Due to the importance of R proteins, elucidation of their signaling pathways is an important research goal. We revealed that OsSPK1, a GDP/GTP exchange factor for the small GTPase OsRac1, is a direct binding protein of the rice R protein Pit, which is a resistance protein to rice blast fungus. OsSPK1 is a key signaling molecule in Pit signaling. Our results provide a critical new insight into molecular mechanisms underlying R protein activation and new knowledge for crop improvement.
Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.
Figure 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.)