated, SpaNc-GFP accumulated gradually at the apex of germlings of more than 24 μm in length. Subsequently the Spk was detected, when the germlings reached about 40 µm in length. From this point on, the localization of the Spk and Spa2-GFP was identical. SpaNcmutant (FGSC #11140) cells displayed distorted hyphae with uneven constrictions, apices with a smaller Spk (1.5 + 0.3 µm; n=30) compared to wild type strain (2.3 + 0.2 µm; n=30), slower growth rate and higher number of branches. The evidence presented here strongly suggests that SpaNc is required for the stability, behavior and morphology of the Spk and maintenance of regular hyphal growth in N. crassa.


13 Confocal and Total Internal Reflection Fluorescence Microscopy (TIRFM) imaging of Fimbrin in Neurospora crassa.
Diego L. Delgado-Álvarez1, Rosa R. Mouriño-Pérez 1, Laurie G. Smith2. 1Departamento de Microbiología. Centro de Investigación Científica y de Educación Superior de Ensenada. 2Cell and Developmental Biology Department. Division of Biological Sciences. University of California, San Diego.

The actin cytoskeleton in filamentous fungi is fundamental for the establishment and maintenance of polarized growth via vesicle delivery, endocytosis, and septal formation. The various functions of actin are determined by its association to its numerous binding proteins (ABPs). Fimbrin is an ABP of the calponin homology domain family. It has two actin binding domains, and causes the formation of microfilament bundles. To determine the localization and dynamics of actin-associated fimbrin we made two fusions, one using the full length protein (Fim) and another using the second binding domain (ABD2), to the N-terminus of GFP in Neurospora crassa. Confocal imaging of Fim-GFP and ABD2-GFP revealed that Fimbrin is distributed throughout the hypha forming cortical patches, which were mainly concentrated in the subapical region forming a broad ring just below the Spitzenkörper. The number of patches diminished in the basal region. Some of the subapical patches become lagged and accumulate in sites where branching will occur. Both proteins localize at the septa, forming a transient pair of rings flanking the contractile ring. TIRFM shows that the patches are highly motile and mainly localized in the membrane.

14 Localization of gamma-tubulin in Neurospora crassa.

Rosa Ramírez-Cota 1, Robert Roberson 2, Salomón Bartnicki-García1 and Rosa Mouriño-Pérez 1 1Departamento de Microbiología. Centro de Investigación Científica y de Educación Superior de Ensenada.2School of Life Sciences. Arizona State University. Tempe, AZ., USA.

Gamma tubulin is the major microtubule (Mt) nucleator. Together with the gamma ring proteins they constitute the Microtubule- Organizing Center (MTOC) at the Mt minus end. In most fungal cells this protein complex is localized at cytoplasmic MTOCs (i.e., spindle pole bodies (SPB)). However, by immunolabeling gamma tubulin was found in the Spitzenkörper (Spk) of Allomyces macrogynus, although it has not been found in the Spk of higher fungi. In order to determine the localization of gamma-tubulin in Neurospora crassa, the protein was tagged with the green fluorescent protein (GFP). A transformant of N. crassawas constructed fusing the gamma-tub gene to the N-terminus of sgfp gene. By laser scanning confocal microscopy, we observed that gamma-tubulin was localized at SPBs. All interphase nuclei had one SPB, although in mitotic nuclei, located mostly in basal regions, two bright fluorescent spots were observed indicating a pair of SPBs. Free gamma-tubulin was also observed in the cytoplasm. Contrary to our expectation, we found that gamma-tubulin is not localized in the Spk ofN. crassa, as has been described for the Spk of A. macrogynuswhere the Spk is believed to function as an MTOC.This evidence suggests that, at molecular level, the hyphal apices of these fungi are organized differently.


15 In vivo localization of Neurospora crassa GS-1, a protein involved in beta-1,3-glucan synthesis.
Jorge Verdín, Salomón Bartnicki and Meritxell Riquelme. Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE). Km 107 Carr. Tijuana-Ensenada, Ensenada 22860, B.C., México.

GS-1 is a protein necessary for beta-1,3-glucan synthase activity and normal hyphal development of N. crassa. GS-1 shares several sequence motifs with Knr4/Smi1 from S. cerevisiae, a cell wall synthesis regulator that coordinates the action of proteins involved in cell wall maintenance and the establishment of cell polarity. To date, the role of GS-1 in beta-1,3- glucan synthesis is unknown. In order to determine the localization and dynamics of GS-1, the N. crassa gene gs-1 (NCU04189.3) was endogenously fused to the green fluorescent protein gene sgfp. Total internal reflection fluorescence (TIRF) and laser scanning confocal microscopy (LSCM) were used to analyze the transformant strain. GS-1::sGFP arranges in an apical ring which corresponds to the outer layer of the Spitzenkörper (Spk). Occasionally, the “Spitzenring” moves retrogradly and disintigrates while a new Spitzenring is assembled in the apex. In subapical regions, discrete fluorescent corpuscles move in anterograde fashion along paths that reach the Spitzenring, where GS-1::sGFP is recruited. Together with the previously reported localization of chitin synthases in the core of the Spk, these results suggest an architectural and functional stratification of the cell wall synthesis machinery within the Spk that should be considered in the models that describe hyphal growth.


16 PAC-1A DYNAMICS IN Neurospora crassa
Callejas-Negrete O. A.1, R. R. Mouriño-Pérez1, R. W. Roberson2, S. Bartnicki- García11. 1Department of the Microbiology, CICESE, Ensenada, Mexico, 2Department of Cellular and Molecular Biosciences, Arizona State University, Tempe, AZ.

The pac-1A gene of Neurospora crassa has a high identity with the A. nidulans nudF gene and the human gene lis1. These genes seem to interact with the dynein/dynactin complex at microtubules (Mts) plus end and are required for nuclear distribution and cell migration. In order to analyze the dynamics of PAC-1A in N. crassa, we recorded by confocal microscopy time-lapse movies of growing hyphae labeled with pac-1A::gfp. We also analyzed the knockout strain of pac-1A. PAC-1A was distributed with different density through the hyphal regions. Single fluorescent particles could be seen in the subapical region; as these particles moved, they lined up forming short filament-like structures. At the hyphal tip, there was a higher accumulation of PAC-1A, although it seemed to disappear when they reached apex. The fluorescent structures moved exclusively in an anterograde fashion following the microtubular cytoskeleton pathway. Fluorescence was not associated with nuclei. FRAP experiments and the fast speed of PAC-1A, indicate that the fluorescent particles move independently of the cytoplasmic flow. In cells treated with 2.5 μg ml-1of benomyl, there were disorganized small fluorescent spots of PAC-1A in the subapical region that did not reach the apex. The deletion of the pac-1A gene causes a severe effect in the cell growth and organelle distribution. The organized displacement of PAC-1A from individual particles at the base of the hypha to the filament-like pattern at the tip, suggests that PAC-1A participates in unique fashion in the dynamics of polarized growth. In ro- 3 mutant containing PAC-1A::GFP, the fluorescent filaments-like structures were not formed, an indication that the behavior of PAC-1A depends on dynactin.


17 Characterization of the nucleotide excision repair genes mus-43 and mus-44 in Neurospora crassa.
Masahito Sato, Takaharu Niki, Takeru Toko, Kouhei Suzuki, Makoto Fujimura, Akihiko Ichiishi. Dept of Life Sciences, Toyo University, Gunma, Japan.

Nucleotide excision repair (NER) is the main mechanism responsible for removing a wide range of bulky DNA lesions, including UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). We identified and characterized the RAD14 and RAD10 homolog genes in N. crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44conferred sensitivity to UV and 4NQO, but not to MMS, MNNG, camptothecin, hydroxyurea, or bleomycin. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18which belongs to a second excision repair pathway. The roles of these genes in the excision of UV damage were characterized by direct assay of CPDs and 6-4PPs. In wild-type cells, binding sites for the antibodies to both CPDs and 6-4PPs decreased rapidly with incubation time after UV irradiation. The mus-18 mutant was slow in removing both types of damage, with approximately 70% of the CPDs and 35% of the 6-4PPs remaining after 3 h. In contrast, the rate of repair of UV-damage in mus-43 and mus-44 mutants were only slightly lower than that in wild-type. Furthermore, there was no decrease in antibody binding sites in the mus-18 mus- 43 and mus-18 mus-44double mutants over the 3 h of the assay, indicating a failure to excise both types of UV damage in these strains. These results suggest that N. crassa has two redundant repair mechanisms -NER and mus-18 excision repair pathways- for UV-induced CPDs and 6-4PPs, and that the mus-18 excision repair pathway is predominant in the removal of these lesions.


18 Biochemical basis of ribonucleotide reductase – associated nonself recognition in Neurospora crassa.
Robert P. Smith, Denis Lafontaine, Kenji Wellman, Leila Haidari, and Myron L. Smith Carleton University, Ottawa, Ontario, Canada,

During the assimilative growth phase of filamentous fungi, cell fusion between conspecifics may result in the formation of heterokaryons, cells that contain genetically distinct nuclei. In Neurospora crassa, proliferation of heterokaryotic cells is governed by at least 13 genes. The het-6 locus of N. crassa contains two genes that have heterokaryon incompatibility function. One of these genes, un-24, has a dual function as it also encodes the large subunit of a class I ribonucleotide reductase (RNR), the enzyme responsible for the conversion of nucleotides into deoxynucleotides. un-24 has two alleles, Oakridge (OR) and Panama (PA), which differ in their carboxy termini. Cells that contain both OR and PA alleles, either as heterokaryons or partial diploids, are non-viable or grow slowly with an aberrant morphology. Western blot analyses of un-24incompatible colonies suggests that toxic OR-PA dimers mediate incompatibility. Interestingly, incompatibility is also observed when 63 aa of the PA C-terminus or 595 aa of the OR form are expressed in strains carrying un- 24OR or un-24PA, respectively. Substitutions of conserved redox-reactive cysteines near the C-terminus that are involved in RNR catalysis results in loss of incompatibility function. We present a detailed model and supporting data of how OR-PA dimerization results in incompatibility-associated cell death.


19 Centromere assembly and maintenance in Neurospora crassa.
Kristina M. Smith, Lanelle R. Connolly, Joseph Mendoza and Michael Freitag. Dept. of Biochem. & Biophys., Oregon State University, Corvallis, USA

Centromeres serve as platforms for the assembly of kinetochores, the attachment points for spindle microtubules that transport chromosomes into daughter nuclei during nuclear division. It remains unclear how centromeres are assembled and maintained in most eukaryotes. The histone H3 variant CenH3 is present in specialized nucleosomes that are associated with centromeric DNA, which is typically AT- and repeat-rich. The centromere-specific protein CenpC connects centromeric nucleosomes to inner kinetochore proteins. Centromeric nucleosomes also carry a unique set of posttranslational histone modifications. One of our goals is to assess the contribution of DNA sequence content versus histone variants and modifications to the placement of functional centromeres. To this end we have generated fusions of CenH3 and CenpC with fluorescent proteins and affinity tags to study their localization and function, and to isolate DNA associated with both proteins by ChIP. Both CenH3 and CenPC GFP fusions localize to a single “chromocenter” in each nucleus. We also address which forces may drive the evolution of centromeric DNA and proteins, a fundamental question in cell biology. Because CenH3 and CenpC are thought to co-evolve with rapidly changing centromeric DNA, we compared the CenH3 and CenpC genes from divergent Neurospora and Gelasinosporaspecies, and used interspecific complementation of a null CenH3 allele to determine requirements for CenH3 function. The Neurospora CenH3 amino-terminal region appears under positive selection, while the carboxy-terminus remains under strong purifying selection. Our findings support the idea that meiotic drive may be involved in centromere evolution in filamentous fungi.


20 Analysis of Protein Interactions in the Neurospora crassa Circadian Clock
Christopher L. Baker, Arminja Kettenbach, Scott A. Gerber, Jennifer J. Loros, Jay C. Dunlap Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

The Neurospora circadian clock is a biochemical oscillator comprising interconnected positive and negative feedback loops. Many of the core clock components have been identified includingfrequency (frq), white collar-1 and 2 (wc-1, wc-2), frequency-interacting RNA helicase(frh). When these, and the other proteins known to interact with them, are considered together we can describe an emerging portrait of a dynamic multi-protein complex required to accurately maintain temporal control. With advances in technology the ability to identify proteins in complex peptide mixtures is becoming a routine procedure. However, when considered at the level of a single purified protein, these methods generate large protein-protein interaction datasets that require laborious biological validation. Here we describe a subtractive proteomics strategy to identify new protein components of the Neurospora crassacircadian clock. Our hypothesis is that by separately purifying multiple known proteins with their interactors, and comparing the resulting lists, that new functional components will appear enriched on more than one list. Additionally, we developed a fast and reliable method to create C-terminal tandem tagged proteins. We have applied this method to three well characterized clock proteins. These data confirm the direct physical interaction between FRQ, WCC, and FRH as well as potentially revealing novel protein-protein interactions within the clock.


21 The Tip+ MAL-3 in Neurospora crassa.
Román-Gavilanes, A. I.1, Lew T.2, Freitag, M.2, Roberson, R. W.3 and Mouriño-Pérez, R. R.1.1Departamento de Microbiología. Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, B. C. México. 2Department of Biochemistry and Biophysics. Center for Genome Research and Biocomputing. Oregon State University. Corvallis, OR. USA. 3 School of Life Sciences. Arizona State University. Tempe, AZ. USA.

Microtuble (Mt) plus end proteins (TIPs +) regulate the polymerization and depolymerization of the Mts during dynamic instability. End binding proteins (e.g., Eb1) are a highly conserved family of Mt plus end stabilizer proteins. MAL-3 represents the member of this family in S. pombe, which sequence was used to identify the encoding gene in Neurospora crassa. MAL3 was molecularly tagged by the fusion of the mal3 gene with the sgfpgene (Mal3::sGFP). Using different fluorescent microscopy techniques (wide field epifluorescence microscopy, laser scanning confocal microscopy and total internal reflection fluorescence microscopy) we analyzed the distribution and dynamics of Mal3::sGFP in N. crassa. We observed abundant fluorescent comet-like structures in all hyphal regions. The comets had a length of 1.6 + 0.4 µm (mean + standard deviation), they moved in an anterograde manner, and had an average motility rate of 1.9 µm s-1, almost 5-folds faster than the cell growth (0.2 µm s-1). In the subapical region, the Mal3 comets displacement was bidirectional and we also observed a few instances of comets moving in retrograde direction from the apex. The latter, suggests that MAL3 supports the polymerization of Mts that are nucleated in the Spk. Membrane organelles stained with FM4-64 did not colocalized with Mal3::sGFP. Cells treated with 5 µg ml of Benomyl did not show fluorescent comets, only a punctuated distribution of the fluorescence in the subapical region and some accumulations around the nuclei. The similarity between Mal3 displacement rate and the Mts polymerization rate and the localization of this protein along the Mts indicate that Mal3 participate in the regulation of rescue during dynamic instability.


22 Development and characterization of a FREQUENCY-LUCIFERASE translational fusion to light up the core of the Neurospora crassa circadian oscillator.
Luis. F. Larrondo, Jennifer J. Loros and Jay C. Dunlap. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755.

In the past years our lab has developed a codon-optimized luciferase gene for Neurospora crassa. This reporter system has proved itself as a valuable tool to reveal known and unknown properties of the Neurospora clock (Gooch et al., 2008). In order to gain a deeper understanding of the molecular details governing circadian oscillations we sought to generate a FREQUENCY- LUCIFERASE translational fusion at the endogenous frq locus. This fusion protein reports in real time the abundance and kinetics of this central core component of the Neuropora circadian oscillator. Initial characterization of this reporter strain shows robust molecular rhythms in FRQ-LUC levels, with a period circa 24 hrs and strong light response dynamics. Using this strain we have developed a high-throughput RIP-based screening protocol to obtain new frq alleles. With this system, non- sense mutations can be effortlessly discarded and full length frq mutants can be easily selected for further characterization.


23 Live Imaging of Chitin Synthase 1 (Chs-1) Traffic in Growing Cells of Neurospora crassa
Eddy Sánchez-León 1; Meritxell Riquelme1; Salomón Bartnicki-García1 and Michael Freitag2 (1) Department of Microbiology. Center for Scientific Research and Higher Education of Ensenada. CICESE. Km 107 Ctra. Tijuana-Ensenada. Ensenada, 22860. Baja California, México. (2) Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon 97331


The biosynthesis of chitin, a minor but very important cell wall structural component, is catalyzed by a diverse group of enzymes named chitin synthases (CHS). In earlier studies we visualized the cellular localization of two CHS, CHS-3 (Class I) and CHS-6 (Class VI), tagged with the green fluorescent protein (GFP) in mature cells of Neurospora crassa. Both fusion proteins were found in specific cell structures: at the core of the Spitzenkörper (Spk), developing septa, endomembranous compartments, and small particles. To study the localization and dynamics of a class III CHS, exclusive of filamentous fungi, we fused the gfp gene to chs1, and expressed it under the control of a strong promoter (ccg1) or the native promoter. N. crassa strains (FGSC 9717; mus-51 delta::bar+; his-3; FGSC 9718, mus-51 delta::bar+) that are deficient in non-homologous end joining were used as host strains for transformation. Live imaging visualization was done by Laser Scanning Confocal Microscopy. There were no significant differences between strains expressing the fusion gene controlled by either promoter or between these strains and the strains previously studied expressing CHS-3-GFP or CHS-6-GFP under the control of ccg1. Observations with Widefield Fluorescence Microscopy revealed a cloud of putative vesicles reaching the Spk. Hyphae exposed to the actin inhibitor Cytochalasin A (100µg/ml) lost fluorescence in the Spk while fluorescent patches accumulated adjacent to the plasma membrane; cells treated with the microtubule inhibitor Benomyl (100µg/ml) retained the fluorescence of the Spk. These studies suggest that transport of CHS-1 to the Spk is dependent on the actin cytoskeleton.


24 Effects of Light on the Circadian Rhythms of Neurospora crassa.
Van Gooch, Brian Bourne, and Julie Fox. Division of Science and Math, University of Minnesota-Morris, Morris, MN 56267.

Recently an optimized firefly luciferase gene has been successfully introduced into Neurospora crassa. The frequency (frq) gene is known to be a core genetic component to the circadian rhythmicity in Neurospora and when the frq promoter is placed in front of the reporter luciferase gene, its transcriptional regulation can be easily monitored by bioluminescence. Classically, Neurospora circadian rhythms have been measured using conidial banding within race tubes in constant dark at 25C. Since conidiation is strongly stimulated by even low levels of light, then measuring the effects of light on the circadian rhythms of Neurospora has been hindered. Using this new frq-luciferase reporter system, one can now more easily monitor molecular level changes of the circadian clock even when exposed to light. Under constant light conditions, we have shown that the rhythmic activity is not expressed (as suggested by other experimenters and a phenomenon that is commonly featured in circadian systems). Entrainment using 12 hours light and 12 hours of dark show very large amplitudes and angular waveforms in comparison to free running rhythms. In addition, entrainment is demonstrated to persist even at extremely low light levels (0.27 nanomoles/sec/m2). In other experiments, we have generated a detailed phase response curve where we see that a 15 minute LED white light pulse causes strong phase shifting (type 0). Finally, we have examined in more detail the phenomenon of “light induction” whereby light stimulates the frq promoter. We have shown that the rate of light induction plateaus quickly as a function of light intensity and that the ultimate level of light induction more strongly depends upon the light duration and the phase of the cycle at which the light exposure occurs.


25 The Role of Coronin in Polarized Growth in Neurospora crassa
Echauri Espinosa R. O.1, Smith L.2, Roberson R. W.3 and Mouriño Pérez R. R.1 1Departamento de Microbiologia. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada, B. C. México. 2Section of Cell and Developmental Biology. University of California San Diego. La Jolla, CA. USA. 3School of Life Sciences. Arizona State University. Tempe, AZ. USA

Coronin is an actin binding protein that plays a major role in regulating actin patch formation. The mutation of the coroningene affects locomotion, growth and actin cytoskeleton organization. Actin is important for both polarized hyphal growth and Spitzenkörper (Spk) function. To determine the role of coronin in polarized growth and Spk function and organization, we have described the cellular features of the coronin null mutant in Neurospora crassaand compare it with the wild type (wt) strain. Growth rate of coronin mutant was around 40% slower than the rate for wt strain. The Spk size and shape of the coronin mutant was smaller with an ovoid shape. Looking at the effects of coronin mutation in other organelles, such as mitochondria, we observed that they had a spherical shape instead of the wt elongated mitochondria. The direction of the hyphal apical growth was affected showing a bumping and zig-zag morphology. Branching rate was 5-folds higher than the wt. Coronin mutant produced half of the conidia than the wt (1.5 X 105 conidia ml-1). The biomass production per day was 40 % less than the wt. The mutation of the coronin gene in N. crassais not essential but strongly reduces the growth rate, affects the direction of polarized growth, increased the frequency of branching and the defects in septal construction.



26 Amplification of glycosyl hydrolase genes in Neurospora crassa
Alan Radford, The University of Leeds, U K.

A previous paper (Radford, A. (2006) FGN. 53: 12-14) described multiple menbers of a number of glycosyl hydrolase families, and further families are now to be found in the CAZy database (Coutinho & Henrissat (1999)). Based on these, the
e-Compendium now contains representatives of thirty-seven different families of glycosyl hydrolases, with up to thirteen members of the largest class, family 61, and twenty-one of the families containing more than one representative. A "search for genes" among other filamentous fungal species databases at Broad for "glycosyl hydrolase" gives comparable numbers of hits for Fusarium, Botrytis and Magnaporthe, but rather fewer for Ustilago, Rhizopus, Candida and Puccinia. Glycosyl hydrolases have many functions, including carbon nutrition (cellulases, amylases and chitinases), wall polymer synthesis in hyphal growth, branching and conidiation, protein glycosylation in the ER and Golgi, etc., so the requirement for multiple functionally divergent glycosyl hydrolases is very ancient. Sequence alignments of families show homologies, but in general sequences are sufficiently diverse to be immune from RIP. Details of the glycosyl hydrolase families will be given, and investigations will be presented to show if RIP has had any detectable role in their evolution.


27 Characterization of the Neurospora VS ribozyme in vivo.
Andrew Keeping and Rick Collins. University of Toronto.

The Varkud satellite (VS) plasmid is found in many Neurospora strains, though no corresponding phenotype has yet been identified. The plasmid encodes a ribozyme that is abundantly transcribed, and of significant interest as a model for RNA catalysis. We have pursued a proteomics approach using difference gel electrophoresis (DIGE) in order to identify differences in the mitochondrial proteome in the presence and absence of the Varkud and VS plasmids. These differences may provide insight into the effect of the VS plasmid and its encoded ribozyme in vivo.


28 Revealing Gene Function Using Phenotypic Analysis of Neurospora crassa Knock-out Mutants
Maria Meza-Lopez, Gloria E. Turner, Richard L. Weiss, Chemistry and Biochemistry, University of California, Los Angeles

Analysis of the genome sequence of N. crassa reveals many proteins of unknown function. To understand the role of these novel proteins in the biology of filamentous fungi and further annotateN. crassa genome, knockout mutants were made using a systematic deletion of each predicted gene in the N. crassagenome. We performed basic phenotypic analysis to assess the loss of each gene on growth, morphology, linear growth rates, and asexual and sexual development in knockout mutants. We selected 20 out of 70 knockout mutants that exhibited phenotypes. The most dramatic phenotype was seen in NCU06205, a knockout mutant missing a tup-1 like protein. This protein is responsible for global transcriptional repression in the budding yeast Saccharomyces cerevisiae, mediating this process through chromatin remodeling. In N. crassa, loss of this regulatory protein slows its growth dramatically and impedes completion of its sexual cycle. Examination of information on the other missing genes revealed representation of several important classes of proteins such as transcription factors, ras proteins, chromatin remodeling factors, and DNA repair proteins. N. crassa is representative of the filamentous fungi that are responsible for agricultural and human diseases. Understanding the biology of this organism will aid researchers who are interested in preventing or controlling fungal pathogenesis and disease.

29 The on-going Neurospora e-Compendium
Alan Radford Biological Sciences, University of Leeds

The e-Compendium at newgenelist/genes/gene_list.htm maintains the data in successive printed Neurospora crassagene compilations dating back through 2001 and 1982 to Barratt et al. (1954), but presented in structured and readily searchable format. Many of the data included, including those on genes currently defined only by phenotype, are not available in the sequence database and associated annotations at the Broad Institute. Moreover, the e-Compendium continues to be developed, as further genes are added as they are described in publications, and correlations are made between sequences, functions and homologs. As of January 2008 the e- Compendium contains data on 2474 sequenced and 441 unsequenced nuclear genes (including 399 tRNA genes, 79 5s rRNA genes and many of the rRNA repeat sequences in the nucleolus organiser region) and 49 mitochondrial genes, together with comprehensive genetic and physical maps of all seven linkage groups. It continues to serve as a readily available and comprehensive reference of gene symbols and names already in use, and therefore as an essential guide for workers naming new genes, in order to avoid duplicate use and resulting ambiguities.


30 Dissecting colony development of Neurospora crassa using mRNA profiling and comparative genomics approaches
Takao Kasuga and N. Louise Glass University of California, Berkeley, 341 Koshland Hall, Berkeley, CA 94501

Colony development, including hyphal extension, branching, anastomosis, and asexual sporulation, is a fundamental part of the lifecycle of filamentous fungi, yet genetic mechanisms underlying these phenomena are poorly understood. We conducted transcriptional profiling during colony development of N. crassausing 70-mer oligomer microarrays. First, six sections representing different ages and developmental stages were excised from a 27 hour old colony and relative mRNA expression levels were inferred using Bayesian analysis. It was found that genes involved in polar growth and cellular signaling were enriched for expression at the periphery of the colony (0-1 hr old). The middle section of the colony (6-9 hr old) was engaged in biosynthesis of amino acids and nucleotides, protein synthesis and energy production. In the older part of colony (18~21 hr old), genes for proteins and peptide degradation and unclassified proteins were actively expressed. At present, less than 50% of N. crassagenes have functional annotation, which imposes the chief limitation on data analysis. We have developed an alternative method by incorporating an evolutionary approach. First, a phylogenetic distribution of each of the N. crassa genes was used to classify genes into mutually exclusive lineage specific (LS) groups using SIMAP database developed by MIPS: (1) Eukaryotes/Prokaryotes-core, (2) Dikarya-core, (3) Ascomycota-core, (4) Euascomycetes-specific, and (5) N. crassa- orphans. We then cross-examined mRNA colony profiles with LS groups. An enrichment for expression of Euascomycetes- specific genes was detected in the periphery of a colony. On the other hand, expression of Eukaryotes/Prokaryotes-core and Dikarya-core genes were enriched in middle section of a colony. In the older part of the colony where the region is engaged in conidiation, an enrichment for expression of N. crassa-orphans genes was observed.


31 Carbon sensing involves all three G protein alpha subunits and multiple putative G protein coupled receptors in N. crassa
Liande Li and Katherine A Borkovich University of California, Riverside, CA, 92521

Our previous studies demonstrate that the G protein coupled receptor (GPCR) GPR-4 and the three Galpha proteins, GNA-1, GNA- 2 and GNA-3, are all important to mass accumulation on poor carbon sources in N. crassa. We also showed that GPR-4 is required for the transient increase in cAMP levels that are observed in wild type glycerol-grown cultures after the addition of glucose. In this study, we demonstrate that the cAMP transient is lost in mutants lacking any of the three Galpha genes, supporting the need for GNA-1, GNA-2 and GNA-3 in carbon sensing. Microarrays were successfully performed for wild type, deltagna- 1 and deltagpr-4 strains. The results indicate that genes are differentially regulated in deltagna-1 and deltagpr- 4mutants under four different carbon conditions compared to the wild type. Many genes are up- or down-regulated with the same or similar patterns in both deltagna-1 and deltagpr-4 mutants, further supporting functioning of GPR-4 and GNA-1 in the same pathway for carbon sensing. We also analyzed the putative GPCRs GPR-5 and GPR-6 that are homologous to the putative nutrient sensor Stm1 from Schizosaccharomyces pombe. Highest expression of gpr-5 and gpr-6 is observed in cultures with glycerol as the carbon source. Phenotypic analysis showed that deltagpr-5 and deltagpr-6 mutants form less mass than wild type when cultured on glycerol plates, and strains lacking gpr-5 and/or gpr-6 in addition to gpr-4 have dry masses similar to deltagpr-5 or deltagpr-4 single mutants. These findings suggest that GPR-4 and GPR-5 are redundant, while GPR-6 plays a minor role in mass formation when grown on glycerol medium. Taken together, the results support the notion that all three Galpha proteins as well as the putative GPCRs GPR-4, GPR-5 and GPR-6, are involved in carbon sensing in N. crassa.


32 High throughput gene knockouts in Neurospora

Patrick D. Collopy1, Hildur V. Colot1, Gyungsoon Park2, Carol Ringelberg1, Lorena Altamirano2, Svetlana Krystofova2, Norma Gorrochotegui- Escalante1, John Jones2, Katherine A. Borkovich2, and Jay C. Dunlap1. 1Department of Genetics, Dartmouth Medical School, Hanover, NH, USA 2Department of Plant Pathology, University of California, Riverside, CA

More than 4,100 gene disruptions have been made in predicted open reading frames in the annotated Neurospora crassagenome using a high-throughput procedure as part of an on-going NIH-funded Program Project (P01)*. Knock-out (KO) cassettes were created using yeast recombinational cloning techniques and electroporation of N. crassa conidia can be performed in a 96-well format. In order to expedite molecular confirmation of KO mutant strains, we have developed a program ( that allows automated identification of the appropriate restriction enzyme to use during Southern analysis. Completed N. crassa KO mutant strains are submitted to the Fungal Genetics Stock Center after targeted gene replacements are confirmed by Southern analysis and the list of submitted strains is available at the Neurospora genome project website

( Use of these tools and our current progress in creating KO mutants will be presented.
*Colot and Park et al., 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. PNAS 103:10352-10357

33 Assembling the Neurospora crassa genome: Construction of a genomic library in a linear plasmid.
Lanelle R. Connolly, Eva Schopf, Kristina M. Smith and Michael Freitag. Dept. of Biochem. & Biophys., Oregon State University, Corvallis, USA

The complete genome sequence of Neurospora crassa has been difficult to assemble, in part because AT-rich DNA may have been lost during the preparation of traditional shotgun, cosmid, and BAC libraries. Much of the missing Neurospora sequence is expected to map to centromeric regions, as there are significant gaps between contigs that are associated with most, if not all, of the seven centromeres. Previous analyses suggested, that - as in many other eukaryotes - Neurospora centromeric regions are AT- rich and contain much repeated DNA. Unlike in mammals or plants, these regions do not harbor satellite repeats, but rather an assembly of retrotransposon relics that have undergone RIP. Various novel direct sequencing approaches promised useful to finish the Neurospora genome. Nevertheless, preliminary results suggest that new sequencing methodology alone will likely not suffice to generate finished sequence of this relatively small eukaryotic genome, simply because the assembly of AT-rich repeats proves to be a major obstacle. To understand centromere formation and maintenance, we wish to complete the assembly of the predicted centromeric regions by alternative means. To this end, we generated a Neurospora genomic library in a linear cloning vector, pJAZZ-OC. This plasmid allows the cloning and maintenance of long AT-rich fragments. Direct and inverted repeats are tolerated. Here we describe the construction and characterization of a pilot library. Preliminary results suggest that we succeeded in cloning many AT-rich inserts that should help to complete the assembly of centromeric sequences of Neurospora crassa.


34 Development of an overexpression screen for new genes involved in the circadian clock.
Hildur V. Colot, Luis F. Larrondo, Jennifer J. Loros and Jay C. Dunlap, Genetics Dept., Dartmouth Medical School, Hanover NH 03755;

With the proliferation of whole genome sequences, the use of multiple approaches for studying the functions of unkown genes has become imperative. We have been engaged in a high-throughput gene deletion project with Neurosporafor a few years and the strains thus created have proved useful to many researchers. As a companion to that project, we have undertaken an overexpression screen using many of the techniques, tools and reagents developed for the knockout project. Overexpression studies allow functional analysis of essential genes and can yield unique insights when compared with underexpression or deletion studies: In genetic terms, deletions provide loss-of-function whereas, ideally, overexpression can mimic gain-of-function alleles. With the goal of finding new genes involved in circadian clock function and/or output, we have placed numerous genes under the control of the inducible qa-2 promoter by using high-throughput gene replacement strains and techniques to replace the native promoters with the qa-2promoter. Both low expression and high/constitutive expression of a given gene can be analyzed in this system. Preliminary data indicate that there is a great deal of variability in the extent to which true overexpression of a gene can be achieved by the inducible promoter; expression levels of a variety of genes with respect to that achievable by qa-2 will be presented.


35 Soluble multiprotein complexes in Neurospora mitochondria
Diane DeAbreu and Rick Collins. University of Toronto.

We have treated highly-purified mitochondria with low concentrations of mild detergents to extract soluble multiprotein complexes. Complexes were separated by blue native gel electrophoresis; individual subunits were dissociated in a second dimension SDS gel and identified by MALDI-TOF peptide fingerprinting mass spectrometry. Hsp60, hsp70, F1 ATPase, pyruvate dehydrogenase, isocitrate dehydrogenase and several other Kreb’s cycle enzymes each were found in discrete complexes.

36 Neurospora comparative genomics using Neurospora discreta and N. tetrasperma genomes
Jason E Stajich1, Thomas J Sharpton1, Christopher Ellison1, Jeremy Schmutz2, Michael Y Zhang3, Kerrie Barry3, Igor Grigoriev3, David Jacobson1, N. Louise Glass1, Donald O Natvig4, John W Taylor1 . 1Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. 2JGI/Stanford, Stanford Human Genome Center, Palo Alto, California 94304. 3DOE Joint Genome Institute, Walnut Creek, California 94598. 4Department of Biology, University of New Mexico, Albuquerque, NM 87131

The sequencing of the Neurospora crassagenome enabled a new era of molecular and evolutionary analysis in filamentous fungi. The availability of whole genome sequences from Neurospora discreta and N. tetrasperma from the Joint Genome Institute elevates Neurosporaas a platform for evolutionary genomics. Using comparative genomics and phylogenomics we have identified gene conservation and changes among N. crassa, N. discreta and N. tetrasperma. Specifically, we identify species-specific genes, significant expansions and contractions in gene family size, and genes under positive selection. Using comparative methods with three Neurospora genomes and the outgroup species Podospora anserina and Chaetomium globosum we have constructed a refined set of Neurospora-specific and shared genes found among these filamentous fungi. In addition to genic content, we have explored the extent of genome structure conservation by cataloging syntenic sequence conservation between the taxa and determining if repeat density correlates with breaks in synteny. Finally, given its importance in N. crassa, we surveyed for signatures of RIP and gene family size in the N. discreta and N. tetraspermagenomes to ascertain the age and influence of this mechanism and the evolutionary dynamics of how RIP has impacted genome evolution.


37 The evolution of the mating-type chromosome in Neurospora tetrasperma: syntenic comparisons to Neurospora crassa and Neurospora discreta
Christopher Ellison, Thomas J Sharpton, Jason E Stajich, David Jacobson, and John W Taylor. Department of Plant and Microbial Biology, UC Berkeley, CA,

The reproductive strategy of Neurospora tetraspermainvolves the packaging of two haploid nuclei of opposite mating type into a single ascospore. This pseudohomothallic condition results in self-fertility. Blocked recombination proximal to the mating type locus during meiosis I is necessary for the correct packaging of alternative mating type alleles into a single heterokaryotic ascospore and previous genetic studies have shown that recombination is suppressed along the majority of the mating type chromosome (75% / 7Mbp). This recombination blockage has been hypothesized to be the result of a series of chromosomal rearrangements. The Joint Genome Institute has recently sequenced the N. tetrasperma genome, including both mating types (mat a [FGSC #2509] and mat A [FGSC #2508]), and the N. discreta genome (mat A [FGSC #8579]), thus allowing the opportunity to test this hypothesis. Using N. crassalinkage groups as a reference, we constructed syntenic alignments between the mating type chromosomes as well as the autosomes. After identifying regions of rearrangement we then compare the degree of rearrangement between 1) N. tetrasperma mat a and mat Achromosomes and 2) autosomes and mating type chromosomes between taxa. Finally, we place the specific locations of rearrangements in the context of the N. tetrasperma genetic map. Elucidating the degree and sites of rearrangement along the N. tetraspermamating type chromosome will lead to a better understanding of the origin of the psuedohomothallic condition as well as the evolution of sex chromosomes in plants and animals.


38 New features over the expression of Neurospora crassa genes in response to extracellular phosphate levels and pH.
Fabio M. Squina 1,3, Juliana Leal 2, Diana E. Gras, 2, Janaina S. Freitas1, Emiliana M. Silva1, Nilce M. Martinez-Rossi2, Antonio Rossi1. 1Departamento de Bioquímica e Imunologia and 2Departamento de Genética of FMRP of Universidade de São Paulo, Brazil. 3Department of Microbiology and Molecular Genetics, Oklahoma State University, USA.

Microorganisms have evolved complex signal transduction networks that enable them to adapt effectively in response to environmental changes in nutrients and extracellular pH. The phosphorus (Pi) acquisition system in Neurospora crassa includes four regulatory genes: nuc-2, preg, pgov and nuc-1. In response to signals of Pi starvation, NUC-2 inhibits the functioning of the complex PREG-PGOV allowing the translocation of transcription regulator NUC-1 into the nucleus and expression of Pi-repressible phosphatases. In an attempt to further understand the molecular responses to exogenous Pi sensing, six subtractive cDNA libraries were constructed to identify genes differentially expressed in the nuc-1RIP and nuc-2A mutant strains of N. crassagrown in low Pi, at acidic and alkaline pH. We identified genes involved in diverse cellular processes such as cellular transport, cellular metabolism, protein biosynthesis, regulation of transcription, development and signal transduction, revealing novel molecular aspects of the functioning of nuc-1 and nuc-2 genes. We also found that transcription of two heat shock protein genes is affected by growth pH while another one is overexpressed when nuc-1 gene is non functional. Financial support: FAPESP, CNPq, FAEPA and CAPES


39 Definition of the DNA-Binding Specificity of the CYS3 Transcription Factor of Neurospora crassa by Binding-site Selection.
John V. Paietta. Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH

The CYS3 transcription factor is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of a coordinately regulated group of genes involved in the acquisition and utilization of sulfur in Neurospora crassa. An approach of using binding site selection from random-sequence oligonucleotides was used to further define CYS3 binding specificity. The derived consensus binding site of AT(G/t)(g/a)C(g/a)C(C/a)AT defines a symmetrical sequence that resembles that of other bZIP proteins such as CREB and C/EBP . By comparison, CYS3 shows a greater range of binding to a central core of varied Pur-Pyr-Pur- Pyr sequences than CREB as determined by gel shift assays. The derived CYS3 consensus binding sequence was further validated by demonstrating in vivosulfur regulation using a heterologous promoter construct. The CYS3 binding site data will be useful for the genome-wide study of sulfur-regulated genes in Neurospora crassa, which has served as a model fungal sulfur control system. The overall pattern of regulation in the Neurospora crassa sulfur metabolic network based on our gene expression

40 A molecular mechanism for light-dependent conidiation: the fluffy gene, a major regulator of conidiation in Neurospora, is activated by light
Maria Olmedo and Luis M. Corrochano Departamento de Genetica, Universidad de Sevilla, Spain

Light stimulates the development of conidia in Neurospora crassa. The products of the wc genes, wc-1 and wc-2, are required for all the Neurospora responses to light, including the activation by light of conidiation. WC-1 is a blue-light photoreceptor and transcription factor that interacts with WC-2 to form a complex (WCC) that binds to the promoters of light-regulated genes. The fluffy gene is a major regulator of conidiation that encodes a 88-kDa polypeptide (FL) containing a Zn2Cys6 binuclear zinc cluster domain that is necessary and sufficient to induce conidiophore development in Neurospora. The major role of FL in conidiation prompted us to investigate the possible regulation by light of fluffy gene transcription. The transcription of gene fluffy is activated by light. Wild type mycelia exposed to 15 min of light increased fluffy mRNA accumulation four-fold, but longer light exposures reduced fluffy photoactivation due to photoadaptation. The photoactivation of fluffy is observed with blue light, but not with red light, as in other light responses of Neurospora. The threshold for fluffy photoactivation is similar to the threshold for other Neurospora light activated genes. The activation of fluffy by light was observed in other conidiation mutants of Neurospora, suggesting that light-dependent activation of fluffy does not require an active conidiation pathway. As expected, strains with mutations in wc-1 or wc-2 did not accumulate fluffy mRNA after light exposure. The promoter of fluffy contains a putative site for WCC binding at position -560 (from the transcription initiation site). We propose that light-dependent binding of WCC to the promoter of fluffy will trigger light-dependent gene transcription. The increased accumulation of FL will induce conidiophore development resulting in an increased production of conidia after light exposure. This simplified model for light-dependent conidiation in Neurospora could help to understand light-dependent conidiation in other Ascomycetes fungi.


41 A Genetic Screen for Negative Feedback Loss in Neurospora Revealed Dual Roles of FRH (FRQ Interacting RNA Helicase) in Circadian Regulation.
Mi Shi1, Christopher L. Baker1, Michael A. Collett3, Jennifer J. Loros1,2, Jay C. Dunlap1.1 Department of Genetics, 2Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755. 3New Zealand Diary Research Institute, Palmerston North, Private Bag 11029, New Zealand

A genetic screen was developed to identify clock components involved in FRQ-based negative feedback in Neurospora crassa. SNP (single nucleotide polymorphism) mapping revealed a chromosomal translocation and a missense frh mutation (named as frhLN) in an arrhythmic strain. Mutant FRHLNprotein binds tightly with FRQ, but not with WCC (white collar complex), which results in a loss of negative feedback and a dominant negative effect on the circadian clock. WCC activity is upregulated in the mutant with a constantly high expression of frq. Interestingly, WCC levels are extremely low in the mutant strain. This result indicates that FRH together with FRQ has dual roles in both negative feedback to inhibit WCC activity and positive feedback to maintain WCC stability.


42 Genome-wide characterization of light-inducible responses reveals a hierarchical light-sensing cascade in Neurospora crassa
Chen-Hui Chen, Jennifer J. Loros, Jay C. Dunlap Department of Genetics, Dartmouth Medical School


To better understand the roles of WC-1, WC-2 and other putative photoreceptors in mediation of light signals and to identify new components in light sensing cascades, we used microarrays with genome-wide coverage to characterize light-inducible transcriptional changes in Neurospora crassa. Several questions in Neurospora photobiology were addressed. First, which genes display altered expression in response to light? Second, how can we categorize light responses in general, and how are they temporarily coordinated? Third, are wc-1 and wc-2 strains truly blind to all light-inducible transcriptional changes? Are there other putative photoreceptors involved in the control of any light response at the level of transcript abundance? Previous studies have demonstrated that Neurospora is very sensitive to light stimuli and can display dramatic transcriptional changes within 15 min or less. Therefore, we sampled more intensively within the first 30 min after exposure to light, namely at 0, 5, 10, 15, 30, 60, 90, 120, 180 and 240 min after lights-on. To eliminate intrinsic systemic variation between arrays and have a direct comparison between experiments, we adopted an unsupervised hierarchical clustering approach to identify bona fide light- responsive genes. An assumption made here is that genes that are truly responsive to light should behave more similar to each other across sequential time points and different strains. Before clustering, the data were normalized so that each measurement reflects the abundance of each transcript relative to the mean value across all arrays. Genes missing more than 25% of their measurements from 90 microarrays were all excluded, which results in 5588 valid spots, providing approximately 53.8% coverage of all 10372 spots on the array. In addition, only genes have at least 4 fold changes (maximum - minimum) across 90 microarrays were selected for further clustering. Totally, 2864 spots passed these selection criteria. After clustering, 316 genes, which are about 5.7% of the total detectable genes, were identified as light-responsive due to their robust and consistent light-responsive pattern. Functional analysis further suggests that there is correspondence between the timing of transcript up regulation and gene function. Moreover, the differential regulation of early and late light responses was further elucidated by the identification of a dedicated transcription factor and motif that specifically control a subset of late light responses. Meanwhile, by comparing light responses in different photoreceptor knockout strains, our data show that most light-induced transcriptional changes in Neurospora are controlled by WC-1 and WC-2, independent of PHY-1, PHY-2, CRY and OPSIN. Intriguingly, the photoadaptation defect noted in vvd strain was observed for nearly all of the early and late-light responsive genes, providing us a unique opportunity for a completely independent validation to the genes we identified as "light responsive".


43 Regulation of the RNAi pathway in Neurospora
Heng-Chi Lee, Swati Choudhary, Mekhala Maiti and Yi Liu Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390 USA


RNA interference (RNAi) is a post-transcriptional gene silencing (PTGS) mechanism conserved from fungi to humans. The RNAi pathway is initiated by recognition of double-stranded RNAs (dsRNAs) by Dicer, which cleaves dsRNA into siRNAs. The siRNA duplexes are then loaded onto the Argonaute protein whereby one strand will be used as the template to guide the Argonaute protein to cleave the complementary mRNA sequences. We showed that the conversion of duplex siRNA into single-stranded siRNA requires the catalyitic activity of the Neurospora Argonaute protein QDE-2. We biochemically purified the argonaute protein QDE-2 protein from Neurosporaand identified QIP, an exonuclease domain-containing protein, as a QDE-2 interacting protein. The gene silencing triggered by dsRNA is greatly impaired in qip mutant. Furthermore, the exonuclease activity of QIP is required for efficiently removing passenger strand from siRNA duplex in vivo. Therefore, QIP acts as a positive regulator of RNAi pathway most likely by rapidly degrading the passenger strand. dsRNA, in addition for being the substrate of dicers, can initiate transcription-based interferon response in mammals. In Neurospora, we found that the expression of qde-2 and dcl-2, two central components of the RNAi pathway in Neurospora, are highly induced by dsRNAs. The dsRNA-induced qde-2 expression is required for normal RNAi efficiency. Genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. These results uncovered a novel dsRNA response program in fungi and suggest that RNAi is part of a broad spectrum and ancient host defense response system against viral and transposon infections. Taken together, our data show that the gene silencing by RNAi pathway in Neurospora is tightly controlled through multiple mechanisms.


44 ATF-1 transcription factor regulates the catalase cat-1 expression through CRE-element in Neurospora crassa.
Kazuhiro Yamashita, Setsuko Watanabe, Akihiko Ichiishi, Fumiyasu Fukumori, Makoto Fujimura. Faculty of Life Sciences, Toyo University, Itakura, Gunma, Japan.


CAT-1 catalase activity increases during the stationary growth phase in hyphae, and CAT-1 is highly accumulated in conidia in Neurospora crassa. The cat-1 gene expression and the enzyme activity were induced by osmotic stress and fludioxonil in the wild type, but not in the os-2 MAP kinase mutant and atf-1 transcription factor disruptant hyphae. Although CAT-1 activity in the os-2 conidia was slightly lower than that in the wild-type strain, the atf-1 conidia lacked CAT- 1 activity, suggesting that production of CAT-1 was largely dependent on the atf-1 gene. To identify its binding sequence, ATF-1 was expressed in Escherichia colias a NusA-His-tag fusion protein. The fusion protein was purified by affinity chromatography and used for the electrophoretic mobility shift assay (EMSA). ATF-1 bound strongly to a cAMP response (CRE) element of the cat-1 promoter located in the region 174 bp upstream of the initiation ATG. ATF-1 also bound to CRE in the promoter region of the ccg-1, which is regulated by OS-2 MAP kinase. These results suggest that ATF-1 regulates cat-1 expression via OS-2 activation in the stress response and also via OS-2-independent signaling during conidiation.


45 Identification of Neurospora crassa Transcription Factors Involved in Glycogen Metabolism Regulation
Gonçalves, R. D.; Cupertino, F. B.; Bertolini, M. C. Departamento de Bioquímica e Tecnologia Química, Instituto de Química, UNESP, Araraquara, SP, Brazil,


In living cells, transcription factors dynamically interact with DNA playing an essential role in gene expression, therefore regulating many cellular functions. With the availability of the Neurospora crassagenome sequence and the knowledge of the transcriptions factors encoded by the fungus genome, a collection of transcription factors knockout strains became available to the scientific community, which allows studying regulation of specific aspects. In our laboratory we have undertook a systematic search for transcription factors that affect glycogen accumulation by taking advantage of the N. crassa knockout strains collection available from the FGSC. It is already known that inN. crassa glycogen content reaches maximal level at the end of the exponential growth phase. However, under heat shock, glycogen content rapidly drops. Such glycogen decrease might result from the regulation at transcriptional level of enzymes involved in the carbohydrate metabolism, since transcription of the gene encoding glycogen synthase (gsn) decreases under this condition. In our screening the glycogen content in the N. crassamutant strains was quantified in the whole cellular extract from mycelium before and after exposure to heat shock (from 30ºC to 45ºC). Approximately 150 mutant strains belonging to different transcription factors classes were analyzed and less than 20 strains showed changes in glycogen accumulation when compared to the wild type (WT) strain. We identified transcription factors mediating regulation of glycogen metabolism during normal conditions, that is mutant strains having hypo and hyperaccumulation of glycogen and transcription factors mediating regulation after heat shock. In this latter case, we identified mutant strains having similar glycogen content before and after heat shock. The majority of the transcription factors identified were annotated as hypothetical proteins and a few of them have been already characterized at the protein level in N. crassa. Supported by FAPESP, CAPES, and CNPq


46 Circadian Rhythm Promoters for an Optimized Bioluminescent Gene in Neurospora crassa.
Julie Fox†, Luis F. Larrondo§, Van Gooch†, Jay Dunlap§, and Jennifer Loros§. †Division of Science and Math, University of Minnesota-Morris, Morris, MN 56267, §Department of Genetics, Dartmouth Medical School, Hanover, NH 03755.


The success of inserting the codon-optimized firefly bioluminescent gene, luciferase, into Neurospora crassa under the control of the circadian regulated frequency (, frq) promoter encouraged us to explore the use of other circadian promoters to control this luciferase reporter. Six new promoter-luciferase constructs were successfully created and studied in wild type, ras- 1 bd , and ras-1 bd , frq 7 Neurospora. The selected promoters included: fluffy, vivid, con-6, con-10, ccg-13 and ccg-4. These clock-controlled genes were chosen based on their underlying biology and so that different peak times of their corresponding mRNA accumulation are represented. The circadian rhythms and bioluminescent outputs of these promoter-luciferase systems seem to correspondingly reflect the function of the individual clock genes. For example, the light regulated con-10 and con-6 reporters display high amplitudes of bioluminescence and appear to be extremely light responsive; in addition, the ccg-4construct is mating type specific. These new bioluminescent constructs may advance our understanding of the molecular mechanisms underlying the transcriptional regulation of these, and others, clock-controlled genes.



47 MAP kinase signaling pathways play crucial roles in fungal development and pathogenesis.
Gyungsoon Park and Kathy Borkovich, University of California, Riverside.

In this study, we characterized three components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; mak-1, MAPK), homologous to that controlling cell wall integrity in Saccharomyces cerevisiae.

Growth of basal and aerial hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. Formation of macroconidia was almost abolished in mek-1 and mak-1 mutants and reduced in mik- 1 strain. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. mik- 1, mek- 1, and mak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAP kinase was not phosphorylated in mik-1 and mek-1 mutants indicating signal cascade among the three kinases. Interestingly, we observed increased levels of mRNA and protein for tyrosinase, an enzyme catalyzing melanin biosynthesis, in the mutants under nitrogen starvation, a condition favoring sexual differentiation. These results implicate the cell MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi.


48 Investigation of functional domains of RRG-1, a response regulator that regulates the OS-2 MAPK cascade.

Carol A. Jones and Katherine A. Borkovich. Department of Plant Pathology, University of California, Riverside, 92521,


Two component signaling pathways are found in bacteria, plants, Archaea, and fungi; however these signaling pathways are not found in humans, Drosophila or C. elegans and are believed to be absent from the animal kingdom as a whole. Two component regulatory systems contain proteins with histidine kinase (HK) and response regulator (RR) domains. Fungi contain hybrid histidine kinases (HHKs) with both histidine kinase and response regulator domains in the same protein. A conserved histidine residue in the histidine kinase domain of the HHK is autophosphorylated in response to some environmental signal. That phosphate is then transferred intramolecularly to an aspartate in the response regulator domain of the HHK. From the RR domain in the HHK the phosphate is then transferred to a histidine in a histidine phosphotransferase (HPT) protein, and finally onto an aspartate on a separate RR protein. Neurospora is predicted to have eleven HHKs, one HPT and two RR proteins. Analysis of a mutant lacking the RR gene, rrg-1, showed that RRG-1 is involved in female fertility, osmotic stress response, fungicide resistance, and conidial integrity. NIK-1/OS-1, and at least one other HHK, function upstream of RRG-1. RRG-1 activates the OS-2 MAPK cascade in response to osmotic stress, fungicide treatment, and other stimuli. The predicted site of phosphorylation for RRG-1 is D921. rrg-1D921N mutants exhibit phenotypes that differ from those of wild type and the rrg-1deletion mutant, in that these strains have delays in conidiation and maturation of female reproductive structures and have slowed apical extension of hyphae. Analysis of N-terminal-truncated versions of RRG-1 will be presented. The results suggest that the N-terminal region of RRG-1 has a specific function during sexual maturation.




49 Conflict between mat-gene trees and species phylogeny of heterothallic and pseudohomothallic Neurospora
Rebecka Strandberg, Audrius Menkis, Timothy James, Lotta Wik and Hanna Johannesson Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden


Ten heterothallic and one pseudohomothallic taxa of Neurospora were used to estimate the phylogenetic relationships of the mating-type genes: mat a-1, mat A-1, mat A-2 and mat A-3. We used parsimony heuristic searches (PAUP) and Bayesian analysis, and both methods resulted in similar, well-supported phylograms. The results showed that phylogenies of mat A-1, mat A-2 and mat A-3 were congruent. Comparison between mat a and mat A gene phylogenies revealed a conflict wherein the placement of the pseudohomothallic N. tetraspermawas incongruent in the different phylogenetic trees. We hypothesize that the origin of this species, and its unique life cycle with constant heterokaryosis for mating-type, may account for this result. In addition, a conflict was observed by comparing phylogenies of different mating-type genes with published species phylogenies derived from non-coding sequences (microsatellite- flanking regions). The placement of N. crassa subgroup C (NcC) and Phylogenetic species (PS2) differed between all mat-genes and the species tree. The reason for this is uncertain, however, the possibility for horizontal gene transfer or a hybridization event should not be excluded and will be discussed.


50 Multiple shifts in reproductive modes in the evolutionary history of Neurospora and Gelasinospora
Kristiina Nygren, Tim Gustafsson, Rebecka Strandberg and Hanna Johannesson Evolutionary Biology Centre, Uppsala University, Sweden


The closely related genera Neurospora and Gelasinosporacover a diverse set of reproductive modes, including heterothallic, homothallic as well as pseudohomothallic species and they are therefore suitable models for studying the evolution of sex and mating systems. Multiple convergent shifts in reproductive modes make it possible to study their genetic mechanisms and consequences. However, the degree to which such events have happened independently in these lineages is largely unknown and in order to infer convergent evolution it is essential to have a robust phylogeny of the whole group. This has proven to be problematic in this group of fungi.

Our aim is to construct a robust phylogeny containing many members of both genera. We here present a phylogeny based on partial sequences from four genes: β-tubulin, elongation factor 1-α, protein kinase C and 28S rDNA; collected from 44 taxa representing most species of Neurospora and Gelasinospora. We use the phylogeny to infer shifts in reproductive modes and test for instance whether selfing (homothallic) lineages form a monophyletic clade. Our data indicate that homothallism evolved from heterothallism in multiple evolutionary events.


51 RFLP analysis reveals more isolates belonging to new Neurospora species
Christopher F. Villalta1, David J. Jacobson2, John W. Taylor3Department of Plant and Microbial Biology, UC Berkeley


Recent studies using phylogenetic species recognition (PSR) and biological species recognition (BSR) found three new Neurospora species, phylogenetic species (PS) 1, 2, and 3 (Dettman et al., Evolution 57: 2703-2720, 2003). However, in that study there were too few individuals found for each species to provide a confident formal description. A more comprehensive search of the existing Perkins culture collection was done to find more individuals of each of these species. A method using RFLPs of the markers used for PSR was devised to easily distinguish members of the new species (PS1-3) from the closely related species N. crassa and N. intermedia. The RFLP analysis was performed on isolates that were either not clearly identifiable by BSR (crosses with tester strains), or were from the same geographic locations as PS1-3. Additional members of all these species were identified: 1 new PS1, 9 new PS2, and 1 new PS3. BSR in and among PS1-3 was investigated by performing intraspecific and interspecific crosses. PS1 and PS2 appear reproductively isolated, successfully mating intraspecifically while not mating successfully with other species of Neurospora. The additional PS3 strain successfully crossed with other PS3 isolates, however, it also successfully crossed with N. crassa confirming PSR but not BSR between PS3 and N. crassa (Dettman et al., Evolution 57: 2721-2741, 2003). We are confident that PS1-3 are indeed new species, and they will be named N. hispaniola (PS1), N. metzenbergi (PS2), and N. perkinsi (PS3).


52 Evolution and diversity of a nonself recognition locus in Neurospora.
Charles Hall1, Julie Welch1, and Louise Glass1. 1Department of Plant and Microbial Biology, University of California, Berkeley, USA.


Among filamentous fungi, hyphal fusion between individuals that differ in allelic specificity at any one of a number of heterokaryon incompatibility (HI) loci called het, results in the termination of heterokaryon formation and cell death. Alleles of some het genes in Neurosporaare highly polymorphic and alleles of alternative specificity are equally frequent in populations and show trans-species polymorphisms. At the well studied het-c, pin-c HI locus, nonself recognition requires allelic (het-c) and non-allelic interactions (between het-c and it’s flanking gene pin-c). The availability of a variety of isolates of N. crassa, N. discreta, and N. tetrasperma as well as genomic sequence from many species of filamentous fungi allow us ask fundamental questions about the evolution heterokaryon incompatibility, specifically at the het- c, pin-c locus. This data allows us to trace the adaptation of het-c and pin-cfrom independent ancestral genes, uninvolved in nonself-recognition to a functional HI locus. The comparison of alleles from multiple isolates combined with experimental determination of HI specificity allows us to study the evolution of new alleles. We will also examine the biological consequences of recombination within het-c, pin-c locus as well as the flanking DNA. Our analysis will broaden our understanding of the evolution of heteroincompatibility systems in fungi.


53 Circadian clock phenotype as a fitness trait; a test under ecologically relevant conditions
Kwangwon Lee Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca NY 14853 USA.


Neurospora crassa has been a successful eukaryotic model system for studying many biological questions using biochemical, genetic, genomic, and molecular tools. However, most of knowledge that we have on Neurospora biology has been learned through experiments under (controlled) artificial laboratory conditions. I developed an experimental system for N. crassa to address how fungal development/behaviors affect fitness under ecologically relevant conditions; natural objects were used as substrates and experimental field conditions were recorded using portable sensors for temperature and light conditions. As a proof of concept for this experimental system, I performed experiments with specific objectives in mind; characterizing the life cycle of N. crassa in natural habitats and testing the hypothesis that circadian clock/light-perception phenotypes affect the fitness of an organism. I found that N. crassa forms abundant microconidia on Ostrya virginiana (Ironwood). On four other species of wood tested in this study, I found different proportions of microconidia/macroconidia. N. crassais a poor competitor in these habitats unless the substrates have been autoclaved. Developmental behaviors, which were not observed on artificial media, were observed in natural objects. For example, sexual spores were released from the perithecia by oozing instead of shooting. I also performed experiments using wild type and a wc-1 mutant to test the role of circadian clock/light- perception in nature. There were six times more conidia produced in the wild type in comparison to the wc-1 mutant. We concluded that clock/light-perception plays a significant role in the fitness of N. crassa in nature.


54 Circadian clock phenotype as a fitness trait; a test by progressive selections
Jung IL Bae, Kwangwon Lee Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca NY 14853 USA.


Circadian clock behavior is a complex trait; there are multiple genes making contributions to different aspects of clock-controlled fungal behaviors. Studies in several model systems have proposed a crucial role of circadian clock in fitness of an organism. However, there was no systematic study on whether the clock plays a role in fitness for a fungal organism. To gain insights on the roles of circadian clock associated with organism's fitness, we relied on population/quantitative genetics approach using Neurospora crassa. First, we generated isogenic population by progressive inter-cross selection (not by mutation), as commonly used in the agricultural traits, e.g. crop yield or milk production. We had a hypothesis that the strains with extreme clock phenotype would show less fitness in comparison to those with ¡°normal¡+ clock phenotype. We focused our study on the circadian period phenotype. We performed four inter-crosses aimed to select for the N. crassa strains with an extreme clock periods, we noticed that 1) population mean values are shifted toward the selective direction, 2) population structure become deviant from the normal distribution, and 3) the interval of periods among inter-cross populations are not changing, between 20 and 26 hours in period. Our data suggest that circadian clock period is important phenotype for fitness. The change of the population composition and the implications of the artificial selection will be discussed.


55 Characterization of the ric8 gene from Neurospora crassa
Sara Martinez and Katherine Borkovich University of California, Riverside Department of Plant Pathology


Heterotrimeric G protein signaling is essential for eukaryotes to sense changes in their environment. The heterotrimer consists of G alpha, G beta, and G gamma subunits, which are bound to G protein coupled receptors (GPCRs). The heterotrimer binds the receptor when G alpha is bound to GDP. Receptor activation causes the G alpha to exchange GTP for GDP, causing dissociation of of G proteins from the receptor. N. crassacontains three G alpha (GNA-1,GNA-2,GNA-3) one G beta (GNB-1), one G gamma (GNG-1) and 10 predicted GPCRs. Another regulator of G proteins, RIC8, was found to play a significant role in asymmetrical cell division and synaptic vesicle priming in C. elegans and mice. RIC8 is thought to function as a GPCR independent guanine nucleotide exchange factor for G alpha. A homolog of RIC8 in N. crassa, RIC8, was identified and has not been characterized in fungi. The ric8gene deletion mutant exhibits severe growth inhibition, inappropriate conidiation in submerged culture, and is female sterile, similar to strains lacking both the gna-1 and gna-3 genes. In addition, introducing constitutively activated alleles of gna-1 and gna-3 partially rescues the defects of ric8 mutants. Localization studies using a GFP tagged allele of ric8show that RIC8 is cytosolic, and semi-quantitative RT-PCR results show that the gene is expressed highly in conidia. These results suggest that RIC8 may play a role in G alpha activation and cellular pathways regulated by G alpha subunits.





56 The effects of prd mutations on the FRQ-less oscillator (FLO).
Sanshu Li and Patricia Lakin-Thomas, Dept. of Biology, York University, Toronto, Canada,


The circadian rhythm of conidiation in Neurospora is assumed to be driven by a FRQ/WC-based oscillator. However, strains carrying null mutations at the frq locus, such as frq10, can show rhythmic conidiation under some conditions. This rhythmicity is driven by a FRQ-less oscillator (FLO), and we are interested in identifying the components of the FLO. We are using the chol-1 mutant, which requires choline for normal growth and has a long conidiation period without choline. The double mutant chol-1 frq10 is rhythmic with a long period, indicating that the long period of chol-1 is driven by the FLO. We introduced the clock mutations prd-1, prd-2, prd-3 and prd-4 into this double mutant to look for interactions with FLO. Double mutants chol-1 prd-1 and chol-1 prd-2, and triple mutants chol-1 frq10 prd-1 and chol-1 frq10 prd-2, are arrhythmic without choline. Double mutants chol-1 prd-3 and chol-1 prd-4, and triple mutants chol-1 frq10 prd-3 and chol-1 frq10 prd-4, have long periods that are similar to chol-1 frq10. In entrainment experiments using temperature pulses at different T-cycles, the triple mutants chol-1 frq10 prd-1 and chol-1 frq10 prd-2 failed to entrain while chol-1 frq10 prd-3 and chol-1 frq10 prd-4 demonstrated entrainment similar to the chol-1 frq10 strain. These results indicate that prd-1 and prd-2 have larger effects on rhythmicity in chol-1 frq10 than prd- 3 and prd-4 and may be candidates for components of the FLO.


57 The Role of Coronin in Polarized Growth in Neurospora crassa
Echauri Espinosa R. O.1, Smith L.2, Roberson R. W.3 and Mouriño Pérez R. R.1 1Departament of Microbiology. Centro de Investigación Científica y Educación Superior de Ensenada. B. C. México.2Section of Cell and Developmental Biology. University of California San Diego. La Jolla, CA. USA. 3School of Life Sciences. Arizona State University. Tempe, AZ. USA


Coronin is an actin binding protein that plays a major role regulating actin patches formation. The mutation of the coroningene affects locomotion, growth and cytoskeleton organization. Actin is very important for polarized growth and assisting the Spitzenkörper (Spk). To determine whether the lack of coronin can affect the presence and features of the Spk and the polarized growth, we characterize the coronin null mutant in Neurospora crassa and compare it with a wild type (wt) strain. The growth rate of coronin was around 40% of the rate for wt (p<0.05). The Spk size and shape of coroninmutant was smaller with a half moon shape. Mitochondria had a spherical shape instead of the wt elongated mitochondria. Hyphal apical growth was affected showing a bumping and zig-zag morphology. Branching rate was 5-folds higher than the wt (p<0.05). Coronin mutant produced half of the conidia than the wt (p<0.05). The biomass production per day was 40 % less than the wt. The mutation of the coronin gene in N. crassa is not essential but strongly reduces the growth rate, affects the direction of polarized growth, increased the frequency of branching and defects in septal construction.


58 Characterization of base excision repair mutants in Neurospora crassa
Maki Kitamura and Hirokazu Inoue, Saitama University, Saitama 338-8570 Japan


There is no report on the base excision repair (BER) in Neurospora crassa. In mutants isolated as mutagen-sensitive mutants, BER-deficient mutants could not be found. From results of genome DNA sequencing and annotation, we identified some BER-homologous genes. In this experiment, 3 homologue genes, mag-1, apn-1, apn-2, were disrupted by homologous gene targeting. And their phenotypes were investigated. They are all sensitive to alkylating agents such as methyl metahnesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl N'-nitro N-nitrosoguanidine (MNNG), but not to UV, hydrogen peroxide (H2O2 ), hydroxylurea (HU). Epistasis analysis showed that apn-1 and apn-2 belong to different group. The relationship between apn-1 and apn-2 looks similar to that between mus-18 and mus-38 in nucleotide excision repair.


59 Redundancy of vib-1 and its paralog during the sexual cycle reveals additional functions of a heterokaryon incompatibility suppressor gene.
Elizabeth Hutchison and N. Louise Glass. Plant and Microbial Biology Department, UC Berkeley, California, 94720


Neurospora crassa hyphae of genetically identical individuals can undergo fusion during vegetative growth, forming a heterokaryon. However, individuals different at any of 11 heterokaryon (het) loci are heterokaryon incompatible; fusion of these strains results in rapid compartmentalization and cell death. vib-1is a suppressor of heterokayon incompatibility (HI) and has two paralogs, NCU04729 and NCU09915. All of these genes have homology to the DNA binding domain of NDT80, a known meiotic transcription factor in S. cerevisiae. Neither of the vib-1paralogs suppresses HI, and the NCU04729 knockout has no apparent phenotype. The NCU09915 knockout is female sterile, and trichogyne assays show that the knockout is not defective in trichogyne-microconidium fusion. Interestingly, the female sterility defect in the NCU09915 deletion strain is complemented by the helper strain (FGSC 4564), even in homozygous crosses. These data indicate that NCU09915 is required for perithecial development, but not karyogamy and meiosis. Consistent with these data, Q-RT-PCR showed that NCU09915 is highly upregulated in the perithecium. Surprisingly, sexual development is impaired in homozygous crosses between NCU09915 and vib-1double knockouts, and cannot be fully complemented by the helper strain. These data suggest that NCU09915 and VIB-1 have overlapping functions during sexual development.


60 Development of neurospora Host Strains with Reduced Codon Bias for Elevated Protein Expression
Christina A. Bormann Chung. Neugenesis Corporation, Burlingame, CA, USA.


The expression of functional proteins in heterologous hosts is a cornerstone of modern biotechnology. Unfortunately, proteins are often difficult to express outside their original host context. They might contain codons that are rarely used in the desired host, come from organisms that use non-canonical code or contain expression-limiting regulatory elements or secondary structure within their coding sequence. Codon optimization often results in expression improvement of poorly expressed genes in Neurospora. To test the hypothesis that codon-usage is limiting for heterologous protein expression, nine tRNA genes that are under- represented in Neuropsora and may be the source of the observed codon bias in this organism have been identified and were engineered into Neurospora strains. The ability to express genes with non-Neurospora codon representation in these strains at high levels is currently being examined.


61 Regulation of crossing over near cog is a complex process
P. J. Yeadon, F. J. Bowring and D. E. A. Catcheside School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia, Australia


There seems to be a crossover hotspot near, but distinct from, the recombination hotspot cog. Heterozygous deletion of a 1.8 kb sequence between his-3 and cog reduces crossing over between his-3 and ad-3 to about 65% of the normal level, while increasing the adjacent his-3 to lys-4 interval to 140%. Homozygous deletion of a 200 bp region, near the 3' end of his-3, in which an excess of crossovers was found during analysis of 150 octads, yields a similar but smaller effect (85% and 125% respectively). Interestingly, heterozygous replacement of the 1.8 kb region with a foreign DNA fragment has little or no additional effect on the his-3 to ad-3 interval, but invariably prevents the increase in the his-3 to lys-4interval. In addition, homozygous replacement of the 1.8 kb region with a foreign sequence does not necessarily restore normal levels of crossing over, depending on the actual sequence used. In conclusion, although the frequency of recombination initiation near his-3 is regulated by the alleles of cog and rec-2(discussed in the presentation by Bowring in session I) in the cross, the frequency and location of resultant crossovers also depends on additional sequences up to 2 kb away. This work was supported by a grant from the Australian Research Council.


62 Strain Improvement for Heterologous Protein Secretion; Exploiting the Power of the FGSC and the Neurospora Genomics Project.

Edward B. Cambareri, David Jacobson, Ken Takeoka, Rebecca Taylor, Kelechi Anoruo and W. Dorsey Stuart. Neugenesis Corporation, 849 Mitten Rd, Burlingame, CA 94010


Neugenesis Corporation is using knock-out strains generated by the NGP to develop improved stains for production of biopharmaceutical proteins. Many monoclonal antibodies and glycoprotein antigens or antigen variants can be expressed and secreted from Neurospora, but are often degraded by proteolytic activities that are co-secreted. New production strains have been generated using KOs generated by the NGP, and morphological mutants from the Fungal Genetics Stock Center. Titers and quality of secreted antibody and Influenza hemaggutinin antigen of have been compared in various production strains . Data will be presented demonstrating dramatic differences in both titer and quality of secreted proteins in these new strains of Neurospora.


63 Relationship between G beta and G alpha Proteins in Neurospora crassa.
Susan Won, Svetlana Krystofova and Katherine Borkovich. University of California, Riverside, USA.


Sexual and asexual development of N. crassa is regulated by heterotrimeric G-proteins. Three G alpha subunits (GNA-1, GNA-2 and GNA-3), one G beta (GNB-1) and one G gamma (GNG-1) have been identified in this organism. Previous work showed that loss of GNB-1 leads to extremely reduced levels of G alpha subunits in N. crassa. In this work, we analyze interactions between GNB-1 and G alpha proteins. Using a genetic approach, delta gna-1 delta gnb-1, delta gna-2 delta gnb-1 and delta gna-3 delta gnb-1 strains, and strains containing constitutively activated G alpha genes in the delta gnb-1background were isolated and their phenotypes analyzed. Using biochemical approaches, Co-IP and pulse-chase assays were used to determine interactions between G protein subunits as well as G alpha turnover in the delta gnb-1 background. The results support an interaction between GNB-1 and all three G alpha proteins in N. crassa.


64 The preparation and use of Neurospora crassa in vitro systems for studying translational regulation. Cheng Wu1, Vladimir Zeenko2, Kate Moore2, Ryan Cleland2 and Matthew Sachs1

1Texas A&M Univeristy, College Station, TX, USA. 2Oregon Health & Science University, Portland, OR, USA.


Cell-free translation systems recapitulate many important features of in vivo translation. They faithfully synthesize polypeptides when programmed with mRNA, therefore enabling biochemical determination of factors and mechanisms contributing to translational processes. For example, co-translational real-time measurement of luciferase enzyme activity arising from translation of a reporter transcript can be used to determine the optimal conditions for in vitro translation to study regulatory effects. Primer-extension inhibition (toeprinting) is used to map the positions of ribosomes on mRNA that are at rate-limiting steps of translation. Pulse-chase studies with [35S]Met can track intermediates in polypeptide synthesis as well as providing qualitative and quantitative data on translation efficiency. Here we describe and compare the efficiency of several different approaches to prepare cell-free translation extracts from N. crassa, and the use of N. crassa extracts to assess the translational effects of several upstream open reading frames present in either N. crassa and Cryptococcus neoformans mRNAs.


65. Progress at the Fungal Genetics Stock Center: A culture collection for model organisms becomes a model for culture collections.

Kevin McCluskey, Aric Wiest, Matthew Kinney and Michael Plamann. University of Missouri- Kansas City


The Fungal Genetics Stock Center has been closely connected to its community since it was founded in 1961. That close connection has allowed the FGSC to provide access to materials that were at the forefront of existing technology. First seen as the deposition of genetically marked strains for genetic mapping, this developed into multiply marked strains, populations for RFLP mapping, cloning vectors, gene libraries and ultimately the collection of targeted knockout strains of Neurospora crassa. Among microbial culture collections, the FGSC is unique in a number of ways including its close connection with its user community as well as its willingness to embrace new resources. The FGSC has also survived the retirement of two Directors. Many collections are not able to do this and it is the integration in the user community that has enabled the FGSC to do so. As much as the community depends on the resources in the FGSC, the FGSC depends on the user community for its continued success.


66. The Exocyst Components Sec5 & Sec15, Localize at the Apical Membrane in Hyphae of Neurospora crassa

A. Beltrán-Aguilar1, M. Riquelme1, and S. Bartnicki-García1. 1Center for Scientific Research and higher Education of Ensenada (CICESE), Department of Microbiology. Km. 107 Ctra. Tijuana-Ensenada, 22860, Baja California, México.


The last stage of the secretory pathway is exocytosis, a process that needs high fidelity protein-protein interactions and the spending of energy to fuse exocytic vesicles to the plasma membrane. The exocyst, first described in secretion termosensitive mutants of Saccharomyces cerevisiae (sec) is an octameric complex (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84) needed for the tethering of secretory vesicles to the plasma membrane, an event previous to the SNAREs assembly. Genome Analysis of Neurospora crassa allowed the identification of homologue genes for all components. We tagged with gfp two putative exocyst components sec5 (NCU07698.3) and sec15 (NCU00117.3), and expressed them in N. crassa FGSC #9717 strain (mus51Δ::Bar;his-3) deficient in Non-Homologous End Joining. We analyzed by Laser Scanning Confocal Microscopy the localization of SEC5::GFP and SEC15::GFP fusions in living hyphae of N. crassa. In both cases the accumulation of fluorescence was found at the very tip of fungal hyphae adjacent to the plasma membrane, just in front of the Spitzenkörper, which correlates with the predicted area of maximum exocytosis. SEC15::GFP occupied a larger area than SEC5::GFP. This work shows for the first time images of two of the eight components of the exocyst labeled with GFP in N. crassa as a way to understand secretion. Our results suggest the presence of an Exocyst in N. crassa and its possible involvement in the apical secretory process.

67. Regulation of Sporulation by G-protein Signaling Pathways in Neurospora crassa

James Kim, Cynthia Larive and Katherine Borkovich. Department of Plant Pathology, University of California, Riverside, CA 92521


Neurospora crassa can sense change in the environment through heterotrimeric G-protein signaling. Heterotrimeric G-proteins couple to G protein coupled receptors and consist of Gα, Gβ, and Gγ subunits. Asexual reproduction (conidiation) occurs in response to nutrient deficiency and desiccation. The Gα protein, GNA-3, is involved in the conidiation process. Our lab showed that a Δgna-3 mutant conidiates inappropriately in submerged culture.

Since conidiation can be a response to starvation, the possibility that GNA-3 might be involved in nutrient sensing was explored. A metabonomics approach was used to create a metabolite fingerprint by utilizing proton-NMR in order to compare amino acid levels and other metabolites that were present in differing levels between wild-type and Δgna-3 mutant.

Principal Components Analysis showed that loss of gna-3 does not significantly alter the metabolite fingerprint of Neurospora crassa. However, levels of trehalose decreased while glucose increased. Also, asparagine, histidine, and glutamate levels decreased while glutamine level increased, the latter a verification of conidiation. This suggests that GNA-3 may be involved in regulating metabolism and/or nutrient sensing in Neurospora.


Index to Poster Authors


Abdullah, Julia                  7, 9

Al- Babili, Salim               1

Al Dabbous, Mash’el        7

Allen, Kenneth                  3

Altamirano, Lorena           32

Anoruo, Kelechi                62

Araujo-Palomares, Cynthia L. 12

Avalos, Javier                    1

Bae, Jung IL,                     54

Baker, Christopher L.        20, 41

Barry, Kerrie                     36

Bartnicki-García, Salomón 8, 14, 15, 16, 23, 66

Beltrán-Aguilar, A.            66

Bertolini, M. C.,                45

Borkovich, Katherine A.   31, 32, 47, 48, 55, 63

Bormann Chung, Christina A. 60

Bourne, Brian                    24

Bowman, Barry                 6

Bowman, Emma Jean        6

Bowring, F. J.                    61

Brody, S.                           8

Brunner Michael               2

Callejas-Negrete O. A.      16

Cambareri, Edward B.       62

Cardani, Amber                 4

Castro-Longoria, Ernestina 8, 12

Catcheside, D. E. A.          61

Chen, Chen-Hui                42

Choudhary, Swati              43

Christopher F., Villalta      51

Cleland, Ryan                    64

Collett, Michael A.            41

Collins, Rick                      27, 35

Collopy, Patrick D.            32

Colot, Hildur V.                32, 34

Connolly, Lanelle R.         19, 33

Corrochano, Luis M.         40

Cupertino, F. B.,                45

DeAbreu, Diane                35

Delgado-Álvarez, Diego L. 13

Draskovic, Marija              6

Dunlap, Jay C.                   20, 22, 32, 34, 41, 42, 46

Echauri Espinosa, R. O.     25, 57

Ellison, Christopher           36, 37

Estrada, Alejandro F.         1

Ferry, M.                           8

Fox, Julie                           24, 46

Free, Stephen J.                 7, 9

Freitag, Michael                19, 21, 23, 33

Freitas, Janaina S.              38

Fujimura, Makoto              17, 44

Fukumori, Fumiyasu         44

Gerber, Scott A.                20

Glass, N. Louise                30, 36, 52, 59

Gonçalves, R. D.,               45

Gooch, Van                       24, 46

Gorrochotegui- Escalante, Norma    32

Gras, Diana E.                   38

Grigoriev, Igor                  36

Gustafsson, Tim                50

Haidari, Leila                    18

Hall, Charles,                    52

Hutchison, Elizabeth         59

Ichiishi, Akihiko               17, 44

Inoue, Hirokazu                 58

Jacobson, David                36, 37, 51, 62

James, Timothy                 49

Johannesson, Hanna          49, 50

Jones, Carol A.                  48

Jones, John                        32

Kasuga, Takao                   30

Keenan, Kelly                   4

Kennell, John C.                11

Kettenbach, Arminja         20

Kinney, Matthew              65

Kitamura, Maki                 58

Krystofova, Svetlana         32, 63

Lafontaine, Denis              18

Lakin-Thomas, Patricia     56

Larrondo, Luis F.               22, 34, 46

Leal, Juliana                      38

Lee, Kwangwon                53, 54

Lee, Heng-Chi                   43

Lew, T.                             21

Li, Sanshu                          56

Li, Liande                          31

Lindamood, Erik                5            

Liu, Yi                              43

Loros, Jennifer J.               20, 22, 34, 41, 42, 46

Maddi, Abhiram                9

Maiti, Mekhala                  43

Malzahn, Erik                    2

Martinez, Sara                   55          

Martinez-Rossi, Nilce M.  38

Maze B.                             11

McCluskey, Kevin             65

Mendoza, Joseph               19

Menkis, Audrius                49

Meza-Lopez, Maria           28

Moore, Kate                      64

Mouriño-Pérez, Rosa R.     13, 14, 16, 21, 25, 57

Natvig, Donald O              36

Ndonwi, N.                        11

Niki, Takaharu                  17

Nygren, Kristiina               50

Olmedo, Maria                  40

Paietta, John V.                 39

Park, Gyungsoon               32, 47

Plamann, Michael              65

Radford, Alan                    26, 29
Keeping, Andrew              27          

Ramírez-Cota, Rosa          14

Ringelberg, Carol              32

Riquelme, Meritxell          12, 15, 23, 66

Rivetta, Alberto                 3

Roberson, Robert W.         14, 16, 21, 25, 57

Román-Gavilanes, A. I.,    21

Rossi, Antonio                  38

Sachs, Matthew                 64

Sancar, Gencer                  2

Sánchez-León, Eddy          23

Sato, Masahito                  17

Scarnati, Matt                    4

Scherzinger, Daniel           1

Schmutz, Jeremy               36

Schopf, Eva                       33

Shao, Xiao                         4

Sharpton, Thomas J           36, 37

Shi, Mi                              41

Silva, Emiliana M.             38

Slayman, Clifford              3            

Smith,  Laurie G.               13, 25, 57

Smith, Robert P.                18

Smith, Kristina M.             19, 33

Smith, Myron L.                18

Squina, Fabio M.               38

Stajich, Jason E                 36, 37

Stout, Angela                    7, 9

Strandberg, Rebecka          49, 50

Stuart,W. Dorsey               62

Suzuki, Kouhei                  17

Takeoka, Ken                    62

Taylor, John W.                 36, 37, 51

Taylor, Rebecca                62

Toko, Takeru                     17

Turner, Gloria E.               28

Verdín, Jorge                    15

Watanabe, Setsuko            44

Watters, Michael               5

Weiss, Richard L.              28

Welch, Julie                       52

Wellman, Kenji                 18

Wiest, Aric                        65

Wik, Lotta                         49

Won, Susan                        63

Wu, Cheng                         64

Yamashita, Kazuhiro         44

Yeadon, P. J.                     61

Youssar, Loubna                1

Zeenko, Vladimir              64

Zhang, Michael Y              36