Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.

<p>(<b>A</b>) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in <i>N. benthamiana</i> leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (<b>B</b>) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from <i>N. benthamiana</i> leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (<b>C</b>) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same <i>N. benthamiana</i> leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (<b>D</b>) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in <i>N. benthamiana</i>, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [<i>p</i><0.05, Tukey's honest significant difference (HSD) test].