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

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.

SUBMITTER: Ko YH

PROVIDER: S-EPMC7229160 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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

Ko Yo-Han YH Choi So-Yeon SY So Kum-Kang KK Kim Jung-Mi JM Chun Jeesun J Kim Dae-Hyuk DH

Scientific reports 20200515 1

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 o ...[more]

PMID: 32415177

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Project description:The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

Dataset Information

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

Publications

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

Similar Datasets

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