Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone

Abstract

Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions. In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F. The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50%o for strains T37 and C58F and was of the order of 1% for strain C58. Control cultures not exposed to acetosyringone did not yield avirulent clones. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors. All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid. In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.The causal agent of crown gall, Agrobacterium tumefaciens, infects plant wounds where it detects the presence of specific compounds of plant origin. The virulence (vir) genes present on the Ti plasmids ofA. tumefaciens are then induced. Expression of the vir genes in the bacterium leads to the transfer to host plant cells of a particular region of the Ti plasmids, called the transferred (T-) DNA. This DNA of bacterial origin becomes stably integrated into the nuclear genome of the plant cells.

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

“…The Ti plasmid was transferred from derivatives of strain D10B/87 to the Ti plasmid-less Agrobacterium recipient C58C1 by conjugation (8). Ti plasmid deletions were detected by separation of linear DNA from plasmid DNA on an agarose gel after lysis in wells (8). These deletions were analyzed by separate digestions of total bacterial DNA with EcoRI, HindIII, and BamHI, and probing by Southern hybridization with overlapping cosmid clones from the vir and T regions of pTiC58 (9).…”

“…Induction of a virB::lacZ fusion was monitored in the presence of 60 M acetosyringone as described previously (8). The Ti plasmid was transferred from derivatives of strain D10B/87 to the Ti plasmid-less Agrobacterium recipient C58C1 by conjugation (8). Ti plasmid deletions were detected by separation of linear DNA from plasmid DNA on an agarose gel after lysis in wells (8).…”

“…Their purity was regularly verified by confirming 100 isolated colonies for pathogenicity on tomato plants (see below). Bacterial cultures in acidic (pH 5.8) minimal medium containing 60 M acetosyringone were initiated from 10 7 cells, as described before (8). Minimal media used to grow the biotype 2 strain D10B/87 were supplemented with 0.1 g of biotin per ml.…”

“…Induction of a virB::lacZ fusion was monitored in the presence of 60 M acetosyringone as described previously (8). The Ti plasmid was transferred from derivatives of strain D10B/87 to the Ti plasmid-less Agrobacterium recipient C58C1 by conjugation (8).…”

“…These mutants had lost the capacity to show the vir gene induction response, and their growth was no longer inhibited by acetosyringone. In most of the C58 mutants that were examined, virA or virG was inactivated by the integration of the endogenous insertion sequence element IS426 (7,8). To determine if the production of nonpathogenic mutants could also be observed in planta, various strains of A. tumefaciens were inoculated on apple plantlets grown aseptically in tissue culture.…”