Investigation On The Pathogenicity Of Xanthomonas Oryzae Pv. Oryzae (Xoo) And Interaction Between The Avirulence Protein AvrXa23of Xoo And A Resistance Gene In Rice

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the mostserious diseases of rice worldwide. Identification of virulence factors of phytopathogens is considered anovel strategy to prevent and control plant diseases.Xoo is a model bacteria for studing the interaction between plant and pathogenic bacteria. Xoo-riceinteraction is a typical model of “gene for gene” relationship. The T3SS effectors are the key pathogenicfactors of Xanthomonas spp. TAL-effectors (Transcription Activator Like effector) consist of one classof the T3SS effectors in Xanthomonas. Currently, more and more bacterial blight resistance genes havebeen identified from cultivated rice and wild rice species, and the molecular recognition code betweenTAL-effector and host target genes has been deciphered, all of these provide theoretical andexperimental basis for investigating the molecular interaction between pathogenic bacteria and rice.There are two parts of research in this dissertation. One is identification of the virulence-relatedfactors from the Xoo strain PXO99; the other part is investigation of the molecular interaction betweenXoo TAL-effector AvrXa23and the R-gene HR2in rice. Results are summarized as follows:The isolation and characterization of virulence factors in Xoo strain PXO99:1. The Tn5-inserted mutants of PXO99were screened on indica rice JG30, which is highly susceptibleto PXO99, by leaf-cutting inoculation. Four virulence-reduced mutants, PXM36, PXM37, PXM69and PXM73were obtained after two rounds of screening;2. PCR and Southern blotting showed a single copy of Tn5-insertion in the genomes of the fourvirulence-reduced mutants;3. PCR walking and sequencing analysis revealed the Tn5transposon insertion sites in the genomes ofthe four virulence-reduced mutants. The disrupted genes in the mutants PXM36, PXM37, PXM69and PXM73were gspE, eglB, hrcQ and gspD, respectively;4. Creation of site specific mutants with a kanamycin-encoding gene cassette inserted at the same siteas that of the Tn5-insertion confirmed the reduced pathogenicity of the four Tn5mutants.Functionally complementary experiments, gene expression assays, and cellulase secretion analysisrevealed the functions of the disrupted genes individually: gspD and gspE are general secretarypathway proteins of type II secretion system (T2SS), the insertion of the gspE and gspD made theT2SS not to be assembled completely, resulting in the reduced virulence. Endoglucanase is thecomposition of cellulase, encoded by eglB gene. Endoglucanase is one of the cell wall degradingenzyme secretion by T2SS, the mutation of eglB contributes to attenuated virulence in the reason ofdecreasing the ability of the cell wall degradation in the rice leaves. HrcQ is the core component ofT3SS, hrcQ mutant completely lost the virulence on host rice and the ability of eliciting HR innonhost tobacco.The molecular interaction between Xoo avirulence protein AvrXa23and rice R-gene HR2:5. The promoter prediction showed that there were four promoters in T189(a TAC clone containsXa23gene) DNA sequences. We constructed the promoter vector driving GUS gene to assay the promoter activity, and we found P4(containing a1.7K DNA fragment) could trigger HR inNicotiana benthamiana leaves unexpectively, so we focus on1.7K vector to research the HRphenomenon;6. Promoter deletion analysis of the1.7K using agroinfiltration transient expression indicated that the216bp and210bp fragments were important for triggering HR;7. One ORF, named HR2ORF, was identified between the216bp and210bp fragments by NCBI-ORFFinder in website. A series of HR2vectors were constructed to assay the ability of eliciting HR, andit was demonstrated that the expression of HR2gene resulted in eliciting HR in tobacco leaves;8. The deletion mutants of the216bp fragment were co-injected into tobacco leaves with AvrXa23carried by Agrobacterium EHA105strain. The results showed that AvrXa23could recognize andbind to the118bp (located in-233~-116upstream of the216bp fragment) DNA sequences, inducedthe HR2expression;9. Based on the results, a model of interaction between AvrXa23and HR2was proposed. AvrXa23specifically recognized and bound to the118bp HR2gene promoter fragment, induced theexpression of HR2, HR2protein accumulation in the infiltrated area, leading to the resistanceresponse. In nonhost Nicotiana benthamiana, the resistance response was reflected in HR trigging.