PMC

Western blots of the supernatant and total cell fractions from different constructs in A. tumefaciens strain A348. Cells were grown at 28°C under vir gene-inducing conditions, separated into supernatant and total cell fractions (noted on the top). The proteins were detected by using specific antibodies (noted on the left). Lane 1, A348; lane 2, A348 (pUFR047); lane 3, A348 (ptac-ros/pUFR047); and lane 4, A348 (ptac-CAT/pUFR047). Total cell proteins, TCP; and supernatant proteins, SP.

Coomassie Brilliant Blue staining and Western blotting of protein gels from different fractions of A. tumefaciens cells containing ptac-osa. (A) Cells were grown under inducing conditions and then collected, and separated into supernatant, periplasmic, cytoplasmic-soluble, and cytoplasmic-insoluble fractions (noted on the top). The fractions were run on SDS/PAGE gels, and stained with Coomassie Brilliant Blue. Lane 1, A348 (pUFR047); and lane 2, A348 (ptac-osa/pUFR047). (B) Western blots of the proteins from different fractions (noted on the top) detected with antibodies (noted on the right). Lane 1, A348 (pUFR047); and lane 2, A348 (ptac-osa/pUFR047). Periplasmic proteins, PP; cytoplasmic-soluble proteins, CSP; and cytoplasmic-insoluble proteins, CIP.

Steady-state abundance and functionality of VirE2::i31 insertion mutants in A. tumefaciens. A348 and At12516 cells carrying these plasmids were assayed for VirE2 protein abundance by immunoblot analysis. (A) Blots were prepared as described in the legend to Fig. 6 with protein samples from plasmid-carrying A348 (top panel) or At12516 (bottom panel) cells. Strains used were as follows: wild type, A348 (top panel) and At12516(pXZB27) (bottom panel); and ΔE2, At12516 (top and bottom panels); the numbers refer to the VirE2 residue immediately preceding the i31 insertion. The small arrow denotes the position of native VirE2. The large arrowhead denotes VirE2::i31 mutants, which migrated to different positions in SDS-polyacrylamide gels. (B) The functionality of the i31 insertion mutants was assessed by inoculation of strains on K. daigremontiana leaves (left leaf, A348 merodiploids coexpressing wild-type virE2 and the indicated virE2::i31 allele; right leaf, At12516 expressing the indicated virE2::i31 allele). The allele for VirE2.39::i31 restored virulence of At12516 cells to wild-type levels. The tumor at the top left was induced by inoculation with wild-type A348, and the tumor at the bottom right was induced by inoculation with At12516(pXZ27). The wound site at the top right shows the avirulence of At12516.

Western blots of A. tumefaciens containing VirE2 fusions with PhoA and GFPmut2. (A) Supernatant fraction from PhoA constructs detected with VirE2 antibodies. A348 (lane 1) was grown under inducing conditions, and A136 (phoA⁻) (ptac-virE1 + E2∷phoA/pUFR047) (lane 2) was grown under noninducing conditions. (B) Cells were grown at 28°C under inducing conditions, separated into the supernatant and total cell fractions (noted on the top), and detected with protein-specific antibodies (noted on the right). Lane 1, A348 (pUFR047); and lane 2, A348 (ptac-virE1 + E2∷gfpmut2).