Abstract
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.
Highlights
Cryphonectria parasitica (Murrill) Barr, causes chestnut blight and has devastated chestnut forests and orchards in North America since the early 20th century [1]
Cryphonectria hypovirus 1 (CHV1) can be transferred from the virus-containing hypovirulent strain to virus-free strain during hyphal fusion resulting in hypovirulence and its associated symptoms, which is an effective good model for naturally occurring biological control by mycovirus, known as virocontrol [8,9]
Molecular analysis of C. parasitica–hypovirus interactions allowed us to ascribe the development of these viral symptoms to aberrant expressions of specific genes in the hypovirulent strain [10,11,12,13,14,15]
Summary
Cryphonectria parasitica (Murrill) Barr, causes chestnut blight and has devastated chestnut forests and orchards in North America since the early 20th century [1]. C. parasitica, which contains a cytoplasmic single stranded RNA (ssRNA) virus, Cryphonectria hypovirus 1 (CHV1), has had lowered virulence, a phenomenon referred to as “hypovirulence” [2,3,4]. CHV1-infected C. parasitica displays diverse hypovirulence-associated symptoms, such as reduced pigmentation, sporulation, laccase production, and oxalate accumulation [5,6,7]. CHV1 can be transferred from the virus-containing hypovirulent strain to virus-free strain during hyphal fusion resulting in hypovirulence and its associated symptoms, which is an effective good model for naturally occurring biological control by mycovirus, known as virocontrol [8,9]. Due to the efficient genetic manipulations of C. parasitica, the availability of a high-quality C. parasitica genome sequence
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