Western blot analysis of the intracellular and extracellular S2 fractions, and tumor assays on tomato stem of VirB2 variants.

<p><i>A. tumefaciens</i> cells grown on acetosyringone (AS)-induced AB-MES (pH 5.5) agar at 19°C for 3 days <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101142#pone.0101142-Lai2" target="_blank">[7]</a> were collected to isolate intracellular proteins and extracellular S2 fractions. C58, <i>A. tumefaciens</i> wild type strain C58; V, empty vector pRL662; Δ<i>virB2</i>, <i>virB2</i> deletion mutant; Δ<i>virB2</i>(pVirB2), expression of wild type <i>virB</i>p-<i>B1</i>-<i>B2</i>-<i>B3</i> in Δ<i>virB2</i>. Western blot analysis with antisera against VirB2 B24 peptide or B23 peptide (for variants in C-PP) or RNA polymerase RpoA, as an internal control. Unprocessed VirB2 precursor is indicated as VirB2p and processed mature VirB2 as VirB2m. Shows representative tumor assay results on tomato stems. Similar results were obtained from at least three independent experiments (3–5 plants for each mutant in each independent experiment). Each region/domain of VirB2 is indicated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101142#pone-0101142-g001" target="_blank">Figure 1</a>.