This project investigates two genes in the fungus Neurospora crassa, one that kills fungal spores and another that prevents spore killing. While these two genes can be thought of as forming a poison and antidote system, their purpose is unclear. Are they beneficial for the fungus or are they detrimental? It seems they have formed a selfish partnership whose killing and resistance properties allow the two genes to be transmitted to every one of the organism's offspring, even when only one parent of a mating pair possesses the genes. The primary goal of this project is to determine how these genes, poison and antidote, achieve this remarkable feat. Additionally, the two genes appear to have driven a major reorganization of the chromosome in which they reside. Therefore, a secondary goal of the project is to investigate the hypothesis that selfish genes are major drivers of genome reorganization in eukaryotic organisms. The primary goal will be pursued by a team of scientists, graduate students, and undergraduate students at Illinois State University in Central Illinois, while the secondary goal will be pursued by the same team in collaboration with evolutionary biologists based in Sweden. In addition, a GK-12 STEM teacher from Central Illinois will assist with the project. Not only will this provide valuable research experience to the teacher, the PI and the teacher will design inquiry-based learning activities for GK-12 classrooms that involve experimentation with N. crassa and other harmless microorganisms.
The two genes under investigation in this study are rfk, the N. crassa spore killing gene, and rsk, the resistance gene. Although the boundaries of the rfk killing gene have not been precisely defined, the killing function has been tracked to a 1500 base pair fragment of DNA on the third chromosome of a strain called Sk 2. The rsk resistance gene is found on the same chromosome. Different rsk alleles exist in nature, and not all of them provide resistance to rfk. Additionally, rsk possesses features typical of protein-coding genes. For example, it possesses a clearly identifiable start codon, stop codon, and open reading frame. This is in stark contrast to the killer gene, which does not have obvious protein-coding features, and thus could produce either a toxic non-coding RNA or a toxic protein. The first aim of this project is to differentiate between these two possibilities and gather evidence on the mechanism of spore killing. First, six previously isolated rfk mutants will be sequenced to produce a catalog of mutations that disrupt killing; second, RNA sequencing will be employed to map transcripts from a functional rfk locus and to determine the global transcriptional changes associated with killing; third, the full-length rfk transcript will be cloned with Rapid Amplification of cDNA End (RACE) technology; fourth, site-directed mutagenesis will be used to determine if random insertion mutations are more disruptive of killing than random point mutations; fifth, a group of six point mutations, which are known to disrupt killing when all are found within the same rfk allele, will be repaired to determine which combination of the six is critical for loss of killing; and sixth, protein chimeras will be produced from resistant and non-resistant versions of RSK to determine which parts of the resistance protein are critical for function. The second aim of this project is to perform the same set of experiments on a different strain called Sk-3. Together, the two aims of this proposal will define the borders of the killing gene, determine if the killer gene product is a toxic RNA or a protein, provide a catalog of mutations that disrupt spore killing, identify transcriptional changes associated with the spore killing mechanism, and identify critical regions of the protein required for resistance, all for Sk-2 and Sk-3, two models of selfish gene function and evolution.
