By transposon (Tn5) insertion mutagenesis, six novel avirulent mutants were isolated in Agrobacterium tumefaciens strain A-208. A binary vector, pIG121-Hm, containing a kanamycin resistance gene (npt II) and a beta-glucuronidase (GUS) gene with an intron, was introduced into these mutants. The resultant mutants were inoculated onto leaves of Kalanchoe daigremontiana and to tobacco BY2 cells to assay GUS activity to monitor the T-DNA transfer into the nuclei of host cells. High GUS activity comparable to that in strain A-208 (parent strain) was detected in three mutants, M-1, M-21 and M-31 mutants, in spite of their inability to induce galls, suggesting that those mutants can transfer T-DNA into host nuclei, but cannot integrate it into chromosomes. M-1 mutant had one insertion of Tn5 in a new gene, abvA gene in pTiT37. Introduction of the 2.3 kb DNA segment containing abvA gene into M-1 mutant complemented completely the avirulent phenotype of M-1 mutant. The abvA gene was distributed not only on the nopaline-type plasmid pTiT37 but also on an octopine-type plasmid pTiA6NC and chromosome (C58) of A. tumefaciens. In M-21 mutant, a Tn5 was inserted into iaaM gene (tryptophan monooxygenase gene), which is involved in the biosynthesis of indoleacetic acid, in the T-DNA region in Ti plasmid. M-31 mutant had one insertion of Tn5 in virA gene in vir-region on Ti plasmid. Introduction of virA gene into M-31 mutant complemented avirulent phenotype of M-31 mutant.
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