Study of cell fusion mutants in Neurospora crassa

We have screened the Neurospora knockout library for mutants affected in the process of anastomosis. Anastomosis is a cell fusion event that occurs between hyphal cells to create an interconnected hyphal network during vegetative growth in the Neurospora life cycle. We found 151 such mutants in the library. In many cases we found that the mutation responsible for the anastomosis-defective phenotype was not tagged by the insertion of the hygromycin-resistance gene used to create the KO library. In 36 of the mutants, the mutation responsible for the anastomosis-defective phenotype was tagged by the hygromycin insertion involved in creating the knockout mutation, as defined by cosegregation experiments. The 36 genes defined by these anastomosis-defective mutants can be roughly divided into four categories. One of these categories includes a number of genes that encode transcription factors. A second category includes genes involved in signal transduction pathways. A third category includes genes that encode elements of the cytoskeleton. The fourth category includes genes that might be directly involved in the cell fusion mechanism. The genes in the last category include small secreted proteins that are somewhat hydrophobic and could function in facilitating the fusion between two plasma membranes. We proceeded with knock-in experiments for two genes in the fourth category, ham-6 and ham-7. After knocking in the wild type copy of ham-6 and ham-7 into the Neurospora ham-6 and ham-7 KO mutants, we restored the wild type phenotype and thus established that ham-6 and ham-7 are involved in the cell fusion mechanism in Neurospora. In the future, the laboratory will be focusing their attention on this fourth category of mutants. The laboratory will try to perform knock-in experiments for the rest of the genes and elucidate the role of these proteins in anastomosis.;Keywords: Cell fusion, Neurospora crassa, anastomosis, whole genome knock-out library, conidial anastomosis tubes, knock-in experiments.