The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

OsCEBiP, a chitin-binding protein, and OsCERK1, a receptor-like kinase, are plasma membrane (PM) proteins that form a receptor complex essential for fungal chitin-driven immune responses in rice. The signaling events immediately following chitin perception are unclear. Investigating the spatiotemporal regulation of the rice small GTPase OsRac1, we find that chitin induces rapid activation of OsRac1 at the PM. Searching for OsRac1 interactors, we identified OsRacGEF1 as a guanine nucleotide exchange factor for OsRac1. OsRacGEF1 interacts with OsCERK1 and is activated when its C-terminal S549 is phosphorylated by the cytoplasmic domain of OsCERK1 in response to chitin. Activated OsRacGEF1 is required for chitin-driven immune responses and resistance to rice blast fungus infection. Further, a protein complex including OsCERK1 and OsRacGEF1 is transported from the endoplasmic reticulum to the PM. Collectively, our results suggest that OsCEBiP, OsCERK1, OsRacGEF1, and OsRac1 function as key components of a "defensome" critically engaged early during chitin-induced immunity.

SUMMARYRho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.

Legumes and nitrogen-fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long-distance negative-feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis-acting region on the NIN promoter. Mutants with deletions of this cis-acting region have increased susceptibility to rhizobial infection and reduced GA-induced CLE-RS1 and CLE-RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA-insensitive phenotypes of an AON-defective mutant of HYPER-NODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA-responsive cis-acting region, and subsequently the GA-induced NIN activates the AON system to regulate nodule formation.

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.

Molecular links between receptor-kinases and Rac/ROP family small GTPases mediated by activator guanine nucleotide exchange factors (GEFs) govern diverse biological processes. However, it is unclear how the Rac/ROP GTPases orchestrate such a wide variety of activities. Here, we show that rice OsRacGEF1 forms homodimers, and heterodimers with OsRacGEF2, at the plasma membrane (PM) and the endoplasmic reticulum (ER). OsRacGEF2 does not bind directly to the receptor-like kinase (RLK) OsCERK1, but forms a complex with OsCERK1 through OsRacGEF1 at the ER. This complex is transported from ER to the PM and there associates with OsRac1, resulting in the formation of a stable immune complex. Such RLK-GEF heterodimer complexes may explain the diversity of Rac/ROP family GTPase signalings.

Rice is one of the most important food crops, feeding about half population in the world. Rice pathogens cause enormous damage to rice production worldwide. In plant immunity research, considerable progress has recently been made in our understanding of the molecular mechanisms underlying microbe-associated molecular pattern (MAMP)-triggered immunity. Using genome sequencing and molecular techniques, a number of new MAMPs and their receptors have been identified in the past two decades. Notably, the mechanisms for chitin perception via the lysine motif (LysM) domain-containing receptor OsCERK1, as well as the mechanisms for bacterial MAMP (e.g. flg22, elf18) perception via the leucine-rich repeat (LRR) domain-containing receptors FLS2 and EFR, have been clarified in rice and Arabidopsis, respectively. In chitin signaling in rice, two direct substrates of OsCERK1, Rac/ROP GTPase guanine nucleotide exchange factor OsRacGEF1 and receptor-like cytoplasmic kinase OsRLCK185, have been identified as components of the OsCERK1 complex and are rapidly phosphorylated by OsCERK1 in response to chitin. Interestingly, OsCERK1 also participates in symbiosis with arbuscular mycorrhizal fungi (AMF) in rice and plays a role in the recognition of short-chitin molecules (CO4/5), which are symbiotic signatures included in AMF germinated spore exudates and induced by synthetic strigolactone. Thus, OsCERK1 contributes to both immunity and symbiotic responses. In this review, we describe recent studies on pathways involved in rice immunity and symbiotic signaling triggered by interactions with microorganisms. In addition, we describe recent advances in genetic engineering by using plant immune receptors and symbiotic microorganisms to enhance disease resistance of rice.

Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we revealed that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane. Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the plasma membrane and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.