Three highly conserved hydrophobic residues in the predicted α2-helix of rice NLR protein Pit contribute to its localization and immune induction.

Nucleotide-binding leucine-rich repeat (NLR) proteins work as crucial intracellular immune receptors. N-terminal domains of NLRs fall into two groups, coiled-coil (CC) and Toll-interleukin 1 receptor domains, which play critical roles in signal transduction and disease resistance. However, the activation mechanisms of NLRs, and how their N-termini function in immune induction, remain largely unknown. Here, we revealed that the CC domain of a rice NLR Pit contributes to self-association. The Pit CC domain possesses three conserved hydrophobic residues that are known to be involved in oligomer formation in two NLRs, barley MLA10 and Arabidopsis RPM1. Interestingly, the function of these residues in Pit differs from that in MLA10 and RPM1. Although three hydrophobic residues are important for Pit-induced disease resistance against rice blast fungus, they do not participate in self-association or binding to downstream signalling molecules. By homology modelling of Pit using the Arabidopsis ZAR1 structure, we tried to clarify the role of three conserved hydrophobic residues and found that they are located in the predicted α2-helix of the Pit CC domain and involved in the plasma membrane localization. Our findings provide novel insights for understanding the mechanisms of NLR activation as well as the relationship between subcellular localization and immune induction.

The Small GTPase OsRac1 Forms Two Distinct Immune Receptor Complexes Containing the PRR OsCERK1 and the NLR Pit.

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 (PM). 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 PM 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.

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity.

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.

Roles of DEMETER in regulating DNA methylation in vegetative tissues and pathogen resistance.

DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5-methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss-of-function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell-specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening.

Small 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.

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function.

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.

In vivo monitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor.

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. RESULTS: We 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. CONCLUSIONS: Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein-protein interactions for other FRET sensors in various plant cells.

Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1.

Small GTPases regulate a large variety of key cellular processes. Plant small Rac/Rop GTPases have recently received broad attention as it is becoming clear that these enzymes regulate various plant cellular processes. OsRac1, a rice Rac/Rop protein, is a key regulator of reactive oxygen species (ROS) production and induces immune responses. Although four structures of plant small GTPases have been reported, all of these were of the inactive form. Here, OsRac1 was purified and co-crystallized with the GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP). The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 1.9 Šresolution.