IDENTIFICATION AND CHARACTERIZATION OF Hrp GENES FROM Xanthomonas Oryzae

Two pathovars, Xanthomonas oryzae pv oryzae and pv. oryzicola, are the critical agents respectively causing leaf blight and leaf streak diseases on rice, and ? possess hrp genes as other gram negative plant pathogenic bacteria. The hrp genes * :efer the pathogenic bacteria to the ability to trigger hyper3ensitive response (HR) on nonhost plants and to be pathogenic (Pth) or parastic on susceptible plants. The hrp gene cluster also encodes conserved proteins composing a type III secretion system apparatus that deliver effect proteins (eg. Avr or harpins or Vir) into host plant cells. In this research, 26 strains of hrjfmutants, further identified into 3 types: HR VPth7harpin, HR/Pth7IIF and HR7Pth~Iharpin, and 4 isolates of dsp mutants are obtained from JXOIII and PX099A representing the wild types of X.o.pv.oryzae , and RS 105, the wild type of X.o.pv.oryzicola. The dcp7disease specific pathogenicity) mutants secrets extracellular polysaccharide (EPS) nonnally, but do elicit HR on tobacco and not be pathogenic on rice even though some have a tightly weak pathogenicity. The signal to trigger HR on tobacco is a harpin-like protein, which is stable at I 000C for I 0mm and sensitive to protease K. LPS (lipopolysaccharide) from HRT/pth /1W and the wild types of the pathovars do not elicit HR on tobacco demonstrated by sonicating or 10% water-phenol a method. Extracellular enzyme (aniylase, pectate lyase, protease, cellulase and lipase) activities of all hrjf mutants are similar to those of the wild types. None ? of the mutants without xanthomonadin is capable of being pathogenic on rice and eliciting HR on tobacco. The extracellular proteins of different hrp mutants induced on a medium (XOM) are different. pUHRX245, a hrp gene clone from JXOIII gene library, and pHURS 130, pHURS 138 and pUHRS662, anotber 3 hrp gene clones from RS 105 gene library, are screened by using biparental conjugation protocol. Restriction endonuclease digestion and constructed subclones indicate that 36.8-kb hrp gene fragment is contained in pUHRX245 and that 39.3-kb hrp gene fragment is harbored 3 ~S11ACT respectively in pUHRS13O, pUHRS 138 and pUHRS662 with the same endonuclease digestion sites. pUHRX245 and pUHRS 1.38 transferred into S 17-1 as a host can restore not only all the hrp mutants to the ability to trigger HR on tobacco but the hrp mutant, M1005, the capability to be pathogenic on rice with the symptom of the leaf streak disease. Series of subclones from 36.8-kb hrp fragment in pUHRX245 show that a 3.3- kb Sad DNA can complement all hrp mutant to HR elicitation on tobacco. Sequencing 3.3-kb fragment demonstrates that contained are an intact hrpXoo gene and hspORFl and hspORF2, which are related to heat shock protein 90 family. The function of hspORFl and hspORF2 is unknown, though they may putatively sense environmental temperature and plant signals. Subclones from 39.3-kb hrp fragment in pUHRS138 demonstrate that a 4.5- kb BamHI-KpnI DNA can restore the HR elicitation on tobacco to all hrp mutants. The fragment sequence confirms that hrpGXOO, and hrpXooc genes are contained in the 4.5-kb DNA. The complementation...