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. 1999 Jan;181(1):186-96.

pSa Causes Oncogenic Suppression of Agrobacterium by Inhibiting VirE2 Protein Export

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Free PMC article

pSa Causes Oncogenic Suppression of Agrobacterium by Inhibiting VirE2 Protein Export

L Y Lee et al. J Bacteriol. .
Free PMC article

Abstract

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

Figures

FIG. 1
FIG. 1
Teratoma formation on Arabidopsis roots. Sterile Arabidopsis root segments were cocultivated with A. tumefaciens for 2 days and then transferred to MS basal medium lacking phytohormones, but containing antibiotics to prevent the growth of bacterial cells. Teratomas first appeared approximately 2 weeks after infection, and the roots were photographed 1 month after infection. Root segments were infected by A. tumefaciens At928 [A208(pBISN1)] (A), A. tumefaciens At929 [A208(pBISN1, pSa)] (B), A. tumefaciens At931 [A208(pBISN1 plus osa)] (C), or A. tumefaciens At932 [A208(pBISN1 plus vector)].
FIG. 2
FIG. 2
Transformation of Arabidopsis roots to kanamycin resistance. Sterile Arabidopsis root segments were cocultivated with A. tumefaciens for 2 days and then transferred to CIM containing antibiotics to prevent the growth of bacterial cells and to select for transformed plant cells. The roots were photographed 1 month after infection. Root segments were infected by A. tumefaciens At928 [A208(pBISN1)] (A), A. tumefaciens At929 [A208(pBISN1, pSa)] (B), A. tumefaciens At931 [A208(pBISN1 plus osa)] (C), or A. tumefaciens At932 [A208(pBISN1 plus vector)] (D).
FIG. 3
FIG. 3
Transient GUS expression in Arabidopsis roots and tobacco leaf discs. Sterile Arabidopsis root segments (A) or tobacco leaf discs (B) were cocultivated with A. tumefaciens for 2 days and then transferred to medium containing Timentin to prevent the growth of bacterial cells. After 2 additional days, the plant material was washed with 0.9% NaCl and stained with X-Gluc. Photographs were taken with a dissecting microscope. Plant tissue was infected with A. tumefaciens At928 [A208(pBISN1)] (a), A. tumefaciens At929 [A208(pBISN1, pSa)] (b), A. tumefaciens At931 [A208(pBISN1 plus osa)] (c), or A. tumefaciens At932 [A208(pBISN1 plus vector)] (d).
FIG. 4
FIG. 4
Extracellular complementation of A. tumefaciens strains harboring pSa or osa. A. tumefaciens At221 (virE2 mutant, a T-DNA donor) and LBA4404 (a VirE2 donor lacking T-DNA) containing various plasmids were coinoculated onto Kalanchoë leaves. The leaves were photographed after 1 month. The A. tumefaciens strains are LBA4404, T-DNA VirE2 donor; At221, virE2 mutant T-DNA donor; At900, LBA4404(pSa); At901, LBA4404(pSa::neo); At902, LBA4404 (osa); At903, LBA4404 (vector); At904, At221(pSa); At905, At221(pSa::neo); At906, At221 (osa); and At907, At221 (vector).
FIG. 5
FIG. 5
Effect of osa on tumorigenesis by the virE2 mutant A. tumefaciens At221. Datura stems were inoculated with various A. tumefaciens strains, and the infected stem sections were photographed 30 days later. Panels: 1, A. tumefaciens At789 (wild-type pTi); 2, A. tumefaciens At793 (no pTi); 3, A. tumefaciens At221 (virE2 mutant); 4, A. tumefaciens At906 (At221 plus osa); 5, A. tumefaciens At907 (At221 plus vector); 6, A. tumefaciens At985 (At221 plus pJB31).
FIG. 6
FIG. 6
Tumorigenesis of various A. tumefaciens strains on VirE2-producing transgenic tobacco leaf discs. Leaf discs of sterile virE2 transgenic tobacco plants were infected with various A. tumefaciens strains. After 2 days of cocultivation, the discs were incubated on MS medium containing Timentin and photographed 1 month later. (A) Panels: 1, infection with A. tumefaciens At789 (wild-type pTi); 2, A. tumefaciens At793 (no pTi); 3, A. tumefaciens At985 [At221 (pJB31)]; 4, A. tumefaciens At989 (virE2 mutant); 5, A. tumefaciens At990 (At221 plus osa); 6, A. tumefaciens At991 (At221 plus vector). (B) Panels 1, no inoculation; 2, A. tumefaciens At928 [A208(pBISN1)]; 3, A. tumefaciens At931 [A208(pBISN1 plus osa)]; 4, A. tumefaciens At932 [A208(pBISN1 plus vector)].
FIG. 7
FIG. 7
Infection of D. stramonium stems with A. tumefaciens strains containing multiple copies of virB9, -10, and -11. D. stramonium seedlings were wounded and inoculated with A. tumefaciens A208 containing pBISN1, pUCD5542, and no additional plasmid (At935) (A); pSa (At925) (B); osa (At926) (C); or vector (At927) (D). The stem sections were photographed 1 month after inoculation.

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