Heterokaryon analysis of a Cdc48-like gene, CpCdc48, from the chestnut blight fungus Cryphonectria parasitica demonstrates it is essential for cell division and growth

Mutation of the Slt2 ortholog from Cryphonectria parasitica results in abnormal cell wall integrity and sectorization with impaired pathogenicity

“…Protoplast preparation and transformation of C. parasitica were performed as described previously 15 , 16 , 52 . Transformants were selected from PDAmb plates supplemented with 150 μg/mL hygromycin B (Calbiochem, San Diego, CA, U.S.A.) or 150 μg/mL geneticin (Invitrogen, Carlsbad, CA, U.S.A.), passaged three to four times on selective medium, and single-spore isolated, as described previously 15 , 53 . PCR and Southern blot analysis were conducted with genomic DNA from the transformants to confirm the replacement and in trans complementation of the CpSlt2 gene.…”

Functional analysis of an essential Ran-binding protein gene, CpRbp1, from the chestnut blight fungus Cryphonectria parasitica using heterokaryon rescue

“…Sequence homology showed that the CpRbp1 gene is highly similar to essential Yrb1 [E-value, 3e-75; amino acid identity, 145/238 (67%)], although not as similar as to RanBP genes from filamentous fungi, and function analyses of CpRbp1 using heterokaryons proved that the CpRbp1 gene is essential for cell viability. In addition, compared to other lethal genes exhibiting the terminal phenotypes of mutated genes 28,30 , no sign of initial germinating processes such as swollen spores was observed in conidia containing CpRbp1-null nuclei, suggesting that the CpRbp1 gene product is involved in an initial fundamental cellular function that regulates many important processes for viability from the beginning.…”

“…However, this application is limited in other fungi and generalization of the technique to other fungal systems would require further studies of other organisms. Although the extent of heterokaryon of C. parasitica in natural environments is largely unknown, the presence of stable heterokaryon has been reported in nature 27 , and well-balanced maintenance of genetically engineered-lethal nuclei has been achieved 28,30 . Therefore, C. parasitica appears to be a good model fungus for studying essential genes based on the ease of detection and single-spore resolution, and more importantly, the balanced proliferation of genetically different nuclei, which is necessary for functional analyses using heterokaryon.…”

“…2d); thus, TdRBP1-Het1 was selected for further analyses. Because the genotype frequency in a heterokaryon can vary depending on growth conditions 28,30 , we examined changes in the genotype frequency of heterokaryon progeny. The genotype frequency was estimated by measuring the ratio of colony-forming units (CFUs) from counted conidia of heterokaryon progeny that were successively transferred every fifth day for up to 20 transfers on selective and nonselective media (Fig.…”

“…The heterokaryon rescue technique allows analyses of whether a gene of interest is essential, as well as even further analyses of the terminal phenotype of the gene 25,26 . Stable heterokaryon formation has been observed in C. parasitica 27 , and a recent study demonstrated that heterokaryon formation of C. parasitica maintained mutant nuclei in which a gene of interest was lethal, providing the possibility of functional analyses of the corresponding lethal gene via complementation 28 . Therefore, although it is highly possible that RanBP1 of C. parasitica is important due to its participation in diverse cellular functions, we attempted to construct a RanBP1-null mutant to determine whether the function of RanBP1 is essential in C. parasitica and to obtain a heterokaryon for further functional analyses of the lethal RanBP1 gene.…”