Abstract
A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.
Highlights
The chestnut blight fungus, Cryphonectria parasitica (Murrill) Barr, eradicated chestnut forests and orchards in North America at the beginning of the last century[1]
Ran-binding protein 1 (RanBP1), drew our attention because it is an important regulator of the Ras-like nuclear small G protein (Ran), which is involved in nucleocytoplasmic transport and has never been analyzed in this fungus
Since our previous proteomic analyses indicated that the protein product of the CpRbp[1] gene was upregulated by Cryphonectria hypovirus 1 (CHV1) infection or tannic acid (TA) supplementation[6], we examined the accumulation of gene transcripts under the corresponding conditions using Quantitative real-time reverse transcription PCR (Fig. 1a)
Summary
The chestnut blight fungus, Cryphonectria parasitica (Murrill) Barr, eradicated chestnut forests and orchards in North America at the beginning of the last century[1]. As an ideal model system, C. parasitica-hypovirus interactions have been considered for investigating fungus-mycovirus interactions This is possible due to the availability of various molecular tools that can be used for genetic manipulation of the fungus including gene replacement, heterokaryon rescue, and gene silencing. Ran is a highly conserved small GTP-binding protein that was initially proved as an essential element of the nucleocytoplasmic transport complex. It has since been implicated in many cellular functions, including various mitotic processes such as the regulation of DNA synthesis, centrosome formation, spindle assembly, nuclear envelope reformation[7], and nuclear envelope structure in yeast[8,9]. To the best of our knowledge, this is the first study on the function of RanBP1 in filamentous fungi using the heterokaryon rescue technique
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