Cloning Of Avirulence Gene AvrXa23 In Xathomonas Oryzae Pv. Oryzae And Functional Analysis Of The Rice Proteins Interacting With AvrXa23

Bacterial blight(BB) is an overwhelming disease caused by Xanthomonas oryzae pv. oryzae(Xoo), seriously restricting the stability of rice yield. BB is a model system used to study the interaction between plants and their pathogens. Studying on the mechanism of disease resistance and bacterial infection is meaningful to prevent and control this disease. In recent years, the availability of genome sequences of rice varieties(Japonica variety Nipponbare and Indica variety 9311) and Xoo races(Korean strain KACC10331, Japanese strain MAFF311018 and Philippines strain PXO99A) has opened the door to reveal mechanisms underlaying the interaction between rice and Xoo. In a previous study, a total of 14 PXO99 A mutants virulent to CBB23 were identified by screening the Tn5-derived mutant library of PXO99 A. Flanking sequence analysis revealed that 3 mutants, P99M2, P99M4 and P99M5, had Tn5 insertions in the conserved region of TALE genes.This study was divided into 2 main parts: cloning of the avrXa23 gene and characterization of the proteins interacting with Avr Xa23 in rice. The main results are as follows:1. We got 34 cosmid clones containing TALE genes by Southern blotting of the PXO99 A genomic library. The 34 cosmid vectors were transformed into P99M5 individually. The cosmid clone C15 could restore the ability of P99M5 in triggering BB resistance of CBB23, suggesting that C15 contains the avirulence gene corresponding to the Xa23 gene.2. DNA sequencing revealed that the cosmid clone C15 harbors full-length of tal C9 a and tal C9 b. Thus, C15 subclones, p HM1-L and p HM1-Mi, containing tal C9 b and tal C9 a respectively, were transformed into complement cells of P99M5. After successful transformation, we got P99M5-L and P99M5-Mi strains. CBB23 is susceptible to P99M5-Mi but resistant to P99M5-L, indicating tal C9 b is the avirulence gene avr Xa23.3. Avrxa23, a 4452 bp full-length DNA sequence encoding a protein containing 1483 amino acids, is a new member of the Xanthomonas TALE gene family. The avr Xa23 was proved to exist in all the Xoo representative races by Southern blotting analysis, indicating that avr Xa23 may be ubiquitous among Xoo strains. Xa23-mediated resistance is induced by Avr Xa23. The distribution of avr Xa23 in all Xoo strains could explain the extremely broad-spectrum resistance of Xa23.4. We constructed a c DNA library of CBB23 leaves. Afterwards, Avr Xa23 was used as bait to screen the c DNA library using yeast two hybrid(Y2H) system, and 323 putative clones were obtained in the preliminary screening. After exclusion of frame shift mutation using sequencing and verification of rescreening, we identified 43 candidate clones interacting with Avr Xa23, encoding 26 different proteins. Consequently, seven of these 26 proteins may be related to disease resistance, these proteins were confirmed by Y2 H and GST pull-down in vitro. Finally, three proteins Os IMPα, Os THIC and Os THI1 were confirmed to interact with Avr Xa23.5. OsIMPα is a nuclear transfer protein, which assists Avr Xa23 into the nucleus. The Os THIC is a HMPP synthase, plays a significant role in the synthesis of vitamin B1. While the Os THI1 is a thiazole synthase that plays an important role in the synthesis of vitamin B1 synthase. Thiamine could induce system acquired resistance(SAR) in plants through the salicylic acid and Ca2+ signaling pathways, functions as an activator of plant disease resistance.6. Subcellular localization experiments showed that Os THI1 was localized in the chloroplast. Os THI1 interacts with N terminus of Avr Xa23. By knocking out Os THI1 gene of CBB4, we got the transgenetic plants c Thi1B4. Compared with CBB4, c Thi1B4 is susceptible to bacterial blight pathogen(PXO61) and contains less vitamin B1.In summary, we successfully cloned the TALE gene avrXa23 corresponding to resistance gene Xa23 and found that avr Xa23 is widely distributed in all tested 38 Xoo races. We found that the nuclear transfer protein IMPα and vitamin B1 synthesis related proteins Os THIC and Os THI1 could interact with Avr Xa23. Thus, we speculated that Avr Xa23 could interact with vitamin B1 synthesis protein to inhibit rice resistance.