Interplay between Self and Non-self recognition mechanisms in Neurospora crassa
Cell Biology
Final Report Abstract
Allorecognition, the ability to distinguish self from non-self is a fundamental requirement for survival in multicellular organisms. In animals and plants it is mostly associated with innate or adaptive immunity. Neurospora crassa has long been used to study cellular fusion and heterokaryon incompatibility (HI), a form of allorecognition in filamentous fungi. Cellular fusion of vegetative cells is an essential process for fungal development, colony establishment and habitat exploitation and can occur between hyphal cells of an individual colony or between hyphal cells of genetically distinct colonies. Cell fusion in filamentous fungi involves chemotropic interactions between interacting partner cells, followed by adhesion, cell wall dissolution and membrane merger, resulting in cytoplasmic and nuclear mixing. HI is one mechanism to restrict the propagation of fusion products between genetically dissimilar individuals by inducing a programmed cell death (PCD) response. Although reported in a large number of multicellular fungi, the molecular mechanisms associated with recognition and death during HI are not well characterized. Allorecognition-induced death in filamentous fungi has been proposed to function as a type of fungal innate immunity system, whereby recognition of non-self reduces the risk of transmission of pathogenic elements between colonies and exploitation by aggressive genotypes. As part of this project we studied cell fusion and allorecognition in germinated asexual spores (germlings) of N. crassa. By analyzing fusion frequencies of germlings between wild isolates of N. crassa populations we defined three allorecognition checkpoints that are based on kind discrimination, and are mediated by polymorphisms different genomic loci. We identified determinants for each checkpoint by bulk segregant analysis (BSA) of progeny from a cross between two wild isolates followed by whole genome re-sequencing. The first allorecognition checkpoint acts during chemotropic interactions and requires genetic differences at a set of greenbeard genes called determinant of communication (doc-1, doc-2, doc-3). If germlings have identical specificity at the doc loci, productive chemotropic interactions occur, while germlings with different doc specificity ignore each other and do not undergo chemotropic interactions. The second allorecognition step acts after chemotropic interactions occurred when two germlings meet and adhere. A set of genes called cell wall remodeling (cwr-1, cwr-2, cwr-3), which we identified by BSA/re-sequencing regulates cell wall breakdown and membrane merger between germlings. If germlings are of different cwr specificity, cell fusion is blocked (Manuscript in preparation). The third allorecognition step occurs after chemotropic interactions, and cell fusion occurred. If germlings are of different specificity at so-called germling regulated death (GRD) loci, allorecognition mediates rapid cell death following membrane merger between germlings. To quantify germling death, we developed a fluorescently activated cell-sorting (FACS) assay using vital dyes. Using BSA/re-sequencing, we identified two divergent and unlinked regions that mediate GRD. The first region includes a gene encoding an essential t-SNARE (sec-9), required for secretory vesicle-plasma membrane fusion and a gene (plp-1) containing a patatin-like phospholipase, NB-ARC and a tetratricopeptide repeat (TPR) domain. Both NB-ARC and TPR domains are found in innate immunity proteins in animals/plants. The second divergent region encodes a hypothetical protein (rcd-1) with two haplotypes. RCD-1 shows similarity to Gasdermin, a protein that causes cell death in vertebrates during innate immunity. Allorecognition in filamentous fungi has been linked to conspecific recognition, while the potential role of allorecognition in the interaction with other organisms has been neglected. It is still unclear if fungal allorecognition systems are involved in defense reactions against pathogens. While there are reports on viral/fungal pathogens of filamentous fungi, studies of bacterial pathogens are rare. In the long run, a better understanding of microbial communities associated with filamentous fungi in the field (including bacterial pathogens) is necessary to elucidate and test the ecological significance of allorecognition as a fungal innate immune system. Increased sampling and sequencing data from microbial communities will overcome this deficit and will reveal if conspecific allorecognition in fungi resulted from exaptation of a fungal innate immune system that evolved to detect pathogens.
Publications
- (2016) Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote. PLoS Biol 14(4): e1002431
Heller, J., Zhao, J., Rosenfield, G., Kowbel, D.J., Gladieux, P., Glass, N.L.
(See online at https://doi.org/10.1371/journal.pbio.1002431) - Molecular mechanisms regulating cell fusion and heterokaryon formation in filamentous fungi. Microbiol Spectrum 5(1):FUNK-0015-2016
Daskalov, A., Heller, J., Herzog, S., Fleißner, A., Glass, N.L.
