The requirement of Ca²⁺ signaling in ETI.

<p>(<b>A</b>) AvrRpm1-, AvrB- and AvrRpt2-induced cell death was detected by Evan's blue staining at different time points after transfection in WT, <i>rpm1</i> or <i>rps2</i> protoplasts. Ctrl is a control vector. Data are shown as mean ± SD. (<b>B</b>) AvrRpm1, AvrB and AvrRpt2 activated endogenous <i>WRKY46</i> expression in protoplasts. The transfected protoplasts were collected 6 hpt for real-time RT-PCR analysis. The expression of <i>WRKY46</i> was normalized to the expression of <i>UBQ10</i>. The data are shown as the mean ± SE from three independent biological replicates. (<b>C</b>) Induction of <i>WRKY46</i> by <i>Pst avrRpm1</i> and <i>avrB</i> infection in plants. Plant leaves were hand-inoculated with control or bacteria at 1×10⁷ cfu/ml. The samples were collected 6 hpi for real-time RT-PCR analysis. The expression of <i>WRKY46</i> was normalized to the expression of <i>UBQ10</i>. The data are shown as the mean ± SE from three independent biological replicates. (<b>D</b>) Induction of <i>WRKY46</i> in dexamethasone (DEX)-inducible <i>avrRpt2</i> transgenic plants and protoplasts. The <i>WRKY46</i> expression was detected 6 hr after DEX treatment with real-time RT-PCR analysis. The expression of <i>WRKY46</i> was normalized to the expression of <i>UBQ10</i>. The data are shown as the mean ± SE from three independent biological replicates. (<b>E</b>) AvrRpm1, AvrB and AvrRpt2 activated <i>WRKY46</i> promoter in protoplasts. The p<i>WRKY46-LUC</i> was co-transfected with <i>avrRpm1</i>, <i>avrB</i>, or <i>avrRpt2</i>, or a vector control in protoplasts and samples were collected at indicated time points. The <i>UBQ-GUS</i> was included as an internal transfection control. The relative luciferase activity was normalized with GUS activity. (<b>F</b>) AvrRpm1, AvrB and AvrRpt2-induced cell death was suppressed by calcium inhibitors in <i>Arabidopsis</i> protoplasts. The <i>avrRpm1</i>, <i>avrB</i>, or <i>avrRpt2</i> was co-transfected with <i>UBQ-GUS</i> and incubated with 1 mM LaCl₃, 1 mM GdCl₃ or 10 µM RR. The samples were collected 16 hpt, and the cell death ratio was presented as the percentage of GUS activity repression in effector-transfected cells compared to control-transfected cells. (<b>G</b>) Effector-induced <i>WRKY46</i> promoter activity was suppressed by calcium inhibitors in protoplasts. The samples were collected 6 hpt. The above experiments were repeated at least four times with similar results.