The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirB-dependent export of VirE2

Abstract

Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.

FIG. 1

Structures of RSF1010 derivatives. Base pair numbering is according to reference . (A) The mobilization region and replication genes of RSF1010. The approximate positions of the genes and DNA sites discussed in this paper are indicated as are the locations and orientations of the corresponding promoters and the DNA sequence surrounding the nic site (arrowhead) at oriT. Relevant restriction sites are shown. (B) Frameshift mutation in mobA. Two base pairs at the AccI site in mobA (underlined) were added by digestion with AccI, blunt-ending with Klenow and deoxynucleoside triphosphates and religation. (C) Transition mutations in oriT. Two base pairs (boldface and underlined) were changed by oligonucleotide-directed mutagenesis to destroy the nic site at oriT; an additional 2 bp were changed (underlined) to create a HindIII site.

FIG. 2

Virulence assay of N. tabacum. N. tabacum cv. Havana 425 leaf explants were infected with A348 strains without and with RSF1010 derivative plasmids for 2 days and were then transferred to antibiotic-containing medium as described in Materials and Methods. The mean numbers of tumors ± standard errors (n = 15 to 17 leaf pieces) were determined after 10 days of incubation.

FIG. 3

Effects of different RSF1010 derivatives on the capacity of Agrobacterium sp. strain LBA4404 to serve as a VirE2 donor. N. tabacum cv. Havana 425 leaf explants were infected with a VirE2 donor strain (LBA4404 without or with RSF1010 derivative plasmids) either in the absence (−) or presence (+) of the T-strand donor strain A348::virE2 for 2 days, as described in Materials and Methods, and then transferred to selection medium. The mean numbers of tumors per explant ± standard errors (n = 18 leaf pieces) were determined after a 10-day incubation on selection medium.