Ogura Keisuke, Matsui Hidenori, Yamamoto Mikihiro, Noutoshi Yoshiteru, Toyoda Kazuhiro, Taguchi Fumiko, Ichinose Yuki
Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama, 700-8530, Japan.
Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.
Biochem Biophys Rep. 2021 Feb 12;26:100944. doi: 10.1016/j.bbrep.2021.100944. eCollection 2021 Jul.
Virulence factor regulator (Vfr) is an indispensable transcription factor in the expression of virulence in the phytopathogenic bacteria . However, the function of Vfr is not known so far. The deletion of resulted in the loss of surface swarming motility and reduced the virulence in . pv. () 6605. In order to identify the target genes of Vfr, we screened the sequences that bind to Vfr by chromatin immune precipitation (ChIP) and sequencing methods using the closely related bacterium . pv. () B728a. For this purpose we first generated a strain that possesses the recombinant gene :: in B728a, and performed ChIP using an anti-FLAG antibody. Immunoprecipitated DNA was purified and sequenced with Illumina HiSeq. The Vfr::FLAG-specific peaks were further subjected to an electrophoresis mobility-shift assay, and the promoter regions of locus tag for Psyr_0578 , Psyr_1776, and Psyr_2237 were identified as putative target genes of Vfr. These genes encode plant pathogen-specific methyl-accepting chemotaxis proteins (Mcp). These genes seem to be involved in the Vfr-regulated expression of virulence.
毒力因子调节蛋白（Vfr）是植物致病细菌中表达毒力所必需的转录因子。然而，目前Vfr的功能尚不清楚。在 中缺失 导致表面群体运动丧失，并降低了其在 pv. () 6605中的毒力。为了鉴定Vfr的靶基因，我们使用密切相关的细菌 pv. () B728a，通过染色质免疫沉淀（ChIP）和测序方法筛选与Vfr结合的序列。为此，我们首先在B728a中构建了一个含有重组基因::的菌株，并使用抗FLAG抗体进行ChIP。对免疫沉淀的DNA进行纯化，并用Illumina HiSeq进行测序。Vfr::FLAG特异性峰进一步进行电泳迁移率变动分析，Psyr_0578、Psyr_1776和Psyr_2237基因座标签中的启动子区域被鉴定为Vfr的假定靶基因。这些基因编码植物病原体特异性甲基接受趋化蛋白（Mcp）。这些基因似乎参与了Vfr调节的毒力表达。
