Study Of Identifying Pathogenic Strains From Xoo Mutant Library Using Rice Suspension Cells

The rice bacterial blight (BB) resistance gene, Xa23, was originally identified from Oryza rufipogon based on the resistance spectrum evaluation, resistance type and inheritance analysis. It is a complete dominant resistance gene with the widest resistance spectrum among all the known BB resistance genes identified so far. To date, no natural Xoo strain that could infect rice harboring Xa23 has been found. We constructed P6 mutant library about 24,000 mutants by Tn5-derived transposon tagging system, and expected to discriminate a pathogenic strain that infect rice harboring Xa23.To date, there were two main methods of identifying pathogenic strain by leaf-cutting or infiltration with a needleless syringe. Two methods not only need lots of time, but also were restricted by rice growth season. In order to identify pathogenic mutants from a P6 mutant library in laboratory, rather than in field or greenhouse, an identifying pathogenic strain system by rice suspension cell line in Lab was established, which offered a new way of identifying other pathogenic strain in cellular level. The results are listed as follow:(1) Using seeds of rice 'Zhongye 1' as explant, from which callus were induced on N6 induced medium with pH 5.8 and lots of embryo callus were generated in the secondary culture at 25℃after 3 months in dark. Using light yellow, grainy and available embryo callus as cell source of the suspension cells, the suspension cells could be obtained in N6 liquid medium at PH: 5.6 after 4-6 times subculture on the rotator shaker under at 25℃, 110-130rpm in dark.(2) The suspension cell death rate induced by compatible strain XMS-25 or XMS-30 was low in 78h, which was not different from CK. It showed that HR was not induced by compatible bacterium. However there was strong HR between the suspension cells and the P6. The change tendency of system death rate and extract liquid color were not obvious before 48h; suspension cell death rate increased gradually and extract liquid color deepened gradually; suspension cell rate reached to the maximum after 60h. Suspension cell death rate and extract liquid color tended to stabilize. It showed that HR was induced by compatible bacterium.Rice bacterial blight (BB) is a model system for study on plant-pathogenic bacteria interactions. The suspension cell line of rice cultivar Zhongye 1 harboring Xa23 gene was established and the interactions between suspension cells and P6 or its virulent mutants were systematically investigated. It was found that there was strong HR between the suspension cells and the virulent mutants of P6, and the color or OD₅₉₅ value of the extracted solution from the suspension cells stained with bromchlorphenol blue could be used as an index to discriminate a pathogenic strain from a non-pathogenic strain. Thus, an experiment system for identifying pathogenic mutants from P6 mutant library using rice suspension cells was established.