Examining the role of the promoter region of het-6 in escape from heterokaryon incompatibility in Neurospora crassa

Abstract

Heterokaryon incompatibility (HI) is a form of nonself recognition that limits the formation of heterokaryons -cells that contain genetically dissimilar nuclei. HI is controlled by several het loci. One of these, het-6 is a supergene complex that encodes two tightly-linked incompatibility genes, un-24 and het-6, with allelic variants Oak Ridge (OR) and Panama (PA), and only two natural haplotypes -un-24 OR het-6 OR or un-24 PA het-6 PA . het-6 encodes a HET domain protein with no known function outside of incompatibility, whereas un-24 encodes incompatibility function and the large subunit of ribonucleotide reductase. Strains incompatible at the het-6 locus can 'escape' from HI by mutation in the het-6 gene (Type I, ~97%), or suppress HI by mutation in a transcription factor called vib-1 (Type II, ~3%). This study examined the possible role of the het-6 promoter region in the initiation of escape, by introducing an ectopic het-6 OR allele into a strain that carried a null endogenous het-6 OR allele (IB20). Self-incompatible transformants exhibited delayed escape and aberrant post-escape behavior. A vib-1 mutant allele was introduced into IB20, and the resulting strain was also transformed with the ectopic het-6 OR . Transformants displayed Type-II like phenotype, but had slower growth rates than a typical Type II strain. Finally, we constructed a plasmid containing the het-6 OR allele in which the putative VIB-1 + binding site was deleted. Transformation of IB20 with this construct generated a transformant with a Type II-like growth phenotype. This study provides insight into the process of escape.iii Acknowledgements First, I would like to thank Dr. Myron Smith, who welcomed me into his lab over four years ago, and has always been a patient teacher and provided invaluable guidance through the completion of this project. I would also like to thank my committee members,