Investigation On The Pathogenic Avirulence Gene Corresponding To The Rice Bacterial Blight Resistance Gene Xa30

Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important pathogenicities of rice, has spreaded in the major rice growing regions in the world. The interaction between Xoo and rice is followed the gene-for-gene hypothesis. Xoo is considered as a model pathogen in the research of molecular plant pathology. According to the"gene-for-gene"hypothesis, the plant disease resistance is determined by the interaction between the rice resistance gene and its corresponding bacterial avirulence gene. In plant pathogenic bacteria, the host specificity is mediated by avirulence (avr) gene-encoded effector molecules that can trigger a race-specific defense response in hosts carrying a corresponding resistance (R) gene. Systematic understanding of the molecular mechanism of pathogenicity of Xoo will present an insight into the study of the complex interaction between rice and Xoo and will give us more opportunities to develop strategies for preventing those important pathogens.To date, complete genome sequences of Japanese Xoo strain MAFF311018, Korean strain KACC10331 and the Phillippine strain PXO99A were published. This provides us more biological information resources for our research, but there are still a large number of unknown genes. The construction of mutant library provides a basis for the studying of Xoo functional genomics. In our previous study, we constructed a Tn5-inserted PXO99 mutant library containing a total of 24,192 clones. According to the T-DNA insertion genomic formula P=1-(1-X/G)~n, the probability of insertion is 99%, and it covered six times of the PXO99 genome.Previous researches in our lab had identified a new BB-resistant germplasm Y238 originally from wild rice, which showed high and broad resistance spectrum to Xoo. Genetic analysis revealed that the resistance of Y238 was controlled by a new dominate gene named Xa30. By using the near-isogenic line CBB30I, Xa30 was mapped on the long arm of rice chromosome 11. PXO99 is a highly toxic strain, which has a broad pathogenicity range, but is incompatible to CBB30I. In order to investigate the interaction between Xoo and Xa30, we attempted to find the cognate Xoo avr-gene of Xa30 by screening PXO99 mutant library using the near isogenic line CBB30I. The main results are as follows:1.The first round screening of the mutant library in the field: The pathogenicity of 15540 mutants were assayed by using leaf-cutting method, each mutant clone was inoculated on one CBB30I plant. 15-18 days after inoculation, more than 210 susceptible leaves were collected for isolating the virulent mutant candidates.2. The second round screening of the mutant library: Every one of the 210 virulent mutant candidates was isolated and inoculated on two CBB30I plants and two susceptible IR24 plants as control. Finally, 23 virulent mutants were confirmed that can cause different degrees of disease symptoms.3. Molecular analysis of the Tn5 inserting in the mutant strains: PCR analysis revealed that all the mutant strains were inserted with Tn5 transposon. Southern bloting analysis indicated that there was a single Tn5-insertion in the eight pathogenic mutants, but XMC-41 was double-copy inserted.4. Isolation of the flanking sequences: The flanking sequences of Tn5 transposon insertion sites in the nine pathogenic mutants were isolated by PCR walking, including DNA digestion, ligation, two rounds of PCR amplification and so on.5. Bioinformatics analysis: The flanking sequences of Tn5 transposon insertion sites in the nine pathogenic mutants were sequenced, then they were aligned with the whole genome sequences of Xoo strains. The results revealed that: In the XMC-103 mutant strain, Tn5 insertion disrupted a gene encoding hypothetical protein. Its downstream gene is talC6a, encoding a TAL effector AvrBs3/pthA protein whose corresponding resistance gene is unknown; In mutants XMC-89, XMC-92, XMC-100, XMC-105and XMC-107, Tn5-transposon inserted at different sites and the disrupted genes encoding different proteins: transcriptional regulator of LysR family, partition protein, sensor histidine kinase, hypothetical protein(its neighboring gene encodes two-component system regulatory protein with HD-GYP domain ) and peptidase, respectively; Tn5-insertion site information from mutants XMC-41, XMC-55 and XMC-101 suggests that penicillin acylase II, maleylacetoacetate isomerase and ATP-dependent RNA helicase might also involve the pathway that avirulence gene mediated incompatibility to CBB30I.