Analysis of cell death induction by XopL in <i>Nicotiana benthamiana</i>.

<p><i>Agrobacterium</i>-strains carrying binary constructs encoding XopL (WT), XopL_D502A (D502A), XopL_K578A (K578A), XopL_A579W (A579W), XopL_Q612A (Q612A), XopL_L619A (L619A), XopL[aa 1–449] (leucine-rich repeat, LRR), XopL[aa 450–660] (C-terminal domain, CTD), both XopL[aa 1–449] and XopL[aa 450–660] expressed in trans (LRR+CTD) or GFP under control of the <i>Cauliflower mosaic virus</i> (CaMV) 35S promoter, were inoculated into <i>N. benthamiana</i> leaves (8×10⁸ cfu/ml). (<b>A</b>) Phenotypes of the inoculated leaf area were documented 6 days post inoculation (dpi). (<b>B</b>) Cell death quantification using electrolyte leakage measurements. Measurements were carried out 2 dpi (light grey bars) and 4 dpi (dark grey bars), respectively. Bars represent the average of triplicates of 5 leaf discs each, error bars represent standard deviations. Asterisks indicate statistically significant differences compared to GFP control (<i>t</i>-test, <i>P</i><0.01). (<b>C</b>) Leaf tissue was harvested 2 dpi, and protein extracts were analyzsed by western blot using a <i>Strep</i>-tag (α-strep) and ubiquitin-specific antibody (α-Ub), respectively. Signals specific for full-length XopL, XopL[aa 1–449] (XopL_LRR)and XopL[aa 450–660] (XopL_CTD) are labeled. Polyubiquitination is indicated by (Ub)_n. Equal loading is shown by Ponceau staining of Rubisco. The experiments were performed three times with similar results.