Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-funct...

Full description

Saved in:
Bibliographic Details
Published in:Journal of general plant pathology : JGPP 2013-11, Vol.79 (6), p.422-430
Main Authors: Arazoe, Takayuki, Ohsato, Shuichi, Arie, Tsutomu, Yoneyama, Katsuyoshi, Kuwata, Shigeru
Format: Article
Language:English
Subjects:
Agriculture
Biomedical and Life Sciences
blasticidin S
conidia
Deoxyribonucleic acid
DNA
Flowers & plants
Fluorescence
Fungal Diseases
Fungi
Genes
Genomes
homologous recombination
Life Sciences
Magnaporthe oryzae
Microbiology
mycelium
plant pathogens
Plant Pathology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.
ISSN:1345-2630
1610-739X
DOI:10.1007/s10327-013-0474-2