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. 2018 Nov 16;115(49):E11551–E11560. doi: 10.1073/pnas.1813058115

Fig. 1.

Fig. 1.

OsSPK1 interacts with the R protein Pit in rice. (A) Schematic representation of Pit and OsSPK1 deletion mutants used in this study. (B) Interaction between Pit and OsSPK1 in a yeast two-hybrid assay. Yeast growth on selective plates without histidine [His (−)] indicates a positive interaction. (C) In vivo interaction between Pit and OsSPK1. Pit-GFP and OsSPK1-Myc were transiently coexpressed in N. benthamiana leaves. Co-IP was carried out with anti-GFP antibody, and the proteins were detected by Western blot with anti-GFP and anti-Myc antibodies. (D) Direct interaction between Pit and OsSPK1 in an in vitro binding assay. Purified MBP or MBP-tagged OsSPK1 (amino acids 1334–1835) immobilized to Sepharose was incubated with His-SUMO–tagged Pit CC. After washing, the bound proteins were eluted by addition of the sample buffer for immunoblotting. (E and F) Localization of OsSPK1 in N. benthamiana leaves. (E) The leaves were injected with Agrobacterium carrying YFP-OsSPK1 (green), and stained with FM4-64 (red; plasma membrane marker). Open arrowheads represent PM and closed arrowheads show the position of YFP-OsSPK1, which does not merge with PM. (Scale bars, 25 μm.) (F) YFP-OsSPK1 (green) and mRFP-HDEL (red; ER marker) were cotransformed into tobacco leaves. Insets are enlargements (3×) relative to white dashed boxes. (Scale bars, 25 μm.) (G) Subcellular distribution of native OsSPK1 in rice cells. Western blots of rice suspension cell fractions separated using a commercial kit. Each fraction was blotted with antibodies against proteins that localize to specific subcellular compartments. UGPase, BiP, and H+ATPase were used as markers for cytoplasm, ER, and plasma membrane, respectively. Cyt, cytosol fraction; EM, endomembrane fraction; PM, plasma membrane fraction; TM, total membrane fraction; Total, total protein extract. (H) BiFC assay of Pit and OsSPK1 in N. benthamiana leaves. Expression of these genes was driven by the CaMV 35S promoter. Empty vector served as a negative control. FM4-64 was used as a plasma membrane marker. (Scale bars, 25 μm.)