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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. <P>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. <P>We identified a transcription factor, VIB-1, which is required for HI and determined that a ?vib-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.<P> 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.<P> Objective 1: Identify direct targets of the transcription factor Vib-1 by performing chromatin-immunoprecipitation and high throughput Illumina sequencing. <P>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.<P>Objective 3: Characterize the function of the proteins encoded by genes that are directly regulated by Vib-1 and are required for PCD. <P>Objective 4: Identify interactions of HET domain proteins.<P> 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.
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". <P> Approach: 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 (?vib-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 ?vib-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