Heterologous expression from Agrobacterium virulence promoters

Abstract

The aim of this work was twofold: to construct plasmids with a gene encoding a pesticidal protein expressed from an Agrobacterium tumefaciens virulence promoter and to determine, in planta, the sites of Agrobacterium vir-induction. A number of methods were employed to detect in situ vir-induction and, to this end, genes encoding β-glucuronidase (GUS) and bioluminescence (lux) were linked in plasmid constructs to Agrobacterium vir-promoters. In each case, expression of the gene was shown to be induced by the v/r-inducing phenolic compound acetosyringone. An existing plasmid, in which the lacZ gene was under control of the virB promoter was utilised to demonstrate v/r-induction occurring at sites of injury on the roots of mung bean seedlings. Pesticidal genes expressed from Agrobacterium vimlence promoters would form the basis of a biological control system. A microbial inoculant harbouring such a construct would produce the pesticidal protein only when in the presence of vi-inducing compounds in plant wound exudates. A chitinase gene, chiB, from Serratia marcescens was characterised and sequenced and, following removal of its promoter region, was linked to an Agrobacterium virB promoter. Plasmids were also constructed in which the chiA gene of S. marcescens was brought under the control of a virB or virE promoter. All the constructs specified acetosyringone- inducible production of chitinase. Chitinase is effective in the biological control of chitin containing organisms such as fungi.