CONSTITUTIVE EXPRESSION OF THE VIRULENCE GENES IMPROVES THE EFFICIENCY OF PLANT TRANSFORMATION BY AGROBACTERIUM

Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system. In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction. A mutation of virG, virGN54D (which codes for a Asn-54 -->, Asp amino acid change in the product), causes constitutive expression of other vir genes independent of virA. We have investigated whether providing Agrobacterium with a plasmid containing virGN54D augments the efficiency of transfer of the T-DNA (transferred DNA). For both tobacco and cotton, we observed an enhancement of transformation efficiency when the inciting Agrobacterium strain carries the virGN54D mutation. We also tested whether supplying Agrobacterium with a similar plasmid containing wild-type virG affects the efficiency of T-DNA transfer. An intermediate efficiency was observed when this plasmid was employed. Using a beta-glucuronidase (GUS) reporter gene to assess transient expression of T-DNA after transfer to tobacco and maize tissues, we observed a higher frequency of GUS-expressing foci after inoculation with Agrobacterium strains carrying virGN54D than with Agrobacterium carrying the wild-type virG. Gene transfer efficiency to maize by an octopine strain was greatly improved upon introduction of virGN54D. Multiple copies of wild-type virG were equally effective in promoting transient expression efficiency in tobacco but were virtually ineffective in maize. We propose the use of virGN54D to improve the efficiency of Agrobacterium-mediated transformation, especially for recalcitrant plant species.