Source: UNIVERSITY OF CALIFORNIA, BERKELEY submitted to
MOLECULAR CHARACTERIZATION OF DETERMINANTS OF SELF AND NONSELF RECOGNITION IN THE MODEL FILAMENTOUS FUNGUS, NEUROSPORA CRASSA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0220412
Grant No.
(N/A)
Project No.
CA-B-MIC-0017-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Glass, N.
Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
Microbial Biology
Non Technical Summary
Filamentous ascomycete fungi are the major cause of diseases of crop plants, including both pre-harvest diseases, such as rice blast caused by the fungus Magnaporthe grisea, or the ubiqitous root pathogen, Fusarium oxysporum and post-harvest rots, caused for example, by Botryis cinerea and Aspergillus niger. In addition filamentous ascomycete also cause serious and often fatal diseases in humans, for example, Valley Fever, caused by Coccidioides immitis, and in immunocompromised patients, such as Aspergillosis, caused by Aspergillus fumigatus and A. flavus. New compounds to combat fungal diseases, especially in humans and animals, often have very toxic side effects, because they are not generally not specific in their action. We have characterized a pathway that induces cell death in filamentous ascomycete fungi, called heterokaryon incompatibility (HI) that they use in nature to combat infection by mycoviruses; HI is a type of fungal "immune system". We have identified a number of proteins that are required for induction of fungal death; these proteins are only found in the genomes of filamentous ascomycete fungi and are absent in plants, humans and animals. These include a fungal-specific transcription factor, Vib-1 and its putative targets, the HET domain proteins. However, it is not clear how Vib-1 mediates death, nor the role of the HET domain proteins in this process. This proposal aims to dissect the direct transcriptional target of Vib-1 and to identify proteins that physically interact with the HET domain proteins, to characterize fully the pathway of HI-induced cell death. The characterization of this pathway may led to the indentification of critical components that we could use as targets for induction of the death pathway in a large number of filamentous fungi, thus convincing to organism to "kill thyself".
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124020103010%
2124020104010%
2124020108010%
2124020110210%
7124020104010%
7124020108010%
7124020110210%
7234020104010%
7234020108010%
7234020110210%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 212 - Pathogens and Nematodes Affecting Plants; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1080 - Genetics; 1030 - Cellular biology; 1102 - Mycology; 1040 - Molecular biology;
Goals / Objectives
The ability to distinguish oneself from another is a ubiquitous phenomenon among all living things. As a filamentous fungal colony grows across a substrate in nature, it will encounter many other microorganisms, including bacteria, fungal colonies of the same or of different species, protist species and insects. Hyphal fusion and nonself recognition between genetically different isolates of the same fungal species can result in compartmentalization and death of hyphae, a phenomenon termed "heterokaryon incompatibility" (HI). HI is a type of programmed cell death (PCD) and is ubiquitous in filamentous ascomycete fungi. We dissect the molecular and cellular basis of HI and PCD in the model filamentous ascomycete fungus, Neurospora crassa. We determined that predicted filamentous fungal specific genes containing HET domains are often involved in HI; the function of the HET domain is unknown. Over-expression of just the HET domain is sufficient to induce PCD in N. crassa. Genes in N. crassa that encode proteins predicted to be involved in apoptosis, which is a conserved pathway of cell death, are not required for PCD induced by HI. These data suggest a novel pathway for induction of death via the HET domain proteins. We identified a transcription factor, VIB-1, which is required for HI and determined that a Δvib-1 deletion mutant is deficient in protein secretion and is unable to utilize complex substrates. These data suggest a link between diffusible factors and the triggering of fungal specific PCD. The identification and characterization of targets of Vib-1 is a specific aim of this project. These secreted compounds could be utilized as specific death inducing compounds for treatment of plant, human and animal diseases caused by filamentous fungi. Objective 1: Identify direct targets of the transcription factor Vib-1 by performing chromatin-immunoprecipitation and high throughput Illumina sequencing. Objective 2: Evaluate strains containing mutations in direct target genes for ability to utilize complex substrates (secretion affected) versus ability to undergo PCD, with special emphasis on any predicted secreted proteins.Objective 3: Characterize the function of the proteins encoded by genes that are directly regulated by Vib-1 and are required for PCD. Objective 4: Identify interactions of HET domain proteins. Expected outputs: We have determined that PCD induced by HI is a fungal specific pathway and that it may involve secretion of either a peptide or a small molecule. We predict the identification of effectors of HI-induced PCD will provide a means to specifically induce PCD in a wide variety of filamentous ascomycete fungi due to the high conservation of this phenomenon in filamentous ascomycete fungi and that this information will be of great interest to companies developing fungal-specific drugs for both human/animal and plant diseases caused by fungi. This project will entail the training of both post-doctoral associates and graduate students in molecular biology, genetics and biochemical methods and will include a number of research publications.
Project Methods
Objective 1: Identify direct targets of Vib-1 by performing chromatin-immunoprecipitation and high throughput Illumina sequencing. Vib-1 epitope-tagged constructs at both the N and C-terminal regions will be constructed using a vector containing green fluorescent protein (GFP), a histidine tag and a TEV protease site. Constructs will be electroporated into a Vib-1 deletion strain (Δvib-1). Complementation of the secretion phenotype will be determined. Nuclear localization of Vib-1-GFP may vary either across the filamentous fungal colony or under different nutritional conditions. The Δvib-1 (vib-1-gfp) complemented strains will be grown under normal vegetative conditions, under HI and on a complex substrate (such as protein). Cells will be cross-linked with formaldehyde, sonicated and the supernatant subjected to differential centrifugation to enrich for nuclei. Nuclei will be lysed and chromatin-immunoprecipitation using anti-GFP antibodies will be performed. Libraries for ChIPSeq (2) will be constructed for high-throughput sequencing. Mapping and statistical analysis of reads to identify enriched sequences will be mapped onto the N. crassa genome sequence using Maq (3) to identify promoter regions bound by Vib-1. Objective 2: Evaluate strains containing mutations in direct target genes for ability to utilize complex substrates (secretion affected) versus ability to undergo PCD, with special emphasis on any predicted secreted proteins. We have a genome deletion strain set for N. crassa consisting of ~10,000 strains with mutations in ~7000 genes. We will recover strains that contain a deletion in the predicted target of the Vib-1 transcription factor identified via ChIP-seq. We will use the knockout cassette provided by Dartmouth for construction of our own mutants via homologous recombination (1). The ability of a deletion strain to undergo HI will be evaluated via crosses to strains containing alternative HET domain genes and will be grown on variety of substrates to assess effects on secretion. Carboxypeptidase secretion in wild-type and in the mutants will be evaluated biochemically. Objective 3: Characterize the function of the proteins encoded by genes that are directly regulated by Vib-1 and are required for PCD. Molecular and phylogenetic methods will be used to evaluate the conservation of target genes, conservation of domains. Mutational analyses will be performed to identify domains/regions required for activity. The predicted proteins will be tagged with GFP or another fluorescent protein and localization either during HI or during growth on a complex substrate will be assessed. Objective 4: Identify interactions of HET domain proteins. Previously, we determined that functional Vib-1 was required for expression of a set of HET domain genes. We will evaluate interactions between proteins encoded by Vib-1 target genes and HET domain proteins via yeast two-hybrid interactions (4). 1.Colot, H. V., et al. 2006. PNAS 103:10352-10357. 2.Johnson, D. S., et al. 2007. Science 316:1497-502. 3.Li, H., J. Ruan, and R. Durbin. 2008. Genome Res. 4.Suter, B., S. Kittanakom, and I. Stagljar. 2008. Curr Opin Biotechnol 19:3

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: The primary target audience of our research is other scientists. During this reporting period, I also made a presentation to the general public on the "Art and Science of Fabulous Fungi" to the general public (Feb 2014), in addition to a presentation to the Scientific Advisory Committee for The Energy BIosciences Institute. I also presented research seminars at the Cellular and Molecular Fungal Biology Conference Gordon Research Conference (June 2014), Dartmouth University (April 2014), The Kluyver Institute (Netherlands Jan 2014), International Mycological Congress (Aug 2014), European Fungal Genetics Conference (March 2014), University of Ilinois Urbana-Champaign (April 2014), Joint Bioenergy Institute (JBEI) (September 2014), University of Georgia (Jan 2014), Texas A&M (Feb 2015) and the Joint Genome Institute (March 2014). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This work supported a number of graduate students and post-doctoral associates. In addition, five undergraduate worked with post-doctoral associates on components of this project. A number of these undergraduate students have been acknowledged in publications that have resulted from this work. How have the results been disseminated to communities of interest? Our work has been published in high impact scientific journals (PLoS Genetics, Molecular Microbiology, Eukaryotic Cell, MBio, Genetics and J Biol Chem). In addition, our work was featured in the NY Times Science Video http://www.nytimes.com/video/science/100000002564178/sciencetake-the-fungus-highway.html What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective #1: We showed that VIB-1 plays a role in nutrient signaling and is required for deconstruction of lignocellulose (Xiong et al., PLoS Genetics 2014; Xiong et al., FGB 2014). To achieve this objective, we took a transcriptional profiling/phosphoproteomics approach to identify vib-1 targets and phosphoproteomic changes associated with plant cell wall deconstruction. We also perform chromatin-immunoprecipitation and high throughput sequencing of epitope-tagged VIB1 strains. These data were quite noisy, however, which will require additional datasets to illuminate direct targets of VIB1. Objective #2: Previously, we showed at mutations in a protein kinase, IME2, restored PCD in a vib-1 deletion strain. We have shown that a putative target of IME2, identified by computational analyses, and which encode another protein kinase, STE20, suppresses PCD induced in a ime2 deletion strain. These data suggest that IME2 regulates a parallel PCD pathway to that regulated by VIB1. These data have been completed and a manuscript is in preparation. Objective #3 AND #4: We determined the genome sequence of 25 wild N. crassa isolates from a single population from Louisiana sugarcane fields. Using a comparative genomics approach, we identified 15 additional HET/nonself recognition loci that display the characteristics unique to loci under balancing selection. Using functional analyses, we showed that allelic differences at one of these loci (het-e) triggers nonself recognition and PCD. Nonself recognition and PCD mediated by allelic differences at het-e were dependent upon functional VIB1. Cell fractionation showed that HET-E localized to the cytoplasm and the endomembrane of the cell, but interactions between alternative HET-E proteins were not detected. This work has been submitted to Molecular Biology and Evolution. Exploring the relationship between cell fusion and PCD: For PCD to occur in filamentous fungi, cell fusion must first occur. We identified components of a fusion pathway that when mutated result in cell lysis and which was dependent upon extracellular calcium concentration. These data suggest linkage between the membrane merger process and PCD, at least in some cases. This led to a publication in Molecular Microbiology (Palma-Guerrero et al., 2014) and a more recent publication in Eukaryotic Cell (Palma-Guerrero et al., 2015).

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Leeder AC, Jonkers W, Li J, Glass NL. Early colony establishment in Neurospora crassa requires a MAP kinase regulatory network. Genetics. 2013 Nov;195(3):883-98. doi: 10.1534/genetics.113.156984. Benz JP, Chau BH, Zheng D, Bauer S, Glass NL, Somerville CR. A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations. Mol Microbiol. 2014 Jan;91(2):275-99. doi: 10.1111/mmi.12459. Znameroski EA, Li X, Tsai JC, Galazka JM, Glass NL, Cate JH. Evidence for transceptor function of cellodextrin transporters in Neurospora crassa. J Biol Chem. 2014 Jan 31;289(5):2610-9. doi: 10.1074/jbc.M113.533273. Palma-Guerrero J, Leeder AC, Welch J, Glass NL. Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa. Mol Microbiol. 2014 Apr;92(1):164-82. doi: 10.1111/mmi.12545. Ellison CE, Kowbel D, Glass NL, Taylor JW, Brem RB. Discovering functions of unannotated genes from a transcriptome survey of wild fungal isolates. MBio. 2014 Apr 1;5(2):e01046-13. doi: 10.1128/mBio.01046-13. Roche CM, Glass NL, Blanch HW, Clark DS. Engineering the filamentous fungus Neurospora crassa for lipid production from lignocellulosic biomass. Biotechnol Bioeng. 2014 Jun;111(6):1097-107. doi: 10.1002/bit.25211. Gon�alves AP, Hall C, Kowbel DJ, Glass NL, Videira A. CZT-1 is a novel transcription factor controlling cell death and natural drug resistance in Neurospora crassa. G3 2014 Apr 8;4(6):1091-102. doi: 10.1534/g3.114.011312. Kubicek CP, Starr TL, Glass NL. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annu Rev Phytopathol. 2014;52:427-51. doi: 10.1146/annurev-phyto-102313-045831. Xiong Y, Sun J, Glass NL. VIB1, a link between glucose signaling and carbon catabolite repression, is essential for plant cell wall degradation by Neurospora crassa. PLoS Genet. 2014 Aug 21;10(8):e1004500. doi: 10.1371/journal.pgen.1004500.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audience includes other researchers, including those that work on filamentous fungi and those that work on other organisms, including protists, nematodes and mammalian cells. In addition, ourteach activities to local bay area mycological socieities are routinely presented in addition to presentations to other interested groups. During the time period of January 1, 2013 to Sept 30, 2013, scientific presentations included Plenary speaker at the 10th Annual Kluyver Symposium in the Netherlands (Jan 2013) Plenary speaker at the European Federation of Biotechnology meeting in Germany (March 2013) Symposium speaker at the Mycological Socieity of America meeting in Austin, TX (August 2013) Outreach presentations included a seminar for an Israeli delegation to the Energy Biosciences Institute (April 2013) Changes/Problems: We have three objectives in the laboratory. The first is to understand the molecular mechanism of self recognition and fusion. The second is to understand how nonself recognition and death is triggered with fusion occurs between genetically dissimilar isolates. The third objective is to dissect the molecular mechanism of plant cell wall deconstruction in N. crassa and how enzymes important for deconstruction are secreted from the cell. What opportunities for training and professional development has the project provided? Undergraduates: John-Allen Smith was involved in general laboratory maintenance and has been working on a research project under the supervision of post-doc Wilfried Jonkers. Graduate students: Monika Fischer, Jason Liu, Christy Roche, Vincent Wu and Sam Coradetti. All students have taken courses conducted by the Computational Genomics Resource Laboratory, in addition to their required courses. All attended scientific conferences last year. All participated in CalDay, either through EBI or PMB. Post-doctoral scholars: Lori Huberman (09/01/2013), Javier Palma-Guerrero, Yi Xiong, Timo Schurg, Morgann Reilly, James Craig, Wilfried Jonkers, Jens Heller, Juihai Zhao and Joanna Beuche. All post-docs attended scientific meetings last year and two of them gave oral presentations and all presented posters. Several post-docs wrote grant applications for external funding, which I edited and discussed. All participated in the Post-doctoral association and several contributed to CalDay. How have the results been disseminated to communities of interest? Fungal Genetics Meeting, Asilomar CA (March 2013) Mycological Society of America meeting (August 2013) Kluvyer Symposium, The Netherlands (Jan 2013) European Federation of Biotechnology (March 2013) American Society of Microbiology (May 2013) Delegation of Israeli scientists, engineers and political scientitists (April 2013) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have used genomic approaches by determining the genome sequence of ~120 wild isolates from a single population of Neurospora crassa from Louisiana. From analyses of these genome sequences, we have identified new genes involved in self and non-self recognition and genes that are important for death of germling upon fusion of cell of unlike genotypes. This is one of the first applications of genome wide association studies to microbial systems. Our focus on genes that limit fusion of asexual spores or death of asexual spores if fusion occurs, will provide additional targets for fungicide development, as asexual spores are most often the infective propagule in the spread of plant disease in the field. 1. Analyses of the genomes of ~120 isolates to identify genes involved in self recognition and fusion. Understanding how genomes encode complex cellular and organismal behaviors has become the outstanding challenge of modern genetics. Unlike classical screening methods, analysis of genetic variation that occurs naturally in wild populations can enable rapid, genome-scale mapping of genotype to phenotype with a medium-throughput experimental design. Here we describe the results of the first genome-wide association analysis (GWAS) used to identify novel loci underlying trait variation in a microbial eukaryote, harnessing wild isolates of the filamentous fungus Neurospora crassa. We genotyped each of a population of wild Louisiana strains at 1 million genetic loci genome-wide, and we used these genotypes to map genetic determinants of microbial communication. In N. crassa, germinated asexual spores (germlings) sense the presence of other germlings, grow toward them in a coordinated fashion, and fuse. We evaluated germlings of each strain for their ability to chemically sense, chemotropically seek, and undergo cell fusion, and we subjected these trait measurements to GWAS. Our results establish protein secretion as a linchpin of germling communication in N. crassa and shed light on the regulation of communication molecules in this fungus. Our study demonstrates the power of population-genetic analyses for the rapid identification of genes contributing to complex traits in microbial species. 2. A fungal colony is a syncytium composed of a branched and interconnected network of cells. Chimerism endows these colonies with increased virulence and ability to exploit nutritionally complex substrates [1]. Moreover, chimera-formation may be a driver for diversification at the species level by allowing lateral gene transfer between strains that are too distantly related to hybridize sexually [2]. However, the processes by which genomic diversity develops and is maintained within a single colony are little understood. In particular, both theory and experiments show that genetically diverse colonies may be unstable and spontaneously segregate into genetically homogenous sectors [3]. By directly measuring patterns of nuclear movement in the model ascomycete fungus Neurospora crassa, we show that genetic diversity is maintained by complex mixing flows of nuclei at all length scales within the hyphal network. Mathematical modeling and experiments in a morphological mutant reveal some of the exquisite hydraulic engineering necessary to create the mixing flows from spatially coarse pressure gradients. In addition to illuminating multinucleate and multigenomic lifestyles, the adaptation of a hyphal network for mixing nuclear material provides a previously unexamined organizing principle for understanding morphological diversity in the more-than-a-million species of filamentous fungi. We inititated a project to evaluate the transcriptional regulatory network associated with the ability of Neurospora crassa to degrade plant cell wall material, which is its natural habitat. 1. Cellulose is recalcitrant to deconstruction to glucose for use in fermentation strategies for biofuels and chemicals derived from lignocellulose. In Neurospora crassa, the transcriptional regulator, CLR-2, is required for cellulolytic gene expression and cellulose deconstruction. To assess conservation and divergence of cellulase gene regulation between fungi from different ecological niches, we compared clr-2 function with its ortholog (clrB) in the distantly related species, Aspergillus nidulans. Manipulation of clr-2 orthologs among filamentous fungi may enable regulated cellulosic enzyme production in a wide array of culture conditions and host strains, potentially reducing costs associated with enzyme production for plant cell wall deconstruction. However, this functionality may require additional engineering in some species. 1. Examination of the role of ACS enzymes in Neurospora crassa Acyl-CoA synthetase (ACS) enzymes catalyze the activation of free fatty acids to CoA esters by a two-step thioesterification reaction. Activated fatty acids (FAs) participate in a variety of anabolic and catabolic lipid metabolic pathways, including de novo complex lipid biosynthesis, FA β-oxidation, and lipid membrane remodeling. Analysis of the genome sequence of the filamentous fungus Neurospora crassa identified seven putative fatty acyl-CoA synthetases (ACS-1 through ACS-7). ACS-3 was found to be the major activator for exogenous FAs for anabolic lipid metabolic pathways, and consistent with this finding, ACS-3 localized to the endoplasmic reticulum, plasma membrane, and septa. Double mutant analyses confirmed partial functional redundancy of ACS-2 and ACS-3. ACS-5 was determined to function in the siderophore biosynthesis, indicating that not all ACSs identified through the bioinformatic analysis are true fatty ACSs. The N. crassa ACSs involved in activation of FAs for catabolism were not specifically defined, presumably due to functional redundancy of several of ACSs for catabolism of exogenous FAs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Coradetti, S.T., Y. Xiong and N. L. Glass, 2013. Analysis of a conserved cellulose transcriptional regulator reveals inducer-independent production of cellulolytic enzymes in Neurospora crassa. Microbiologyopen 2:595-609
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Palma-Guerrero, J., C. R. Hall, D. Kowbel, J. Welch, J. W. Taylor, R. B. Brem and N. L. Glass, 2013. Genome wide association identifies novel loci involved in fungal communication. PLoS Genetics 9:e1003669
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Roper, M., A. Simonin, P. C. Hickey, A. Leeder and N. L. Glass, 2013. Nuclear dynamics in a fungal chimera. Proc Natl Acad Sci USA 110:12875-12880.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Roche, C. M., H. W. Blanch, D. S. Clark and N. L. Glass, 2013. Physiological role of acyl-CoA synthetase homologs in lipid metabolism in Neurospora crassa. Eukaryot Cell 12:1244-1257
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Glass, N. L., M. Schmoll, J. H. Cate and S. Coradetti, 2013. Plant cell wall deconstruction by ascomycete fungi. Annu Rev Microbiol 67:477-498
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Znameroski, E. A. and N. L. Glass, 2013. Using a model filamentous fungus to unravel mechanisms of lignocellulose deconstruction. Biotechnol Biofuels 6:6. doi: 10.1186/1754-6834-6-6.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Activities: We have included an investigation into the relationship between cell fusion and cell death in Neurospora crassa. In the past year, I mentored three post-doctoral fellow on programmed cell death, Juihai Zhao, Joanne Bueche and Jens Heller. Jens Heller is the cross-over post-doctoral associate with the cell fusion group, which includes post-doctoral associates, Dr. Javier Palma-Guerrero and Wilfried Jonkers, as well as rotation graduate student, Monika Fischer. In addition, Jane Ly, is an undergraduate student working with Dr. Bueche on this project. Events: Drs. Zhao and Dr. Bueche presented their work on cell death in N. crassa as a poster at the International Neurospora Conference (March 201s). Dr. Palma-Guerrero was an invited speaker at the Neurospora conference (March 2012). I was an invited plenary speaker at the Neurospora satellite meeting at the European Fungal Genetics meeting (March 2012) entitled "The Social Network: Deciphering Fungal Language during Conidial Germination". Products: As a service to the community, we have RNA-seq data from 112 wild isolates from N. crassa as well as genome sequence for an additional 25 isolates. These data are kept as a database in our laboratory server that individual researchers wish to evaluate for their specific phenotype among the 112 strains are given access to for analyses. Dissemination: We have collaborations with a computational biologist (Dr. Rachel Brem, MCB, UCB) and a population geneticist (Dr. John Taylor, PMB, UCB) to develop our database of 112 isolates, for which we have evaluated by cell fusion and cell death phenotypes as an example for this use of these data to the larger filamentous fungal research community. We have also continued our collaboration with Dr. Sven Saupe by sequencing the genome of additional Podospora anserina strains, as well as Dr. Arnaldo Videria, as one of his graduate students spent 3 months in my laboratory in 2012. We have collaborated with Dr. Feng Yang at the Pacific Northwest National Laboratories on phosphoproteomics in N. crassa. PARTICIPANTS: N. Louise Glass (PI) provided mentoring, data evaluation and manuscript preparation. Joanna Bueche (co-author on 2012 paper in Genetics), post-doctoral associate investigating the function of the IME-2 in cell death in N. crassa. Juihai Zhao, a post-doctoral associate working on the full genome analyses and function of non self recognition loci (het loci). Dr. Javier Palma-Guerrero works on plasma membrane merger during cell fusion in N. crassa, and is the senior post-doctoral associate of the team. Dr. Wilfried Jonkers works on the cell fusion phosphoproteome and proteins involved in cell signaling and chemotropic interactions. Dr. Charles Hall, a post-doctoral associate and computational biologist, worked with us on the development of the community database that includes sequence analyses of the 112 wild isolates. We collaborate with Dr. Rachel Brem (MCB) and Dr. John Taylor (PMB) on this project. David Kowbel, a research associate and Juliet Welch, a visiting scientist, have also contributed to the database or strain collection. Our Partner Organizations: CNRS France. We have a long time collaboration with Dr. Sven Saupe in Bordeaux France on comparative analyses of heterokaryon incompatibility/programmed cell death loci in various filamentous fungi. University Porto, Portugal. We have a long time collaboration on comparative analyses of drug induced versus heterokaryon incompatibility induced programmed cell death in Neurospora crassa with Dr. Arnaldo Videira. TARGET AUDIENCES: The scientific research community is our target audience, particularly the filamentous fungal research community. Publication of a number of articles that reveal mechanistic aspects of heterokaryon incompatibility/programmed cell death versus cell fusion. I have also given presentations to amateur mycologists involved in the Bay Area Mycological Society on communication in fungi. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge: We characterized the relationship between IME-2, a protein kinase and VIB-1, a transcription factor that is required for programmed cell death in N. crassa. We have used genomic approaches to identify all nonself recognition loci in N. crassa and to experimentally test hypotheses regarding function. We have identified the phosphoproteome of N. crassa, the first for a filamentous fungus, with a focus on identifying phosphorylation targets during cell fusion. We showed that the network associated with the growth habit of filamentous fungi is required for optimal nutrient and organelle distribution in a colony of N. crassa. These are new and important concepts for the function of the hyphal network in filamentous fungi. Change in actions: Other investigators continue to use our datasets for their own research. Change in conditions: None

Publications

  • Hutchison, E.A, J. Bueche and N. L. Glass, 2012. Diversification of a protein kinase cascade: IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa. Genetics 192:467-482.
  • Simonin, A., J. Palma-Guerrero, M. Fricker and N. L. Glass, 2012. The physiological significance of network organization in fungi. Eukaryot Cell 11:1345-1352.
  • Richards, F., N. L. Glass and A. Pringle, 2012. Cooperation among germinating spores facilitates the growth of the fungus Neurospora crassa. Biol Letts 8:419-422.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Activities: Mentored Elizabeth Hutchison, who graduated in spring 2011, with a Ph.D. Completed two research papers and am currently editin two additional research papers from Dr. Hutchison's thesis research. Recruited two additional post-doctoral associates, Drs. Joanna Bueche and Juihai Zhao to the Heterokaryon Incompatibility/Programmed Cell Death project. Successfully obtained additional funding for this project from the National Institutes of Health. Events: Dr. Hutchison presented her work on heterokaryon incompatibility/programmed cell death at the Fungal Genetics Conference, Asilomar CA March 2012. Products: We have developed a database for Neurospora researchers that includes RNA-Seq data for 100 wild isolates of N. crassa. In addition, we determined the DNA sequence of 4 Podospora anserina strains. The tools have been made available to the research community and are being used to identify new heterokaryon incompatibility/programmed cell death loci. Dissemination: We have continued our collaborations with the research group of Professor Sven Saupe (Podospora anserina), CNRS, Bordeaux France and Professor Arnaldo Videira (N. crassa), University of Porto, Portugal. Professor Videira submitted an IDF to the University of California-Berkeley Office of Technology and Licensing on our collaborative work. PARTICIPANTS: N. Louise Glass (PI) provided mentoring, data evaluation and manuscript preparation. Elizabeth Hutchison (graduate student) performed experiments, evaluated data and prepared manuscripts. Juliet Welch (part-time technician) maintained the Neurospora culture collection and grew strains for RNA-Seq analyses. Partner Organizations: CNRS France. We have a long time collaboration with Dr. Sven Saupe in Bordeaux France on comparative analyses of heterokaryon incompatibility/programmed cell death loci in various filamentous fungi. University Porto, Portugal. We have a long time collaboration on comparative analyses of drug induced versus heterokaryon incompatibility induced programmed cell death in Neurospora crassa with Dr. Arnaldo Videira. Dr. John Taylor, University of California-Berkeley. We developed the database of RNA-Seq/genomic DNA sequence of wild Neurospora isolates as a collaborative effort. TARGET AUDIENCES: Target audiences: Scientific research community. Publication a number of articles that reveal mechanistic aspects of heterokaryon incompatibility/programmed cell death. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: We identified new components of the heterokaryon incompatibility/programmed cell death pathway and identified new cellular components associated (mitochondria), not previously associated with heterokaryon incompatibility/programmed cell death in filamentous fungi. By comparative genomics, we identified ~10 potential heterokaryon incompatibility/programmed cell death loci and confirmed that one of them induces cell death. These both constitute significant progress in our understanding of heterokaryon incompatibility/programmed cell death and are being incorporated into two separate publications. Change in actions: Other investigators are using these datasets to identify heterokaryon incompatibility/programmed cell death loci and mechanisms in plant pathogenic fungi. Change in conditions: Our preliminary data allowed us to secure additional funding from the National Institutes of Health, to characterize heterokaryon incompatibility/programmed cell death as a potential drug target for human fungal diseases.

Publications

  • Fernandes, A.S., A. P. Goncalves, A. Castro, T. A. Lopes, R. Gardner, N. L. Glass and A. Videira, 2011. Modulation of fungal sensitivity to staurosporine by targeting proteins identified by transcriptional profiling. Fungal Genet Biol 48:1130-1138.
  • Ellison, C.E., C. Hall, D. Kowbel, J. Welch, R. B. Brem, N. L. Glass and J. W. Taylor, 2011. Population genomics and local adaptation in wild isolates of a model microbial eukaryote. Proc Natl Acad Sci USA 108:2831-2836.
  • Hutchison, E. and N. L. Glass, 2012. Programmed cell death and heterokaryon incompability in filamentous fungi In Biocommunication of Fungi. G. Witzany, Ed. Springer Science, The Netherlands. (in press)


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Programmed cell death in filamentous fungi is a rapid, fungal specific process that has potential as a target for the control of plant, human and animal diseases caused by filamentous fungi. PCD in filamentous fungi requires genes that are filamentous fungal specific and apparently evolved independently from mechanisms for death transduction in other eukaryotic cells. I have mentored three undergraduate students during the past year, Nick Salem, Rachel Dean and Viktoria Betin, four graduate students, Betsy Hutchison, Anna Simonin, Elizabeth Znameroski and Sam Coradetti. In addition, I mentor 8 post-doctoral associates, Javier Palma-Guerrero, Abigail Leeder, Trevor Starr, Jianping Sun, Charles Hall, James Craig, Marcus Roper and Morgan Reilly. We train a number of undergraduates in molecular biology techniques, some of whom continue to pursue independent research in my laboratory. One previous undergraduate (Ms. Mabel Yang) is a co-author on a published research paper during the past year (FG&B 2010). We have continued a collaboration on PCD with Dr. Arnaldo Videria, a Professor at the University of Porto. Dr. Videira spent a sabbatical in my laboratory, where he investigated PCD induced by stress treatments. This collaboration led to a publication in 2010 and one currently submitted. Students presented their work at the Neurospora Meeting (Mar 2010) and the Mycological Society meeting (July 2010). During the past year, I presented our work at a meeting at the International Fungal Biology Meeting at Ensenada, Mexico, the University of California-Davis, Plant Pathology Department, The Vienna University of Technology, Vienna Austria, The International Mycological Congress (IM9) in Edinburgh, The Cellular and Molecular Fungal Biology Gordon Research Conference and the Genencor Corporation. PARTICIPANTS: Most significant to this project is mentoring of undergraduates Nick Salem and Rachel Dean and graduate students Betsy Hutchison, Anna Simonon and post-doctoral associates Dr. Charles Hall and Javier Palma-Guerrero and volunteer scientist, Juliet Welch. In addition, I mentor graduate students Elizabeth Znameroski and Sam Coradetti, post-doctoral associates Abigail Leeder, Trevor Starr, Jianping Sun, James Craig, Marcus Roper and Morgan Reilly and technician David Kowbel. TARGET AUDIENCES: Our target audiences are researchers that work on filamentous fungi, particularly, researchers that work on both plant pathogenic and human pathogenic fungi. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We recently determined that there is a link between cell fusion processes in filamentous fungi and programmed cell death. In particular, N. crassa can distinguish self versus nonself at a distance during interactions of germinated asexual spores. These observations indicate that fungi communicate whether there are the same or different, which directly affects fusion frequency and PCD. We have developed a new paradigm for how nonself recognition triggers programmed cell death that is an apparently fungal-specific process that requires genes/proteins that are only found in filamentous fungi. We evaluated the evolution of nonself recognition loci on a genomic level and identified new loci involved in nonself recognition and cell death by comparative genomic approaches. We developed technology to transit from expression profiling using microarrays to high throughput sequencing methods using Illumina/Solexa technology. We identified and are in the process of characterizing additional mutants/genes required for PCD in Neurospora crassa. Our work has provided a model for PCD in filamentous fungi, which has informed researchers working on this issue with plant pathogenic fungi. PCD is associated with plant infection and also affect population structure of filamentous fungi, such as pathogen of rice, Magnaporthe grisea (researchers mutated orthologous genes in this organism and assessed their role in pathogenicity) and in human pathogens, such as Candida albicans (we compared expression profiles induced by exposure to stress in Neurospora versus Candida). Filamentous fungi are a major cause of plant and animal disease and yet our knowledge about these organisms has lagged far behind that of bacteria and yeast. Our research is involved in understanding a unique and biologically relevant process in filamentous fungi that results in programmed cell death. This pathway is an attractive fungal-specific target for drug development for treatment of fungal plant and animal diseases.

Publications

  • 1. Castro, A., C. Lemos, A. Falcao, A. S. Fernandes, N.L. Glass and A. Videira, 2010. Rotenone enhances the antifungal properties of staurosporine. Eukaryot Cell 9:906-14.
  • 2. Hutchison, E. A and N. L. Glass, 2010. Meiotic regulators Ndt80 and Ime2 have different roles in Saccharomyces and Neurospora. Genetics 185:1271-1282.
  • 3. Simonin, A., C. G. Rasmussen, M. Yang and N. L. Glass, 2010. Genes encoding a striatin-like protein (ham-3) and a forkhead associated protein (ham-4) are required for hyphal fusion in Neurospora crassa. Fungal Genet Biol 47:855-868.
  • 4. Galazka, J.M. C. Tian, W. T. Beeson, B. Martinez, N. L. Glass and J. H. D. Cate, 2010. Cellodextrin transport in yeast for improved biofuel production. Science 330:84-86.
  • 5. Li, S., J. Du, J. Sun, J. M. Galezka, N. L. Glass, J. D. H Cate H. Yang and H. Zhao, 2010. Overcoming glucose repression in mixed sugar fermentation by co-expressing a cellobiose transporter and a β-glucosidase in Saccharomyces cerevisiae. Molec BioSystems 6:2129-2132.
  • 6. Hall, C., J. Welch, D. J. Kowbel and N. L. Glass, 2010. Evolution and diversity of a fungal self/nonself recognition locus. PLoS One 5(11):e14055.
  • 7. Greenwald, C. J., T. Kasuga, N. L. Glass, B. D. Shaw, D. J. Ebbole and H. H. Wilkinson, 2010. Temporal and spatial regulation of gene expression during asexual development of Neurospora crassa. Genetics 186:1217-1230.
  • 8. Aanen, D.K., A.J.M. Debets, N. L.Glass and S.J. Saupe, 2010. Biology and genetics of vegetative incompatibility in fungi. In Cellular and Molecular Biology of Filamentous Fungi. K.A. Borkovich and D. Ebbole, Eds. American Society of Microbiology, pp. 274-288.
  • 9. Read, N. D., A. Fleissner, M. G. Roca and N. L. Glass, 2010. Hyphal Fusion. In Cellular and Molecular Biology of Filamentous Fungi. K.A. Borkovich and D. Ebbole, Eds. American Society of Microbiology, pp. 260-273.