Screening Of Xanthomonas Oryzae Pv. Oryzae Tn5-mutants That Overcome The Xa32 Mediated-resistance In Rice And Identification Of The Avirulence Gene AvrXa32

Bacterial blight (BB) is one of the most important bacterial diseases of rice in the world. The interaction between Xanthomonas oryzae pv. oryzae (Xoo),the cause agent of BB, and rice represents as an excellent model to study the plant pathogenic bacteria-host interaction. To date, the genomes of two different rice subspecies japonica variety Nipponbare and indica variety 9311 and three different races of Xoo PXO99, KACC, MAFF have been sequenced. The BB, therefore, becomes the important crop disease for witch both the host plant and the pathogenic bacteria genome sequences are available on the Internet. All of these provide plentiful biological information resources for investigating the molecular interaction between pathogenic bacteria and host plant.The avirulence gene, which is the key factor interacting with host plant, determine the incompatibility or compatibility through the interaction between the bacterial and host. The Xoo, with a given avr-gene, is incompatible on rice harbouring corresponding R-gene and compatible on the rice plants without the R-gene. Study the avr-gene corresponding to the R gene is helpful to reveal the mystery of molecular interaction between the bacterial and the host plant, clarify the molecular mechanism of the host resistance and all of these are meaningful to the rice breeding for disease resistance.In our previous study, Xa32 is approved to be a wide spectrum, complete dominant, host all-growth-stage effective BB resistance gene from wild rice. Screening the PXO99-Tn5 mutants that can overcome the Xa32-mediated resistance in rice is meaningful for studing the resistance mechanism of Xa32 and helpful to study how the PXO99 has a wide host range of rice genotypies. To date, all the identified avr-genes in Xoo are belonging to the gene familiy encoding TAL effectors. Using the genomic information, therefore, to obtain the TAL effectors adjacent genes and analysis on their function and structure is helpful to discover the TAL effectors distribution in the PXO99 and predicate the avr-gene combine with the flanking sequences analysis. In order to obtain the avrXa32, we have done a large scale screening of PXO99-Tn5 mutant, gain the flanking sequences of the Tn5 insertion sites and made analysis using bioinformatics. The main results are as follows:1. The pathogenicity of PXO99-Tn5 mutants were surveyed using leaf-cutting method. Each mutant clone was inoculated on 1 plant of rice introgression line C4064 (harbouring Xa32). 15-18 days after inoculation, leaves of more than 122 susceptible plants were collected for isolating the virulent mutant candidates. Each of the 122 virulent mutant candidates was isolated and re-inoculated on 2 plants of C4064 and 2 plants of susceptible genotype JG30 as control. Finally, 6 virulent mutants KXM2, KXM12, KXM22, KXM24, KXM66 and KXM71 were confirmed that they can cause disease symptoms on the C4064 plants.2. Molecular analysis of the mutant strains. PCR analysis revealed that all the 122 candidate mutant strains were inserted with Tn5. Southern blotting analysis indicated that there was a double Tn5-insertion in KXM22, and other 5 pathogenic mutants were single-copy inserted.3. The flanking sequences of Tn5-insertion sites in the 4 of the 6 pathogenic mutants were isolated by PCR walking, including DNA digestion, ligation, two rounds of PCR amplification.Analysis and prediction of the mutant genes disrupted were performed using bioinformatics. The flanking sequences were aligned with the whole genome sequences of Xoo strains and the results showed that the inserted sites of KXM2, KXM12, KXM22 and KXM71 were genes encoding ISXoo6 transposase, Na+ symporter, ISXo8 Transposase and inter between UDP-N-acetylmuramate adenylate kinase, respectively.4. Through analysis on function and structure of the adjacent genes, we found that the adjacent genes of TAL effectors have remarkable genetic features: trans-posable elements such as ISs, transposes and phage-related genes were located in the neighbouring areas. These ISs might promote duplication and rearrangement of avr-genes, and likely contributed to their proliferation in the Xoo genome, at last might cause the variation of physiological races. The 2 Xoo mutants KXM2 and KXM22, were disrupted in the sites encoding ISXoo6 transposase and ISXo8 Transposase, respectively, which downstream might be TAL effectors. These may cause the change of Xoo pathogenicity on the C4064 plants.