|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1997 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1996 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Three avirulent mutant strains, (B-119, M-3, and M-14 strains) were produced from A208 strain (virulent) of Agrobacterium tumefaciens by using transposon (Tn5) tagging method. With the all mutants, one Tn5 was inserted in chromosome but not in Ti-plasmid. In B-119 strain, a Tn5 was inserted in a new chromosal virulence gene (acvB). One Tn5 was inserted in chvA with M-3 strain, and in chvB with M-14 strain. acvB and chvA genes were cloned using B-119 and M-3 strains, respectively, and their function were analyzed.
acvB gene were distributed in all strains of A.tumefaciens tested, while it was absent in Rhozobium, Pseudomonas, and Bacillus. Such distsribution of acvB gene among bacteria suggested that acvB gene is responsible for a character unique to A.tumefaciens, possibly T-DNA transfer to host plant. A homolg of acvB gene was located on an octopine type Ti-plasmid.
A large amount of AcvB protein was prepared by expressing acvB gene in E.coli, and antibody for AcvB was prepared. By the
experiment using AcvB-antibody, it was shown that AcvB protein (47 kDa) is localized in periplasm of A.tumefaciens. And AcvB protein was shown to have ability to bind a single stranded DNA in vitro.
In spite that B-119 and M-3 strains are avirulent, T-strands were produced normally in both strains in respons to induction with acetosyringone at pH 5.5. B-119 sstrain could transfer T-DNA to protoplasts of tobacco BY2 cells without cell walls, but not to the cells with cell walls. On the other hand, M-3 strain could transfer T-DNA neither to BY2 cells nor to their protoplasts.
When induced with acetosyringone, a protein of about 19 kDa appeared in outermembranes of A 208 and B-119 strains, but not in ones of M-3 strains. The 19 kDa protein is likely to be pili protein (pilin) from its molecular weight and its localization in outermembrane.
Taken together the results described above and some other informations, the following mechanism are conceivable for virulence of B-119, and M-3 strains ; with B-119 strain, AcvB protein is missing in periplasm. With M-3 strain, ChvA protein is absent in innermembrane. The absence of these protein in the respective mutant may cause the degeneration or disappearence of pili which is possibly a chamnel for T-DNA transfer from A.tumefaciens to host plant cell. Less