VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals

Abstract

The vir genes of Agrobacterium tumefaciens tumor-inducing (Ti) plasmids direct the transfer of oncogenic portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells (T-DNA) into plant cells and are coordinately induced by plant-released phenolic chemical signals. We have used DNA microarrays, representing all genes of the octopine- and nopaline-type Ti plasmids, to identify all Ti-plasmid-encoded genes in the vir regulons of both plasmids. Acetosyringone (AS) induced the expression of all known members of the vir regulons, as well as a small number of additional genes. Unexpectedly, AS also caused a modest induction of virtually every Ti plasmid gene. This suggested that the copy number of the Ti plasmid might increase in response to AS, a hypothesis confirmed by DNA dot blotting. VirA and VirG were the only Vir proteins required for this copy number increase. Promoter resections and primer extension analysis of the repABC promoter region showed that expression of the promoter closest to repA (promoter P4) was induced by AS. We also identified a sequence resembling a consensus VirG-binding motif approximately 70 nucleotides upstream from the P4 transcription start site. Mutating this sequence blocked the AS-induced copy number increase of a RepABC-dependent miniplasmid, indicating that phospho-VirG increases copy number solely by enhancing repABC expression.

Fig. 1.

Genetic maps of the vir regions of the octopine- (A) and nopaline-type (B) Ti plasmids. Genes shown in red are members of the vir regulon, whereas genes shown in black are IS elements, and genes shown in gray encode uncharacterized proteins. Horizontal arrows indicate known or suspected operons.

Fig. 2.

Transcription profile of AS-induced and uninduced cultures of A348(pCC113)(pSM243cd) (Left) and C58(pCC113)(pSM243cd) (Right). Ti plasmid-encoded genes are indicated by using diamonds, whereas genes thought to be members of the putative SOS or heat-shock regulons are indicated by using stars and triangles, respectively, and other non-Ti plasmid-encoded genes are indicated by using circles.

Fig. 3.

S1 nuclease protection assays for A348 (A) and C58 (B). In each panel, the left lane shows the radio-labeled oligonucleotide in the absence of nuclease digestion, while the middle and right lanes show nuclease-resistant oligonucleotides after hybridization with mRNA from uninduced or AS-induced cultures, respectively. The rpoA and fusA genes are chromosomally encoded and judged likely to be constitutively expressed, whereas agaD and hupT are Ti plasmid-encoded and serve as controls for Ti plasmid genes that are not directly VirG-regulated.

Fig. 4.

DNA dot blots of strains A348(pCC113)(pSM243cd) (Upper) and C58(pCC113)(pSM243cd) (Lower) cultured in the presence or absence of 100 μM AS. Genomic DNA samples were serially diluted in 3-fold increments and spotted onto nylon membranes. Membranes were hybridized with ³²P-end-labeled probes specific for each replicon, which were synthesized by PCR on rpoD, kdpD, bapA, and virD5 genes by using oligonucleotides described in Table 3 and radiolabeled by treatment with EcoRI and the Klenow fragment of DNA polymerase in the presence of [α-³²P]-dATP.

Fig. 5.

Localization of the AS inducible rep promoter. Each plasmid contains repC-lacZ translational fusions and various amounts of rep promoter DNA or rep promoter-lacZ transcriptional fusions and were introduced into strain NTL4(pHC090), a Ti plasmid-less strain with a plasmid expressing virA and virG from their native promoters. Base positions at the endpoints of resections refer to distances from the repA gene translation start site. The resulting strains were cultured overnight in the presence or absence of 100 μM AS in acidified AB minimal medium (pH 5.5) and tested for β-galactosidase-specific activity (33).

Fig. 6.

DNA sequence of traI-repA intergenic region. Arrows indicate the 5′ ends of oligonucleotides used for PCR amplification. Nested deletions of the 5′ end of this region were made by using reverse primer PR and various forward primers, as indicated, whereas nested deletions of the 3′ end were made by using forward primer KP1FP and various reverse primers, as indicated. The locations of promoters P1-P4, two tra boxes, and the putative vir box are indicated. The translation start sites of the divergent repA and traI genes are double-underlined.

Fig. 7.

Primer extension assay using mRNA from strain NTL4(pHC090)(pKPRR5) after overnight incubation in the presence or absence of 100 μM AS. Two radiolabeled oligonucleotide primers were added, one of which hybridizes to repA mRNA, and the other of which hybridizes to the lacZα gene (of pHC090), which serves as a constitutive control.

Fig. 8.

Increase in copy number of repABC-dependent plasmids by phosphoVirG. Strain NTL4(pHC090)(pKP23) was incubated overnight in the presence or absence of 100 μM AS. Purified plasmid DNA was digested with KpnI, which linearizes the minireplicons (upper band of each lane) and cuts pHC090 at two sites, generating two lower bands of each lane. The two pHC090-derived fragments were used to normalize the band intensities of repABC-dependent plasmids. Plasmids pHC121 and pHC122 are derivatives of pKP23 with the indicated alterations in the vir box motif.

Fig. 9.

Summary of the regulation of the repABC operon. According to this model, wound-released signals act through VirA to phosphorylate VirG, which activates promoter P4, whereas 3-oxo-octanoylhomoserine lactone-TraR complexes activate promoters P1-P3 (10). All four promoters are autorepressed by RepA/RepB complexes, which bind to an operator directly downstream of promoter P4 (11).