Functional Analysis of an Essential GSP1/Ran Ortholog Gene, CpRan1, from the Chestnut Blight Fungus Cryphonectria parasitica Using a Heterokaryon.

Functional analysis of a GSP1/Ran ortholog, CpRan1, from Cryphonectria parasitica was conducted. Genotype analysis revealed that the putative CpRan1-null mutant was a heterokaryotic transformant harboring two different types of nuclei, one with the wild-type CpRan1 allele and the other with the CpRan1-null mutant allele. The mycelial growth and colony morphology of the heterokaryotic transformant was normal. Microscopic analysis of the resulting conidia (aseptate and monokaryotic asexual spores) demonstrated that although normal germinating spores were observed from conidia harboring a nucleus with the wild-type CpRan1 allele, a number of residual conidia that did not germinate existed. Complementation analysis using protoplasts from the heterokaryon with the wild-type CpRan1 allele confirmed that the CpRan1 gene is essential to C. parasitica. Complementation analysis using the various CpRan1 chimera constructs allowed us to perform a functional analysis of essential amino acids of the CpRan1. Among the four suggested essential amino acids, Lys-97 for ubiquitination was determined to not be an essential residue. Moreover, the CpRan1-null mutant allele was successfully complemented with mouse Ran gene, which suggested that the biological function of Ran gene is evolutionary conserved and that our heterokaryon rescue can be applied for the functional analysis of heterologous genes.