Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid - PubMed (original) (raw)

Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid

V S Kalogeraki et al. J Bacteriol. 1998 Nov.

Abstract

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

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Figures

FIG. 1

FIG. 1

Octopine- and nopaline-type Ti plasmid vir regions. Solid parallelograms represent the regions of homology described by Engler and colleagues (11). The dashed parallelogram indicates the region conserved between these plasmids. Restriction fragments newly sequenced in this study are indicated with white boxes.

FIG. 2

FIG. 2

Sequence similarity between Orf10 of pTiC58 and the VirF of pTiA6. Straight lines represent polypeptide sequence with no similarity. Boxes represent conserved regions; black boxes represent regions of stronger similarity than gray boxes (also see Table 2 for similarity scores).

FIG. 3

FIG. 3

Nuclease S1 protection assays. Lane 1, oligonucleotide without S1 digestion; lane 2, protection by RNA purified from strain VIK10 cultured without acetosyringone; lane 3, protection by RNA purified from strain VIK10 cultured with acetosyringone; lane 4, protection by RNA purified from strain VIK11 cultured with acetosyringone.

FIG. 4

FIG. 4

Low-stringency Southern hybridizations using internal PCR fragments for virK (top), virL (middle), and virM (bottom). The last lane contains molecular mass standards that hybridize to the same probe.

FIG. 5

FIG. 5

Replacement of the virH locus by a gene mediating resistance to spectinomycin. (A) Double recombination required for this exchange. (B) Southern hybridizations of the parental strain R10 (wt [wild type]) and two identical mutants (1 and 2). The first of these was designated strain VIK28. The extreme left lane contains molecular mass standards that hybridize with the same probe. A PCR fragment lying within orf6 was used as a probe.

FIG. 6

FIG. 6

Carbon monoxide difference spectra of VirH1 and VirH2. Bacterial extracts were prepared as described in Materials and Methods and scanned across the visible spectrum in the presence and absence of carbon monoxide.

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