Cloning And Functional Analysis Of Pathogenicity Effectors Of Xanthomonas Oryzae

The gram-negative plant-pathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo)and Xanthomonas oryzicola pv. oryzicola (Xooc) are the causative agents of bacterialblight and bacteria leaf streak in rice, respectively, which lead to economicallyimportant yield losses. These pathosystems have become well-established models forunderstading molecular interactions of plant-pathogens.The interactions between Xanthomonas oryzae pv.oryzae and rice are controlledin a gene-for-gene manner. However, the basis of the gene-for-gene specificity is notwell understood. In this study, a DNA fragment (359 bp) of the avrXa3 genecontaining three nuclear localization signal (NLS) motifs present in all members ofavrBs3/pthA family was used as a probe to screen the genomic library of JXOâ…¢strainof Xoo. Our results demonstrated that diverse members of the family existed in thepathogen genome. The avrBs3/PthA (avr/pth) genes could individually or collectivelyreside in one locus. The positive avr gene clones were transferred into the compatiblerecipient PXO99^A. Pathogenicity tests in near isogenic lines of rice confirmed thatthere were four R genes (Xa2, Xa3, xa5 and xa8) matching the four avr genes (avrXa2,avrXa3, avrxa5 and avrxa8) genes in the genome of Xoo strain JXOâ…¢. TheavrBs3/PthA gene (1.7 kb) present in p54 could be an avirulence gene specificallyinteracting with Xa3 gene present in IRBB3, hereby nominated as avr/pth3. Thesequence indicated that there are only 1.5 repeat numbers of the 102 bp repeat unit inavr/pth3. Alignments of 12^th-13^th amino acids together in the repetitive regionsencoded by this gene with those by other representatives in the AvrBs3 familyrevealed unique repeat arrangement which might reflect varieties of avirulence genesin Xoo. Furthermore, the recurrent parent rice line IR24 could contain several R genesresistant to Xoo bacterial blight. Some avr/pth genes and their functions identified inthis study shed new light on the mechanisms of gene for gene specificity in the Xoo-rice system, which will be definitely useful for further identifications of unknownR genes in rice against the bacterial blight.avrBs2,pga and popC in Xanthomonas oryzae contain a conserved sequencemotif in their promoter region (plant-inducible promoter, PIP-box,TTCGC-N15-TTCGC). It is speculated that HrpX controls their expression bybinding to this promoter element. However, the function of these three genes inpathogenicity is not being understood, avrBs2,pga and popC genes were PCR-generated by using the degenerated primers which were designed on the basic ofknown relative genes in other xanthomonads. Sequencing confirmed that the genesfrom Xoo in this study were as same as those reported in the genome sequence ofMAFF311018. avrBs2,pga and popC genes were first confirmed existing in Xooc.Comparison of the putative proteins with those of xanthomonads loaded in thedatabase of the NCBI revealed that the proteins of X. oryzae had 74%-98% identitiesto those in other Xanthomonas species. Based on the strategy for mutation byhomology recombination, the avrBs2, pga and popC mutants of two pathovars ofX.oryzae were generated and the single cross event in mutation was confirmed byPCR and Southern hybridization. The ability to cause water soaking symptoms in riceseedlings, the lesion lengths in adult rice, and the induction of hypersensitive responsein tobacco by those mutants were tested individually and respectively. The resultsdemonstrated that the mutation of the avrBs2, pga and popC genes, either in Xoo or inXooc, did not lost the ability to induce hypersensitive response in nonhost tobaccoand kept the same lesion lengths in rice as their parental wild strains. However, themutation in the avrBs2 gene reduced the ability to form water soaking symptoms indee seedlings compared with those by the wild type strains of Xoo and Xooc.Interestingly, the growing ability of and the water soaking symptom by the pgamutant of Xooc in rice were enhanced when compared with those by the wild typestrains. It is suggested that the polygalacturonase encoded by the pga gene of Xoocwas of the inhibitor for the innate immunity in rice.