MULTIPLE COPIES OF VIRG ALLOW INDUCTION OF AGROBACTERIUM-TUMEFACIENS-VIR GENES AND T-DNA PROCESSING AT ALKALINE PH

Previous work indicated that the virulence (vir) genes of the octopine -type Ti (tumor-inducing) plasmid pTiA6 of Agrobacterium tumefaciens a re induced by phenolic plant signal molecules only in acidic medium (p H <6.5). Upon induction of the vir genes, the T-DNA (transferred DNA) is processed from the Ti plasmid and is transferred to plant cells. We report here that several vir genes of pTiA6 can be induced to high le vels by acetosyringone in both minimal and rich media at a pH greater than 7.5 when multiple (5-10) copies of virG are present in A. tumefac iens. In AB minimal medium, the extent of induction (measured by the e xpression of vir gene::lacZ fusions) at alkaline pH (>7.5) can be as h igh as 41% of that at acidic pH (5.5). In LB rich medium with a pH gre ater than 8.0, vir gene induction could be up to fourfold that of the level in the corresponding acidic medium. The induction of octopine-ty pe vir genes at alkaline pH depended on the source of virG gene presen t in multiple copies within the bacterial cell: In some instances, mul tiple copies of virG from the nopaline-type Ti plasmid pTiC58 did not affect induction at alkaline pH, whereas multiple copies of virG from the octopine-type Ti plasmid pTiA6 or the agropine-type Ti plasmid pTi Bo542 did. After 12 hr of induction, virD and virG induction by acetos yringone at both acidic and alkaline pH correlated well with the produ ction of processed T-DNA intermediates. The correlation was poor after induction for 24 hr. The induction of virE did not correlate with T-D NA processing at either early or late times. These data show that the presence of multiple copies of virG in A. tumefaciens can alter the pH -sensitivity profile of vir gene induction, suggesting that virG, as w ell as virA, may play a role in the pH response to plant phenolic sign al molecules.