水稻白叶枯病菌hrp调节基因hrpXoo的克隆与序列分析

Cloning and Sequencing Analysis of an hrp Regulatory Gene, hrpXoo, of Xanthomonas oryzae pv. oryzae

用化学方法诱变水稻白叶枯病菌PXO99A 菌株 ,获得 6株hrp-突变体 ,此突变体除丧失在非寄主烟草上激发过敏反应和在感病寄主水稻上的致病能力外 ,有些缺乏激发烟草产生HR的信号物质 ,有些在胞内存在此信号物质 ,而不能泌至胞外。来自Xanthomonasoryzaepv .oryzaeJXOIII粘粒基因文库的hrp基因克隆pUHRX2 4 5 ,所携hrp基因片段大小为 36 .8kb。系列亚克隆 36 .8kbhrp基因片段及各亚克隆对hrp-突变体功能互补作用的结果显示 ,3.3kbSacI片段为最小酶切功能片段。序列测定和分析结果表明 ,3.3kbhrp片段含hrpX oo基因和含与热激蛋白 90家族有关的 2个开放阅读框hspORF1和hspORF2。HspORF1在蛋白质数据库中未发现序列蛋白。HspORF2与Hsp90 Xo的同源性达 99%。hrpXoo与黄单胞菌中已报道的hrpX基因有很高的同源性(90 %以上 )。在黄单胞菌中高度保守的编码α 螺旋 转 α 螺旋结构的 6 0 bp核苷酸序列 ,在交叉功能互补时是必需的。不含此结构的hrpXoo (1.1kb)片段 ,可使JXOIII的hrp-突变体在水稻上具致病性和在非寄主烟草上激发产生HR ,但不能使来自PXO99A 和RS10 5的hrp-突变体恢复在烟草上激发产生HR的功能。黄单胞菌中已知HrpX的同列比较显示 ,X .oryzae和X .campestris种间在 88、196和 2 4

Six hrp - mutants obtained from the PXO99 A strain of Xanthomonas oryzae pv. oryzae (Xoo) by using diethyl sulfate (DES) as a mutagenic chemical. These mutants lost their pathogenicity on susceptible rice, Shanyou63, and the ability to trigger hypersensitive response (HR) on a nonhost plant of tobacco. Some of the mutants lack a substrate that induces HR on tobacco, while others possess the substrate within cells but do not secret it out. An hrp gene clone, pUHRX245, screened from a gene library of Xoo by biparental conjugation, originally contained 36.8 kb foreign DNA of Xoo genome. A series of subclones from 36.8 kb hrp fragment in pUHRX245 showed that a 3.3 kb Sac I DNA complemented all hrp - mutants to hypersensitive response (HR) elicitation on tobacco, but only restored the pathogenicity to hrp - mutants, JCM2482 of Xoo and M1005 of X.o.pv.oryzicola (Xooc), with symptoms of rice leaf blight and streak, respectively. The fragment sequenced confirms that hrpXoo genes and two open reading frames, hspORF1 and hspORF2, related with heat shock protein 90 family, are contained in 3.3 kb DNA. There was no alignment of HspORF1 in protein database while HspORF2 was homologous to Hsp90 Xo with an identity over 99%. The hrpXoo gene was highly homologous to hrpX genes already reported in other Xanthomonas . A 60 bp nucleotide sequence coding for a structure of an alpha helix turn alpha helix is highly conserved in Xanthomonas . A 1.1 kb upstream DFNA of the hrpXoo without the 60 bp fragment conferred pathogenicity on rice and HR induction on tobacco only to hrp - mutant of a JXOIII strain of Xoo but not to hrp - mutants either of PXO99 A or of RS105 strain of Xooc. Sequence comparison of hrpXoo with other hrpX genes of Xanthomonas already loaded in GenBank showed at least 90% identity. The differences in amino acids between HrpXoo and the other five HrpX proteins suggest that the alternation of amino acids at sites 88,196 and 247 might reflect the differences between X. campestris and X. oryzae, and that the amino acid change at the site 175 in HrpX represent the difference between Xanthomonas that develop systemically in vascular bundles and locally in infected tissues after infection.