Figure 4. NlSEF1 in rice and its molecular characterization.

(A) Ca²⁺-dependent mobility of purified NlSEF1 under SDS-PAGE conditions. The NlSEF1 protein was incubated for 30 min at 25 °C with each of the following solutions: lane 1, 0.5 mM CaCl₂; lane 2, 0.15 mM CaCl₂; lane 3, 0.05 mM CaCl₂; lane 4, 0.015 CaCl₂; lane 5, 0.5 mM EDTA. (B) Western blot analysis of protein NlSEF1 in rice infested by BPH nymphs. Lane 1, the extracts from the salivary glands of BPH; lanes 2–4, the extracts from rice plants of Mudgo that were infested by nymphs (lanes 2 and 3) or kept non-infested (lane 4). (C) Fluochemical intracellular Ca²⁺ determination in leaves infested by BPH. The green fluorescence refers to binding of Fluo-3 AM with Ca²⁺. A portion of rice leaf incubated with 5 μM Fluo-3 AM solution that was infested by newly emerged BPH female adults which had been injected with the dsRNA of NlSEF1 (dsSEF-BPH, dsSEF) or kept non-manipulated (C-BPH) for 1, 3 and 6 h. Metric bar = 25 μm. (D) Mean fluorescence intensity at feeding sites of newly emerged female adults of dsSEF-BPH (dsSEF) or C-BPH. Asterisks indicate significant difference between treatments (P < 0.05, t-test).