The ability to recognize self from nonself in order to maintain cellular identity is a key aspect of intra- and inter-organismal interactions. In this study, the objective was to identify new genes involved in self/nonself recognition in the filamentous ascomycete Neurospora crassa. In N. crassa, hyphal fusions can produce either viable, compatible heterokaryons, or incompatible heterokaryons that undergo programmed cell death (PCD). N. crassa nonself recognition is mediated by eleven heterokaryon (het) loci, and genetic differences at any one of the eleven het loci is sufficient to induce PCD. One of the most well characterized het loci is the het-c pin-c locus. Allelic and nonallelic interactions between these two genes contribute to nonself recognition. In addition, het-c pin-c-mediated incompatibility and PCD is suppressed by mutations in the transcription factor vib-1. The aims of this study were to identify new genes involved in incompatibility using transcriptional profiling and comparative genomics, and to further characterize the transcription factor vib-1 and its paralogs.
The first chapter focuses on transcriptional profiling results from compatible and incompatible heterokaryons. In general, metabolism and protein synthesis genes were down-regulated in incompatible heterokaryons, while phosphatidyl inositol and calcium signaling genes were upregulated. In addition, I provide evidence that reactive oxygen species (ROS) are produced upon induction of incompatibility, and that ROS frequently co-localize to woronin body structures. Data from transcriptional profiling suggested that N. crassa may not be using a conventional cell death pathway and indeed, mutations in aif-1 and metacaspase homologs did not affect cell N. crassa PCD. In addition, though kinase signaling pathways were enriched in the incompatible dataset, mutations in two N. crassa kinases, mak-2 and ste-12, did not affect heterokaryon incompatibility (HI). I did identify two genes, NCU06177 (Ca2+/calmodulin dependent protein kinase C) and NCU05693 (contains a dynamin domain), that when mutated caused a small but significant increase in incompatibility-induced cell death. Finally, a significant overlap also existed between the incompatibility PCD dataset as well as a dataset from N. crassa cell death induced by phytosphingosine, indicating that N. crassa may be using a common cell death pathway for different cell death input signals.
The second chapter describes the characterization of the transcription factor vib-1 and its two paralogs, fsd-1 and NCU04729. All three paralogs are homologous to the S. cerevisiae major middle meiotic transcription factor NDT80. NDT80 is necessary for the prophase to metaphase transition, and mutants in NDT80 do not complete meiosis. Phylogenetic analysis of fungal NDT80 homologs revealed that most filamentous fungi actually have two or more NDT80 homologs, suggesting that at least some of the fungal homologs may have diverged in function from that of meiosis. Surprisingly, though two of the N. crassa NDT80 homologs (vib-1, fsd-1) affected sexual development, none of the homologs were necessary for completion of meiosis.
Upon nitrogen starvation, N. crassa forms spherical hyphal structures called protoperithecia, which can be fertilized. Mutants in vib-1 affected the number of protoperithecial structures formed, and Δvib-1 Δfsd-1 strains were affected for the timing of protoperithecial development. In addition, microarray analysis revealed a role for fsd-1in spore maturation, consistent with NDT80 data from S. cerevisiae. Further, I show that a homolog of the kinase IME2, another major yeast meiotic regulatory protein, is also not necessary for meiosis in N. crassa. IME2 is necessary for NDT80 expression and activity in S. cerevisiae, but this role is not conserved in N. crassa. Interestingly, I also observed that ime-2 negatively regulates protoperithecial formation upon nitrogen starvation, and that vib-1 mutations are epistatic to ime-2 mutations in this regard. Thus, the meiotic regulatory pathway of N. crassa functions differently than that of yeast, and our study gives insight into how filamentous fungi regulate meiosis.
Due to our previous observation that vib-1 and ime-2 interacted genetically, the aim of the third chapter was to determine if ime-2 affects other vib-1 mutant phenotypes, such as protease production and cell death due to heterokaryon incompatibility. Single mutants for ime-2 were not affected for het-c pin-c mediated incompatibility, but interestingly, Δvib-1 Δime-2 strains exhibited wild type levels of cell death. In addition, Δvib-1 Δime-2 strains produced wild type levels of extracellular proteases. These data indicate that ime-2 and vib-1 are functioning in a common genetic pathway. Further, I determined that vib-1 is phosphorylated, and I created phospho-mimic or phospho-dead alleles of VIB-1 for these sites. These alleles will be tested for functionality in HI in future studies. Our data also implicate ime-2 in the targeting or processing of a specific mitochondrial matrix protein, ARG-4. Based on our data, I propose a model in which two redundant cell death pathways exist in N. crassa (with one possibly functioning through the mitochondria) and that ime-2 negatively regulates both of these pathways.
Finally, in chapter four, I took a comparative genomics approach to the identification of new het loci. Specifically, I focus on genes that contain a HET domain, a protein domain that has been implicated in incompatibility and fungal PCD. To identify new het loci, I looked for HET domain genes that exhibited evolutionary characteristics of nonself recognition loci, such as balancing selection and trans-species polymorphism. Twenty-three of the 55 HET domain genes exhibited these characteristics, and I tested three of these candidate genes (NCU09037, NCU04694, and NCU07511) for function in incompatibility. NCU09037 has two distinct alleles that are under balancing selection, and I show that it can function as a het locus. Further, only allelic interactions are necessary for NCU09037-induced incompatibility. I did not observe an HI phenotype in strains expressing alternate alleles of NCU07511, and I am currently testing the HI function of NCU04694. Thus, I show that nonself recognition loci can successfully be predicted from their evolutionary features.