水稻白叶枯病黄单胞菌rpfF基因的定位及其在致病调控中的作用

Characterization of rpfF Gene Involved in Production of A Diffusible Regulatory Factor of Xanthomonas oryzae pv. oryzae

检测到水稻白叶枯病菌能产生一种“跨菌调控因子”，在固体平板上，这种因子由白叶枯病菌分泌到细胞外后可以扩散并进入甘蓝黑腐病菌，调控甘蓝黑腐病菌致病因子的表达．本研究从水稻白叶枯病菌中克隆鉴定了一个与这种“跨菌”调控因子合成有关的基因ｒｐｆＦ，通过转座子诱变和标记置换（ｍａｒｋｅｒｅｘｃｈａｎｇｅ）技术，构建了ｒＰｆＦ突变体，突变体丧失了产生这种“跨菌”调控因子的能力，在水稻植株上的致病能力明显减弱，ＤＮＡ序列分析得知ｒｐｆＦ基因编码的产物和大肠杆菌的３－酮脂酰－ＣＯＡ硫解酶（３－ｋｅｔｏａｃｙｌ－ＣＯＡｔｈｉｏｌａｓｅ）同源。

Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterial pathogen which causes bacterial blight of rice, a cosmid clone pGXN3000 containing DNA from Xoo wild type strain was isolated and shown to complement the non-pathogenic mutant 8523 of Xanthomonas campestris pv. campestris (Xcc) defective in synthesis of extracellular enzymes which carries rpfF gene (regulatory of pathogenicity factor F) mutation. This suggests that the recombinant plasmid pGXN3000 contains a rpfF homologous gene. By subcloning of pGXN3000, the rpfF gene of Xoo strain was located on a 1.3 kb EcoR I fragment.By Tn5 mutagenesis and marker exchange, a Xoo rpfF gene mutant was constructed. This mutant is defective in exopolysaccharide (EPS) production and shows reduced pathogenicity on rice. These results indicate that rpfF gene of Xoo regulates the synthesis of EPS positively and plays an important role in process of pathogenicity.Wild type Xoo strain produces a diffusible molecule which can restore the extracellular protease synthesis of 8523 in vitro,while the Xoo rpfF mutant does not. DNA sequence analysis shows that rpfF gene product is homologous to 3-ketoacyl--CoA thiolase of E. coli.