Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae
Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae
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DOI：
10.1007/s10327-013-0474-2
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发表时间：
2013-11-01
影响因子：
1.2
通讯作者：
Kuwata, Shigeru
中科院分区：
文献类型：
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作者：
Arazoe, Takayuki;Ohsato, Shuichi;Kuwata, Shigeru
为了研究稻瘟病菌的有丝分裂同源重组(HR)，我们建立了HR检测系统。该系统由两个非功能增强型黄色荧光蛋白(YFP)和杀菌素S脱氨酶融合基因(YFP：：BSD)组成。如果基因组中的两个非功能基因之间发生有丝分裂HR，功能基因YFP：：BSD的恢复是可望的。YFP荧光检测和BS抗性检测均能检测到YFP：：BSD基因的表达。当两个非功能基因同时转化水稻疫霉基因组时，6个同时含有这两个基因的转化系在生长过程中都有部分菌丝表现出YFP荧光和BS抗性。从6个品系的菌丝体中收集的分生孢子中，约有10%有YFP荧光，这表明在菌丝生长过程中，HR持续存在。为了确定HR介导的表型变化是否以及如何在DNA水平上发生，我们通过对BS抗性菌丝基因组DNA进行PCR-RFLP和测序分析，证实了恢复功能的YFP：：BSD基因和非功能重组基因的存在。综上所述，这些发现表明，我们建立了一套成功的水稻疫霉HR检测系统，该系统可用于其他植物病原菌，并且有丝分裂HR确实存在于稻瘟病菌中，并构成了真菌有丝分裂HR的第一个实验证据。
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.