Session I: From Genes to Populations

Tony Griffiths, Chair 


Control of DNA Methylation in Neurospora
Eric Selker, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229

Most methylated regions of Neurospora are products of RIP (repeat-induced point mutation), a premeiotic homology-based genome defense system that litters duplicated sequences with C:G to T:A mutations and typically leaves them methylated at remaining cytosines. I will present our current understanding about how A:T-rich DNA, such as that resulting from RIP, triggers methylation. Our efforts to elucidate the control of DNA methylation in vegetative cells have revealed mechanistic ties between modifications of DNA and histones. The DIM-2 DNA methyltransferase is directed by heterochromatin protein 1 (HP1), which in turn recognizes trimethyl-lysine 9 on histone H3, placed by the DIM-5 histone H3 methyltransferase. Results of in vitro and in vivo studies indicate that DIM-5 recognizes at least residues 8-12 of histone H3 and is sensitive to methylation of lysine 4 and phosphorylation of serine 10 in histone H3, supporting our suggestion that histones serve to integrate diverse signals to control DNA methylation. Additional support for this notion comes from our studies on mutants defective in other histone modification enzymes. DNA methylation and HP1 localization do not depend on RNAi machinery in Neurospora but do depend, in part, on deacetylation and dephosphorylation of histones. Conversely, DNA methylation can lead to deacetylation of histones, which may aid in propagation of DNA methylation and the associated silenced chromatin state.

Structural studies of protein (histone) methylation
Xiaodong Cheng. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322;

There is a rapidly growing appreciation that the study of covalent modification to proteins and transcriptional regulation will likely dominate the research headlines in the next decade. Protein (de)methylation plays a central role in both of these fields, as several different residues (Arg, Lys) are methylated in cells and methylation plays a central role in the "histone code" that regulates chromatin structure, impacts transcription, and responds to DNA damage. In some cases, a single lysine can be mono-, di-, or trimethylated, with different functional consequences for each of the three forms. I will review structural aspects of methylation of histone lysine residues by two enzyme families with entirely different structural scaffolding (the SET proteins and Dot1p) and methylation of protein arginine residues by PRMTs, and discuss, somewhat speculatively, their mechanisms.

Meiotic Recombination initiated by the cog hotspot in Neurospora.
Frederick Bowring, Jane Yeadon, Hui-Yin Lee, Sue Conway and David Catcheside. School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, SA 5001. Australia.

Meiotic recombination in Neurospora crassa is initiated at hotspots regulated by transacting genes. Our current focus is on recombination initiated by the hotspot cog, occurring within the his-3 locus and in flanking regions stretching proximally to lys-4 and distally to ad-3. We have analysed recombination by measuring the frequency of His+ progeny from crosses heterozygous for auxotrophic his-3 alleles, and by following segregation in octads heterozygous for lys-4, his-3, ad-3, cot-1, am, snp markers between his-3 and cog and additional snps both proximal and distal of his-3. Octad analysis revealed additional hotspots that, like cog, appear to be particularly active in initiating recombination, and showed that all reciprocal exchanges whose location could be determined also experienced conversion nearby. We have constructed knockouts of spo11, msh-2, msh4 and ku70. Each mutant shows disturbance of chromosome behavior during meiosis. Although chromosome pairing is severely affected in crosses homozygous spo11 and aneuploidy rife amongst the progeny, we were surprised to find recombination events initiated by the cog hotspot are somewhat elevated over normal amongst those infrequent spores that are sufficiently genetically balanced to be viable. One interpretation of these data is that Neurospora is able to initiate recombination, at least at cog, by mechanisms other than Spo11-induced double strand breaks.

DNA repair and genomic instability in Neurospora
Hirokazu Inoue (Saitama University, Saitama, Japan)

Mutants which show high sensitivity to mutagen(s) have been isolated and characterized in Neurospora. Based on spectra of mutagen sensitivity and epistatic relationship, they were classified into 5 groups; excision repair, recombination repair, post replication repair, damage-checkpoint and mismatch repair. Some of those mutants have mutator phenotype and/or growth defect phenotype. Majority of spontaneous mutation is generated in replication process. Recently 2 different recQ genes encoding proteins with 3’-5’ helicase motif were identified in Neurospora. A double recQ mutant showed genomic instability. Mutator or growth defect phenotype of a recQ double mutant was suppressed by mutation of either of nonhomologous end joining (NHEJ) or homologous recombination (HR), respectively. Roles of HR and NHEJ in double-strand breaks repair, gene targeting and spontaneous mutagenesis are discussed.

Neurospora comparative biology is enabled by phylogenetics and species recognition.
John Taylor1, Dave Jacobson1,2, Elizabeth Turner1, Luz Beatrice Gilbert,1 Jeremy Dettman3.1UC, Berkeley, 2Stanford U. 3U. Toronto, Mississauga.

Comparative biology depends on accuracy in recognition of genetically isolated groups of organisms, in analysis of relationships among these groups, and in dating of evolutionary events that create modern groups. In Neurospora, outbreeding species form a single evolutionary lineage of 15 phylogenetic species in two lineages, biological and phylogenetic species recognition are nearly equivalent, and genetic isolation precedes reproductive isolation. Recognition and comparison of Neurospora species is unmatched in its scope in fungi and represents a tool to enable careful comparative biology. Research using this tool began with an examination of the evolution of microsatellites. Current projects include: 1) Phylogenomics, where outbreeding Neurospora are providing a means of testing the claims made for this approach in studies of yeast. 2) Evolution of reproductive isolation, where previous knowledge of phylogenetic relationships and reproductive isolation among clades facilitated the choice of strains for QTL analysis of reproductive isolation in hybrid matings. 3) Intraspecific or intraclade variation, where studies of variation in fecundity and phenotypes associated with biological clocks or reproduction are being used to dissect genetic control of variable phenotype.

Evolutionary genetics of reproductive isolation barriers separating Neurospora crassa and N. intermedia.
Elizabeth Turner & John W. Taylor, Plant & Microbial Biology, University of California, Berkeley, CA 94720

Reproductive isolation (RI) barriers between different pairs of N. crassa and N. intermedia strains range from mild (reduced numbers of viable progeny) to severe (failure to develop perithecia). For the N. crassa NcC clade, which is endemic in southern India, the severity of RI from N. intermedia is biogeographically structured: crosses to sympatric N. intermedia strains show more severe barriers. This pattern is consistent with reinforcement, the evolution of stronger RI barriers by natural selection against hybridization. The potential fitness advantage of the putative reinforcement barrier, the early abortion of hybrid perithecia, was demonstrated in experiments showing that early abortion can dramatically increase the overall fecundity of NcC females when they have additional opportunities to mate with conspecific males. We are undertaking quantitative trait locus (QTL) mapping to study the evolutionary genetics of RI barriers separating N. crassa and N. intermedia using a mapping population derived from a cross between strains of the N. crassa NcA and NcC clades. We have identified loci that are significantly associated with the strength of RI in mating assays between these f1 hybrids and N. intermedia tester strains from different geographic regions, including a QTL on linkage group VI responsible for about 30% of the variance in development of hybrid perithecia fertilized by N. intermedia sympatric to NcC.

Artificial selection for ascospore size in Neurospora crassa
Heather H. Wilkinson, Department of Plant Pathology, Program for the Biology of Filamentous Fungi, Texas A&M University.

We are interested in ecological and evolutionary genetic bases for life-history trait variation in natural Neurospora crassa populations. Studies thus far have focused on breeding well-characterized isolates from a Louisiana sugar cane field and discerning the patterns of heritability associated with variation in a wide variety of developmental phenotypes. As a test of concept in the present study, crosses that yielded the smallest or the largest ascospores in that F1 population were used to artificially select, in both directions, for ascospore size. In total, selection on both large and small ascospores lineages has been carried out to the F4 generation. We are exploring 1) the degree to which evolution of the mean of one trait influences the evolution of the mean of another (e.g. ascospore shape); and 2) the degree to which selection on the mean of a trait influences the shape of the distribution around the mean (e.g. standard deviation, skewness, kurtosis). The implications to the functional ecology of a trait will be discussed.

Workshop: How to utilize new tools and resources for Neurospora developed in the Program Project

Jay Dunlap, Organizer

Knockouts Workshop
Hildur Colot and Patrick Collopy.

This workshop will present protocols and guidelines for making your own knockout strains in Neurospora.  In order to achieve high-throughput production of knockout strains as part of the Program Project, we have created novel procedures and software tools, as well as adapting, simplifying and streamlining existing techniques.
We will briefly describe the overall scheme and then elaborate on certain details, including primer design, yeast recombinational cloning for assembling the deletion cassettes, the use of magnetic beads for isolation of yeast DNA,  the creation of mus-51 and mus-52 strains, 96-well electroporation into Neurospora, a modified transformation medium, mini-slants for spot-testing, the use of magnetic beads for Neurospora DNA preps, and a custom-written application for designing Southern blots.
We have performed significant portions of the work on a pipetting robot.  However, the protocols were first developed manually and can be done without the robot with small numbers of samples or in a 96-well format.  We will provide protocols for performing all the procedures without the need for any specialized equipment, along with information on more expensive options suitable for high throughput. 
Finally, we will give a brief tour of the relevant web-based resources, including lists of primers used, lists of knockout strains completed and in progress (including access to the LIMS we use to track our work), updated protocols, and the programs for both primer design and Southern design.  The web sites include:

Introduction to Neurospora Microarray Methods
Jeffrey Townsend, Takao Kasuga, Baikang Pei

The detection of large and small yet statistically significant differences in gene expression in spotted DNA microarray studies is an ongoing challenge. We will begin with discussion of experimental protocols that are designed for investigations of differential gene expression using resources available in the Neurospora community. We have recently developed Neurospora microarrays comprising predicted 10,526 Neurospora genes and made them publicly available through FGSC. Since the release of the arrays and accompanying protocols at
we have received feedback from the Neurospora community. We will discuss growth conditions, RNA extraction, cDNA synthesis, hybridization and acquisition of microarray data. We will provide protocols, alternative methods and troubleshooting methodology. From there we will explore ways to design experiments. Multifactorial experimental designs using DNA microarrays are becoming increasingly common. We will highlight experimental design criteria that will maximize inferential and statistical power. In experimental design, opportunities for transitive inference should be exploited, while always ensuring that comparisons of greatest interest comprise direct hybridizations. We will briefly overview productive methods for analysis for completed datasets, including Bayesian (BAGEL) and ANOVA methods. Understanding the difference in gene expression that is detectable as significant is a vital component of experimental design and evaluation. The gene expression level at which there is an empirical 50% probability of a significant call is presented as a summary statistic for the power to detect small differences in gene expression. Lastly, we will provide an introduction to a filamentous fungal microarray database in construction where data may be deposited, examined, and analyzed.


Session II: Genomics and Program Project Report

Kathy Borkovich, Chair

Enabling a community to dissect an organism: Functional  analysis of Neurospora as a model filamentous fungus
Jay Dunlap1, Hildur Colot1, Kathy Borkovich2, Gloria Turner3, Dick Weiss3, Mike Plamann4, Bruce Birren5, Matt Sachs6, Louise Glass7, Jeffrey Townsend9, Mary Anne Nelson8, Jennifer Loros1 1Dept. Genetics, Dartmouth Medical School, Hanover, NH 03755 2Dept. Plant Pathology, UC Riverside, Riverside, CA 3Dept. Microbiology, UCLA, Los Angeles, CA 4Dept. Biology, Univ. Missouri, Kansas City, MO 5 MIT Center for Genome Research, Cambridge, MA 6 Oregon Health Sciences University, Portland, OR 7 Dept. Plant and Microbial Biology, UC Berkeley, Berkeley, CA 8 Dept. Biology, Univ New Mexico, Albuquerque, NM 9 Dept. Molec. Cell. Biology, Univ. of Connecticut, Storrs, CT

The overall goal of the four interdependent projects in this Program Project is to carry out functional genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has become a model for the assemblage of over 250,000 species of non-yeast fungi. The timeline for this effort envisioned periodic repots to the community and public assessment of progress towards our goals and benchmarks. Building from the completely sequenced 43 Mb Neurospora genome the first Project is pursuing the systematic disruption of genes through targeted gene replacements, preliminary phenotyping of these strains, and their distribution to the scientific community at large. Project #2, through a primary focus in Annotation and Genomics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project #3 will be used to collect baseline expression data the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project #1. cDNA libraries generated in Project #4 will illuminate alternative splicing, alternative promoters, antisense transcripts and help to document the overall complexity of expressed sequences in Neurospora, as well as driving the assembly of a SNP map.

Genome informatics and Neurospora crassa functional studies
Matthew R. Henn1, Dave DeCaprio1, Heather M. Hood2, Steve Rounsley1, Matthew Crawford1, Phil Montgomery1, Gloria E. Turner3, Chad Nusbaum1, Matthew S. Sachs2, James E. Galagan1, Bruce W. Birren1. 1Broad Institute of MIT & Harvard, Cambridge, MA 02141, 2Oregon Health & Science University, Beaverton, OR 97006, 3Department of Chemistry and Biochemistry, University of California, Los Angeles California 90095

The next challenges for understanding the Neurospora crassa genome sequence are to refine the genome annotation and to actively capture, improve, and integrate with the sequence the wealth of information that exists within the research community. To this end, the Broad Institute as part of the NIH Program Project, "Functional Analysis of a Model Filamentous Fungus,” has constructed community annotation and phenotype ontology infrastructures that for the first time provide the research community the ability to link information about genetic features with the Neurospora genome, to refine gene structures, and to curate all entries in a searchable database. To increase the accuracy of the gene model, gene calling using EST-based algorithms was also implemented. To maximize the value of EST sequencing, the Broad’s Neighborhood Quality Score algorithm was adapted to work with fungal EST sequences and we have begun comparing Mauriceville strain cDNAs with the Oak Ridge strain genomic sequence to identify single nucleotide polymorphisms (SNPs) for genetic mapping. The Broad Institute is also improving the quality of the N. crassa genome for subsequent release. One barrier to finishing the genome is regions recalcitrant to cloning. Initial results from pyrosequencing using 454 technology has improved coverage of uncaptured regions. In addition, an optical map, which represents a restriction map of the entire genome, has helped anchor significant portions of the genome leading to better representation of complete chromosomes.

Addition by subtraction: Novel insights into Neurospora biology obtained from transcription factor knockouts
Gyungsoon Park. University of California, Riverside.

We have developed a high-throughput method for creating Neurospora knockout mutants. In this procedure, yeast recombinational cloning is used to create constructs that are then transformed into Neurospora strains deficient in nonhomologous end-joining DNA repair (mus-51 and mus-52 mutants). Here we present the results of our initial application of the procedure, with mutational analysis of 103 transcription factor-encoding genes. The methodology is robust, with a >90% success rate for producing the desired knockout mutant. The resulting mutants were screened for a variety of phenotypes and 43% exhibited discernible defects. The genes producing phenotypes are variously involved in growth of basal hyphae (25 genes), aerial hyphae height and/or macroconidiation (27 genes), and differentiation of protoperithecia or perithecia (15 genes). This analysis demonstrated roles for many uncharacterized transcription factors and also revealed novel functions for genes that had been previously studied. Several transcription factors are required for than one aspect of growth or development, suggesting roles in integration of multiple upstream signals. The observation that half of the genes did not produce obvious defects when mutated may result from functional redundancy, which has been reported for other transcription factor genes. The availability of this collection of Neurospora transcription factor mutants will enable future investigations aimed at elucidating the complexity of gene regulation in filamentous fungi.

A High-Density SNP Map for Neurospora crassa.
Randy Lambreghts1, Mi Shi1, David DeCaprio2, James E. Galagan2, Bruce W. Birren2, Jay C. Dunlap1, and Jennifer J. Loros1. 1Department of Genetics, Dartmouth Medical School, Hanover, NH 03755; 2 Broad Institute, Cambridge, MA 02141

A collection of SNPs (single nucleotide polymorphisms) in the Mauriceville strain of Neurospora crassa, relative to the Oak Ridge standard, was established, randomly distributed over the entire genome with an average density of one every 100 kb. To this end, an EST library was constructed from Mauriceville germinating conidia and sequenced. By alignment with the published Oak Ridge genomic sequence, a set of putative SNPs was formulated, out of which a subset was selected based on quality of the sequencing information as well as amenability to CAPS (cleaved amplified polymorphic sequence), some of which were experimentally validated by PCR amplification of genomic DNA followed by differential restriction digest. We present a map containing confirmed SNPs, each one supplemented by a primer pair and a restriction enzyme that has been shown to consistently distinguish between the Oak Ridge and Mauriceville version. Given the possibility of high-throughput assignment of a large number of SNP markers to individual progeny, we expect this information to be invaluable for the rapid mapping of conventionally derived mutations, as well as in the further assembly and orientation of the genomic sequence and in the analysis of complex traits such as QTLs.

Exploring regulatory networks in Neurospora
Chaoguang Tian, Takao Kasuga and N. Louise Glass
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720

Exploring regulatory networks is one of the main functions of post-genomics research. As part of the Neurospora Functional Genomics Program Project grant, we have constructed full genome oligonucleotide microarrays for the filamentous fungus Neurospora crassa using gene annotation provided by The Broad Institute and Munich Information for Protein Sequences (MIPS). Our goal is to define transcriptional regulatory networks in N. crassa, as a model organism for filamentous fungi. By the genome-wide microarrays, we can define transcription factor targets by profiling transcription factor mutant strains. There are at least five predicted DNA-binding transcription factors families in N. crassa: BHLH, BZIP, C2H2, GATA and ZnII(Cys)6. Using phylogenetic analyses of the transcription factor gene families within ascomycete fungi, we have chosen to profile mutants in transcription factor genes that are phylogenetically diverse, plus their closest paralogs; the vast majority of these transcription factors and their regulatory networks are completely uncharacterized. Thirty-five transcription factor mutant strains (generated by the Neurospora Functional Genomics Program Project Grant) have been initially selected for transcriptional profiling. We compare the gene expression profiles of transcription factor knock out strains to wild type across a fungal colony and under different stress conditions. Putative target genes of the transcription factors are subsequently subjected to cis-element analysis. By performing comprehensive transcriptional profiling, cis-element analysis and chromatin immunoprecipitation, the transcription regulatory network of a model filamentous fungus can be constructed.


Session III: Cell Morphogenesis and Assembly
Oded Yarden,


Functio Variabilis: Unravelling the diverse roles of ion transport in hyphal morphogenesis.
Roger R. Lew Biology Department, York University, Toronto, Canada.

During tip growth (hyphal extension), Neurospora relies upon an internally generated Ca2+ gradient to maintain polar growth of hyphae. The tip-high Ca2+ gradient is created by the action of a stretch-activated phospholipase C which produces inositol trisphosphate (IP3) that then activates a Ca2+ channel to cause Ca2+ release at the tip. Ca2+ sequestration behind the tip occurs into endoplasmic reticulum and into a unique population of tip-localized mitochondria. As a walled cell, Neurospora usually relies upon hydrostatic pressure (turgor) to cause tip extension. Upon hyperosmotic stress, an ensemble of ion transporters (the H+ ATPase, and K+ and Cl- transporters) are activated by a MAP kinase cascade to maintain turgor at about 500 kiloPascals. Besides turgor, there are intrahyphal osmotic differences that cause mass flow of cytoplasm which is normally directed towards the growing tips. Thus, ion transport at a number of organelles plays key roles in multiple functions during hyphal growth: Ca2+ gradients mediate tip polarity, trans plasma membrane ion gradients generate the turgor driving force, and intrahyphal ionic gradients mediate cytoplasm migration toward the growing tips.

How is calcium sequestered and does vacuolar calcium play a role in morphogenesis?
Barry Bowman

See poster # 10

The cellular and genetic determination of Woronin body formation in apical hyphal compartments of Neurospora crassa
Gregory Jedd. Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604

Woronin bodies are peroxisome-derived organelles that are centered on a crystalline core of the HEX-1 protein and function as emergency patches of the septal pore. To fully exploit this function, all hyphal compartments need to inherit Woronin bodies and my group has been studying the cellular and genetic control of this process. Time-lapse confocal microscopy has shown that Woronin bodies form in the apical hyphal compartment where they are generally transported in an apically directed manner. These vesicles undergo maturation entailing membrane fission and associate with the cell cortex roughly coinciding with septum formation. Cortical Woronin bodies are immobilized and thereby retained in sub-apical compartments, and by the continuous execution of this process all vegetative compartments are endowed with a roughly uniform number of Woronin bodies. To investigate the genetic control of this process, we determined the requirements for hex-1 gene expression and defined an important role for intron splicing in hex1 mRNA export or stability. We next examined the localization of YFP expressed from hex-1 regulatory sequences and observed a tip-high fluorescent gradient that diminishes towards sub-apical compartments. To directly assess the spatial distribution of various species of mRNA, we developed a method to fractionate the fungal colony into a series of zones corresponding to apical and increasingly sub-apical compartments. Examining RNA from these regions we found that hex-1 mRNA is highly enriched in apical hyphal compartments, whereas other transcripts accumulated in sub-apical compartments. When the hex-1 structural gene was expressed from regulatory sequences of an abundant, sub-apically localized transcript, Woronin body formation was re-determined to this region of the colony. Together, these results define the genetic differentiation of apical hyphal compartments and show that polarized gene expression is a key determinant of apically localized Woronin body-genesis1.
Tey, W.K., North, A.J., Reyes, J.L., Lu, Y.F., Jedd, G. (2005) Polarized gene expression determines Woronin body formation at the leading edge of the fungal colony. Mol. Biol. Cell. 16, 2651-2659.

Glycosphingolipid structure and biosynthesis in Neurospora crassa.
Steven B. Levery1, Chaeho Park2, Beau Bennion1, Elizabeth Owuor1, Isabelle E.J.A. François3, Kathelijne K.A. Ferket3, Bruno P.A. Cammue3, Karin Thevissen3. 1Department of Chemistry, University of New Hampshire, Durham, NH, USA; 2Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA; 3Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Heverlee-Leuven, Belgium.

Glycosphingolipids (GSLs) have been implicated in a number of studies as targets of plant defensin binding to the fungal membrane. For example, sensitivity of the yeasts Pichia pastoris and Candida albicans toward RsAFP2, a defensin isolated from seeds of Raphanus sativus (radish), was found to be dependent on GCS, the gene encoding glucosylceramide synthase (UDP-Glc:ceramide beta- glucosyltransferase). Although Neurospora crassa is not a phytopathogen, it has been used as a system to investigate plant defensin-phytopathogen interactions. Chemically mutagenized N. crassa strains selected for resistance to RsAFP2 were found to have dramatically altered glycolipid expression profiles. Characterizing the true nature of these alterations has required detailed structure elucidation of GSL components isolated from wild type and defensin-resistant mutant N. crassa strains. Key general characteristics of fungal GSL expression at the gene and metabolic levels will be discussed and compared with what we have learned so far from studies of GSL expression in N. crassa.

Biogenesis of mitochondria: Pathways and machineries involved in the import of proteins
Walter Neupert, Institute of Physiological Chemistry, University of Muenchen, Germany

The life of almost all proteins of the mitochondrion begins at ribosomes in the cytoplasm of the cell. In order to reach their active states as constituents of protein assemblies in one of the various mitochondrial subcompartments (the outer membrane, the intermembrane space, the inner membrane or the matrix) they interact with protein translocases of the mitochondria. So far six such molecular machines have been identified: two in the outer membrane (the TOM and TOB complexes), one in the intermembrane space (the Mia1/Erv1 machinery), and three in the inner membrane (the Tim23, TIM22 and OXA1 complexes). In addition, a number of chaperones and co-chaperones in the matrix are required for folding in the matrix space as well as a series of assembly factors for the formation of cofactor containing supramolecular protein complexes of the mitochondria.
The two most recently identified protein translocases are the TOB complex and the Mia40/Erv1 machinery. The TOB complex is responsible for the membrane integration of β-barrel proteins of the mitochondrial outer membrane, such as Tom40 and porin. The TOB complex interacts with the precursors of these proteins during or after their translocation across the outer membrane by the TOM complex. The Mia40/Erv1 machinery is essential for the import into the intermembrane space of members of a family of small proteins with CX3C and CX9C motifs. Oxidative folding leading to formation of disulfide bonds appears to be a step which provides a driving force for translocation across the outer membrane via the TOM complex.
A further focus of our interest is the ATP driven import motor of the mitochondria which is coupled to the TIM23 translocase. Tim44 is a central component which is peripherally associated with the membrane integrated components Tim23 and Tim17, and which organizes the import motor. Tim44 recruits mtHsp70 which binds and, by a ratchet-like mechanism, takes into the matrix unfolded precursor polypeptides that have passed the TOM complex and the channel of the TIM23 translocase. The import motor contains two further Tim44 associated essential proteins, the J-protein, Tim14, and J-like protein, Tim16. The way how they control the activity of the import motor appears to be key to the understanding of the function of the TIM23 complex which is responsible for the import of the vast majority of nuclear encoded inner membrane and matrix proteins of mitochondria.

Function and expression of Tob55/Sam50 in Neurospora crassa: An essential protein for assembly of beta-barrels into the mitochondrial outer membrane
Dr. Frank Nargang, Dept. of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol as mitochondrial precursors, and imported into the organelle. The translocase of the outer mitochondrial membrane (the TOM complex) recognizes and imports all classes of mitochondrial precursors either into, or across, the outer membrane. Further sorting of precursors to the correct mitochondrial sub-compartment is achieved via additional translocase complexes housed in the membranes of the organelle. One of these translocases is The TOB (Topogenesis of Outer Membrane Beta-barrel proteins) or SAM (Syntheses and Assembly Machinery) complex. The major protein of the TOB complex is Tob55. We have constructed a knockout of N. crassa tob55 and have shown that it is essential for the viability of the organism. Reduced levels of Tob55 result in a specific deficiency in the assembly of beta-barrel proteins into the outer membrane. Our studies have also revealed that the N. crassa Tob55 exists in at least three isoforms which appear to be generated via alternative splicing. We are currently investigating the ability of different isoforms to rescue Tob55 function.

The NAD(P)H dehydrogenases of Neurospora crassa mitochondria
Arnaldo Videira. Instituto de Biologia Molecular e Celular (IBMC) and Instituto de Ci ncias Biomédicas de Abel Salazar (ICBAS), University of Porto, Portugal

The proton-pumping NADH dehydrogenase or complex I is a major entry point of electrons into the mitochondrial respiratory chain. It catalyses electron transfer from NADH to ubiquinone through a series of protein-bound prosthetic groups. Complex I deficiencies have been implicated in various mitochondrial diseases. Complex I from the filamentous fungus Neurospora crassa contains at least 39 polypeptide subunits of dual genetic origin, mostly conserved in mammals, suggesting that the enzyme is involved in other cellular processes beyond bioenergetics. Mutations in different subunits have been generated in the last years, including mutations of conserved amino acid residues of iron-sulphur proteins as found in human diseases, in order to reveal the role of the proteins in complex I assembly and function. Complex I is likely regulated by transitions between active (A) and de-activated (D) forms and one of the proteins involved in this phenomenon has been identified. In addition to complex I and depending on the organism, several non-proton-pumping alternative NAD(P)H dehydrogenases may also be present in the inner mitochondrial membrane. The fungus N. crassa contains four alternative NAD(P)H dehydrogenases: the main external NAD(P)H dehydrogenase, an external calcium-dependent NADPH dehydrogenase, a third external enzyme and the single internal NADH dehydrogenase. These proteins appear to have both alternative and complementary functions. Overall, mitochondrial respiratory chain NAD(P)H dehydrogenases have important roles in fungal development.


Session IV: Cell Signaling and Gene Regulation

Nora Plesofsky, Chair

The arginine attenuator peptide: a regulator of translation and mRNA levels.
Matt Sachs, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, OR

The Neurospora crassa arg-2 gene and its fungal homologs encode the arginine-specific carbamoyl-phosphate synthetase (CPS-A) small subunit. Excess arginine decreases translation of arg-2 through the action of an evolutionarily conserved upstream open reading frame (uORF) in the mRNA. This uORF encodes a cis-regulatory element, the arginine attenuator peptide (AAP), which stalls ribosomes in the presence of arginine, thereby decreasing ribosome access to the downstream CPS-A reading frame. We performed an extensive analysis of the sequence requirements for AAP-mediated translational control of ribosome stalling using the N. crassa cell free translation system. The results showed that some but not all of the evolutionarily conserved residues in the AAP sequence were crucial for regulation. Furthermore, we showed using yeast that AAP-mediated ribosome stalling at the uORF stop codon causes the mRNA to be destabilized by inducing the nonsense mediated mRNA decay (NMD) pathway. Preliminary evidence using an N. crassa mutant defective in NMD indicates that the amount of cellular arg 2 mRNA is also controlled at the level of mRNA stability by NMD.

Involvement of the Neursopora mitochondrial tyrosyl-tRNA synthetase and DEAD-box proteins in splicing group I and group II introns.
Alan Lambowitz, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin TX 78712.

Group I and group II introns are commonly found inserted in fungal mtDNA genes. These introns self-splice in vitro, but require proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. Protein factors that function in splicing group I introns were first identified by mutational analysis in Neurospora crassa. The Neurospora mitochondrial tyrosyl-tRNA synthetase (CYT-18) binds specifically to group I introns RNAs and promotes RNA splicing by stabilizing the catalytically active RNA structure. The N. crassa CYT-19 protein functions in conjunction with CYT-18 and is DEAD-box protein that acts as an ATP-dependent RNA chaperone to disrupt stable inactive structures that are kinetic traps during CYT-18-assisted RNA folding. Studies with CYT-18 and CYT-19 have suggested general paradigms for how proteins function in RNA folding and facilitate RNA-catalyzed reactions. They also show how cellular RNA bindings proteins can evolve to function in RNA splicing, and more generally, how essential proteins can acquire new functions.

G proteins and histidine kinases differentially regulate sexual development
Katherine A. Borkovich, Department of Plant Pathology, University of California, Riverside.

Heterotrimeric G protein pathways and two-component regulatory systems control environmental responses in fungi. Our laboratory has demonstrated roles for both of these pathways in regulation of sexual development in Neurospora. The G protein coupled receptors (GPCRs) PRE-1 and PRE-2, along with their cognate pheromone ligands, MFA-1 and CCG-4, are essential for initial recognition between opposite mating type females and males during mating. The heterotrimeric Ga protein GNA-1 and the Gbg dimer GNB-1/GNG-1 are also necessary for female fertility, but in a mating type independent manner. Loss of the G protein subunits or the receptor leads to trichogyne "blindness" in females, while males that do not produce pheromone are unable to attract trichogynes. A change in identity can be accomplished by heterologous expression of the receptor or pheromone in cells of opposite mating type and co-expression of a cognate receptor-pheromone pair leads to self-stimulation. The GPCR GPR-1 is required for proper formation of beaks and ostioles during perithecial development; the coupled Ga is not currently known, but available genetic data points to GNA-1 as the most likely candidate. We have begun analysis of two-component regulatory systems with characterization of the response regulator RRG-1. rrg-1 mutants have an early block in female fertility, in that they do not produce protoperithecia. This defect correlates with loss of stimulation of a downstream mitogen-activated protein kinase pathway in Neurospora

Cytology of conidial anastomosis induction, homing and fusion in Neurospora crassa
M. Gabriela Roca and Nick D. Read. Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh, EH9 3JH, UK.

Conidia of Neurospora crassa form conidial anastomosis tubes (CATs) which are morphologically and physiologically distinct from germ tubes, and under separate genetic control. The dynamic behaviour of nuclei, mitochondria and microtubules during macroconidial germination and fusion was analysed using a range of vital dyes and GFP labelling. We found that: (1) mitosis occurs much more rapidly in germ tubes (~ 15 min) than in ungerminated macroconidia (~ 3.5 h); (2) mitochondria are concentrated within germ tube and CAT tips; (3) both nuclei and mitochondria are physically connected to microtubules; (5) CAT fusion occurs after mitosis has been undergone in ungerminated macroconidia or germ tubes; (6) nuclei do not enter CATs prior to fusion; (7) the nuclei within germ tubes or CATs do not exhibit any special localization which can be associated with the pattern of CAT induction, homing or fusion; (8) microtubules extend through fused CATs from both conidial germlings which have fused with each other; (9) microtubules pass through fused CATs prior to mitochondria which are then followed by nuclei; and (10) organelle fluxes between fused conidial germlings are typically several orders of magnitude slower than those between fused hyphae in the mature colony.

Glycogen metabolism and stress response in Neurospora crassa. Regulation of the gsn gene.
Prof. Dr. Maria Célia Bertolini. Instituto de Química, UNESP, Departamento de Bioquímica e Tecnologia Química R. Prof. Francisco Degni, s/n 14800-900, Araraquara, SP Brazi

The synthesis and degradation of glycogen molecules are carried out by the concerted action of a set of enzymes, the main control being of the activities of glycogen synthase and glycogen phosphorylase, respectively. Glycogen synthase catalyzes the formation of the α-1,4-glycosidic linkages of glycogen by addition of UDP-glucose units into glycogen, and is considered to be the limiting-rate step of the glycogen synthesis. This enzyme is regulated both by allosteric modulation, and by reversible phosphorylation. In addition to reversible changes in the glycogen synthase activity, glycogen levels are also correlated with physiological conditions. In Saccharomyces cerevisiae, glycogen accumulation is induced by conditions that stress the cells, such as heat shock. Such condition induces transcription of the gene encoding glycogen synthase (GSY2), which explains the glycogen accumulation. We have isolated the gene encoding the Neurospora crassa glycogen synthase (gsn) and demonstrated that the gene transcription is repressed when cells are exposed to temperatures varying from 30 to 45ºC (heat shock). In addition, glycogen levels rapidly decrease in the same growth condition. Analysis of the gsn promoter region allowed us to identify multiple regulatory elements, including many HSE (Heat Shock Element) and two STRE (STress Responsive Element), which are usually found in promoters of genes responsive to stress conditions. Gel shift assays (EMSA) using nuclear extract prepared from N. crassa heat shocked mycelia showed the presence of protein(s) that bind specifically to the STRE elements of the gsn promoter region. Our main purpose is to identify the protein(s) that bind to the STRE elements in order to study how these elements function in the regulation of gene transcription. Approaches coupling EMSA and mass spectrometry have been used to reach the goal. Results will be presented concerning the strategies we have used to identify the protein(s).

Heterokaryon incompatibility
N. Louise Glass, Karine Dementhon, Isao Kaneko and Qijun Xiang. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic non-allelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain. These data suggest non-allelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions. Functional VIB-1, which is a putative transcription factor, is required for expression of pin-c, het-6 and tol, all of which encode HET domain proteins. These observations explain why mutations at vib-1 suppress het-c, het-6 and mat incompatibility.

MAK-2 MAP Kinase and cAMP signaling pathways interact to control aerial growth and conidiophore development
Dan Ebbole, Texas A & M University.

The mak-2 and pp-1 mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate genes having reduced expression in the mak-2 mutant.
Expression of some of these genes is protoperithecia specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants.

Session V: Clocks, Light, and Oxygen

Deborah Bell-Pedersen, Chair


Molecular mechanism of light responses in Neurospora
Qiyang He, and Yi Liu. University of Texas Southwestern Medical Center, Dallas, TX.

Blue light regulates many molecular and physiological activities in a large number of organisms. In Neurospora crassa, a eukaryotic model system for studying blue-light responses, the transcription factor and blue-light photoreceptor WHITE COLLAR-1 (WC-1) and its partner WC-2 are central to blue-light sensing. Neurospora’s light responses are transient, i.e. following an initial acute phase of induction, light-regulated processes are down-regulated under continuous illumination, a phenomenon called photoadaptation. The molecular mechanism(s) of photoadaptation are not well understood. Here we show that a common mechanism controls the light-induced transcription of immediate early genes (such as frq, al-3, and vvd) in Neurospora, in which light induces the binding of an identical large WC-1/WC-2 complex (L-WCC) to the light response elements (LREs) in their promoters. Using recombinant proteins, we show that the WC complexes are functional without the requirement of additional factors. In vivo, WCC has a long period photocycle, indicating that it cannot be efficiently used for repeated light activation. Contrary to previous expectations, we demonstrate that the light-induced hyperphosphorylation of WC proteins inhibits bindings of the L-WCC to the LREs. We show that, in vivo, due to its rapid hyperphosphorylation, L-WCC can only bind transiently to LREs, indicating that WCC hyperphosphorylation is a critical process for photoadaptation. Finally, phosphorylation was also shown to inhibit the LRE-binding activity of D-WCC (dark WC complex), suggesting that it plays an important role in the circadian negative feedback loop.

Clocks and Light and Oxygen! Oh My!
Luis Larrondo, Bill Belden, Allan Froehlich, Jay Dunlap and Jennifer Loros. Dept. of Biochemistry, Dartmouth Medical School,

The ascomycete Neurospora crassa has a long standing as a model organism for investigating both circadian rhythms and photoreception (J. J. Loros and J. C. Dunlap, Circadian Rhythms, Photobiology and Functional Genomics in Neurospora. Ch.4, pp 53- 74 in Volume XIII The Mycota. "Fungal Genomics". Editor Alistair J P Brown, 2005). The circadian system of N. crassa involves a number of interlocked molecular feedback loops that regulate the time-of-day-specific expression of a number of output genes, thereby generating distinct phenotypes, including the clock-dependent rhythm of macroconidiation. Light is a major entraining signal to the clock, coordinating the organism with the diurnal environment. Known players involved in circadian and light regulation of these processes include the products of the frq, wc-1, wc-2, and vvd genes. Both VVD and WC-1 have been shown to be blue light photoreceptors; WC-1 is required for circadian entrainment as well as the autoregulatory feedback loop involving the frq gene and VVD has been shown to participate in light signaling to the clock. The recent cloning of a mutation, bd, that allows the easily visualized rhythm in conidiation suggests an imbalance of reactive oxygen species in this mutant strain. Recent work on the clock, light signaling and Ras will be discussed. This work was supported by grants from the National Institutes of Health MH44651 AND NIGMS1P01 GM 068087-01 to J.C.D. and J.J.L. and R37GM34985 to J.C.D., the National Science Foundation MCB-0084509 to J.J.L., and the Norris Cotton Cancer Centre core grant at Dartmouth Medical School.

Coordination of negative and positive functions of FREQUENCY in the circadian clock
Michael Brunner. Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D 69120 Heidelberg

The Neurospora circadian clock protein FREQUENCY (FRQ) has a function in the negative and the positive limb of interconnected feedback loops. In the negative feedback loop FRQ inhibits its transcription factor White Collar Complex (WCC), and in the positive loop it supports accumulation of WCC. These contradictory functions of FRQ are confined to distinct subcellular compartments and coordinated in temporal fashion. Negative feedback occurs early after the onset of FRQ expression and requires nuclear FRQ. Nuclear FRQ promotes hyperphosphorylation of WCC leading to its inactivation. Support of WCC accumulation depends on cytosolic FRQ and occurs about 8 h after the onset of FRQ expression when high amounts of FRQ have accumulated. The transcriptional function of FRQ in the negative limb and its posttranslational function in the positive limb are independent and associated with distinct regions of FRQ. Phosphorylation of serine residues within the PEST-2 region triggers the maturation FRQ of toward a cytoplasmic activator.

Quelling in Neurospora: an overview
Carlo Cogoni, Dip. Biotecnologie Cellulari ed Ematologia, Sez. Genetica Molecolare, Policlinico Umberto I Universita' degli Studi di Roma 'La Sapienza'
Roma, Italy

The introduction of transgenes or double-strand RNAs (dsRNAs) into a variety of eukaryotic cells can trigger a series of post-transcriptional gene silencing mechanisms in which dsRNA intermediate molecules, after being processed into short interfering RNA molecules (siRNA), were identified as strong elicitors of mRNA degradation. Two PTGS mechanisms have been identified in Neurospora: quelling and MSUD (meiotic silencing by unpaired DNA). Quellling occurs during the vegetative phase of the life cycle and was the first PTGS mechanism characterized in this organism.
Several components of the quelling machinery have been identified by using either forward or reverse genetic approaches. The identification of genes required in the silencing process together with findings from other organisms has led to a current model for quelling. In Neurospora, as in other organisms, it would seem that quelling is serving to limit the expansion of transposons since an introduced Tad element, a LINE-1-like retrotransposon, has an elevated expansion in the absence of the quelling components QDE2 and DICER.

Natural variation and the multigenic nature of circadian behavior in Neurospora

Kwangwon Lee. Department of Plant Pathology Cornell University

We studied two under-explored areas in Neurospora biology, natural variation and quantitative trait loci (QTL) analysis. Natural variation in circadian period, phase and temperature compensation optimizes the ability of an organism to synchronize its biological processes to a local environment.  Among a world-wide collection of 144 Neurospora crassa accessions circadian rhythms of asexual conidiation revealed significant variation centered on a 22 hour period and morning specific phase.  Consistent with the phenotypic variation, there is significant genotypic variation among circadian components, WHITE COLLAR-1 (WC-1), WHITE COLLAR-2, FREQUENCY, and VIVID.  Furthermore, we found significant association between circadian parameters and molecular variation in the circadian genes.  WC-1 mediates interactions between the circadian clock and the environment, acting both as a core clock component and a blue light photoreceptor.  Our data show that a putative activation domain in WC-1 is highly polymorphic in length, revealing a significant association between circadian period, latitude of origin and wc-1 genotype.  We suggest that environment specific natural variation at WC-1 fine-tunes circadian period.
In an attempt to characterize the polygenic nature of the circadian clock, we performed QTL analyses in three mapping populations, which were generated by crossing natural accessions, with 188 F1 progenies. At least 80 loci were determined by simple sequence repeat markers for their map position in each population, covering the genome with about 1000 cM. The clock QTLs identified from three experimental populations will be discussed.

Chromatin-Remodeling Enzymes and Circadian Rhythms.
William J. Belden, Jennifer J. Loros, and Jay C. Dunlap.

Neurospora crassa contains a circadian feedback loop that is controlled by daily oscillations in transcription of the frequency gene. The transcription factors White Collar-1 (WC-1) and White Collar-2 (WC-2) activate frq expression in a circadian and light dependent manner, and are thought to act together as heteromeric complex. To better understand regulated events at the frq promoter, we deleted genes homologous to the swi/snf family of ATP-dependent chromatin-remodeling enzymes. The 19 putative chromatin-remodeling factors were knocked out by gene replacement and characterized; one was essential for growth, another was ascospore lethal and a third had a defect in circadian regulated spore formation, and is now designated clockswitch (csw-1). To further define events at frq, we used ChIP and nuclease accessibility assays to examine how nucleosome modifications might regulate circadian relevant transcription. The WC proteins do not act solely as an obligate complex because in vivo binding of WC-2 to the frq promoter occurs in a rhythmic fashion with the peak in binding occurring coincident with the peak in frq transcription. A basal level of WC-1 is associated with the promoter at all circadian times and only slight increases are observed when frq is transcribed. There is marked reduction in the level of acetylated histone H3 upon light induced transcription. Chromatin rearrangements at frq are seen when the gene is expressed and ChIP assays indicate that CSW-1 is localized to this region, suggesting a direct role for this chromatin-remodeling enzyme in regulating frq expression.

Cell differentiation as a response to oxidative stress
Leonardo Peraza1, Nallely Cano2, Mauricio Rios1, Jesús Aguirre2, and Wilhelm Hansberg1 1Departamento de Bioquímica and 2Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM) Apartado postal 70- 242, México, 04510 D. F.

Different stress conditions in Neurospora lead to an unstable state in which formation of reactive oxygen species surpass the antioxidant capacity of the cell. Cell differentiation is one possible response to this inevitably transient hyperoxidant state. The hypothesis predicts that disruption of anti-oxidant enzymes should intensify cell differentiation processes; deletion of pro-oxidant enzymes should inhibit them. Catalase and NADPH oxidase genes were disrupted to find that cat-3 mutant increased asexual and sexual sporulation, cat-2 mutant increased submerged conidiation, nox-1 decreased asexual and inhibited sexual sporulation, and nox-2 inhibited ascospore germination (1). sod-1 deletion mutant presented circadian conidiation, enhanced aerial and submerged conidiation, and diminished protoperithecia formation. sod-1 phenocopies bd, which is a dominant mutation probably in ras-1. Both strains seem to produce a cyclic oxidative stress that leads to cyclic conidiation. 1) Aguirre J; Rios-Momberg M; Hewitt D; Hansberg W (2005) Reactive oxygen species and development in microbial eukaryotes. Trends in Microbiology 13:111-118.

Please note, some talks were selected from posters and their abstracts appear here.

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