SummarySmall signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics‐ and proteomics‐based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis‐related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions.

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.

Background:The plant small GTPase OsRac1 plays an important role in rice innate immunity. Results: The crystal structure and NADPH oxidase-binding mode of active-form OsRac1 were determined. Conclusion: The structure explains the mechanism by which OsRac1 regulates reactive oxygen species production and activates the immune response. Significance: A new insight into the activation of plant immunity by small GTPase is revealed.

Background Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles.ResultsWe have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images.ConclusionsOur protocol should be adaptable for monitoring activation for other plant small GTPases and protein–protein interactions for other FRET sensors in various plant cells.