利用酵母双杂法鉴定水稻白叶枯病菌Ⅲ型效应物寄主靶标初探

Preliminary Study on Screening of Host Target Proteins Interacting with Type Ⅲ Effectors from Xanthomonas oryzae pv. oryzae via Yeast Two-Hybrid System

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白叶枯病是世界水稻生产中最严重的细菌性病害之一。前期研究发现Xoo PXO99A中一个编码Ⅲ型效应物的PXO_03420(hpaF)基因与病菌游动性、致病性和致敏性有关,为探讨hpaF基因编码产物在病菌与日本晴水稻中的互作关系,本研究采用Matchmaker酵母双杂交系统构建了日本晴水稻叶片高质量cDNA文库,文库重组率为87.6%,滴度为2.57×106pfu/mL。将文库与构建的Y187/pGBKT7_03420诱饵进行酵母双杂交,共得到26个阳性克隆。通过对阳性克隆测序所得结果进行结构域分析,初步鉴定HpaF与水稻叶片中具有WHY或PRK结构域的两个蛋白存在互作关系。本研究结果为进一步研究hpaF基因的功能奠定了基础。

Xanthomonas oryzae pv. oryzae（Xoo） is one of plant pathogenic bacterias that causes bacterial leaf blight in the world. Preliminary study revealed that a type Ⅲ secretion effector PXO_03420（hpaF） was involved with the motility, pathogenicity and allergenicity of Xoo PXO99A. In this report, a high quality cDNA library of Oryza sativa L. japonica. cv. Nipponbare infected by PXO99Awas generated using the Clontech＇s Matchmaker yeast two-hybrid system. The titer of the amplified library was 2.57×10^6pfu/mL and the recombinant rate was 87.6%. This cDNA library was screened by yeast two-hybrid system using pGBKT7-hpaF as bait plasmid and 26 positive clones were obtained. Those positive clones were tested by quantitative determination of β-galactosidase activity and sent for sequencing. Analysis of sequencing result showed that hpaF of Xoo PXO99Aencodes the LRR protein that interacts with two rice＇s WHY or PRK domain proteins. This work laid a solid foundation for the further study on pathogenic molecular mechanism of hpaF.