title: Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.
abstract: The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Chen
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Guo-Ao
authors.initials: GA
authors.lastname: Ning
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Huan-Bin
authors.initials: HB
authors.lastname: Shi
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Chu-Long
authors.initials: CL
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Lu
authors.affiliation: Analysis Center of Agrobiology and Environmental Science, Zhejiang University, Hangzhou, China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Mao
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Xiao-Xiao
authors.initials: XX
authors.lastname: Feng
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China. Electronic address: zhenzhusu@126.com.
authors.forename: Zhen-Zhu
authors.initials: ZZ
authors.lastname: Su
authors.affiliation: State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China; China Tobacco Gene Research Center, Zhengzhou Tobacco Institute of CNTC, Zhengzhou, China. Electronic address: fuchenglin@zju.edu.cn.
authors.forename: Fu-Cheng
authors.initials: FC
authors.lastname: Lin
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D001203:Ascomycota / Q000235:genetics* / Q000502:physiology; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D012275:Oryza / Q000382:microbiology; D010935:Plant Diseases / Q000382:microbiology; D014170:Transformation, Genetic
keywords: Agrobacterium tumefaciens-mediated transformation; Endophytic fungus; Gene deletion mutagenesis; Harpophora oryzae; Insertional mutagenesis; Sulfonylurea resistance