Correction: A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity.

[This corrects the article DOI: 10.1371/journal.pgen.1006049.].

A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity.

Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.

Overexpression of a rice BAHD acyltransferase gene in switchgrass (Panicum virgatum L.) enhances saccharification.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a promising bioenergy feedstock because it can be grown on marginal land and produces abundant biomass. Recalcitrance of the lignocellulosic components of the switchgrass cell wall to enzymatic degradation into simple sugars impedes efficient biofuel production. We previously demonstrated that overexpression of OsAT10, a BAHD acyltransferase gene, enhances saccharification efficiency in rice. RESULTS: Here we show that overexpression of the rice OsAT10 gene in switchgrass decreased the levels of cell wall-bound ferulic acid (FA) in green leaf tissues and to a lesser extent in senesced tissues, and significantly increased levels of cell wall-bound p-coumaric acid (p-CA) in green leaves but decreased its level in senesced tissues of the T0 plants under greenhouse conditions. The engineered switchgrass lines exhibit an approximate 40% increase in saccharification efficiency in green tissues and a 30% increase in senesced tissues. CONCLUSION: Our study demonstrates that overexpression of OsAT10, a rice BAHD acyltransferase gene, enhances saccharification of lignocellulosic biomass in switchgrass.

Correction: Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses.

[This corrects the article DOI: 10.1371/journal.ppat.1004809.].

Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses.

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.

Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification.

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Towards establishment of a rice stress response interactome.

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%-60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein-protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance.

An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity.

Cell-surface pattern recognition receptors (PRRs) are key components of the innate immune response in animals and plants. These receptors typically carry or associate with non-RD kinases to control early events of innate immunity signaling. Despite their importance, the mode of regulation of PRRs is largely unknown. Here we show that the rice PRR, XA21, interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice lines silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice lines overexpressing XB24 are compromised for immunity. XB24 ATPase enzyme activity is required for XB24 function. XA21 is degraded in the presence of the pathogen-associated molecular pattern Ax21 when XB24 is overexpressed. These results demonstrate a function for this large class of broadly conserved ATPases in PRR-mediated immunity.

A conserved threonine residue in the juxtamembrane domain of the XA21 pattern recognition receptor is critical for kinase autophosphorylation and XA21-mediated immunity.

Despite the key role that pattern recognition receptors (PRRs) play in regulating immunity in plants and animals, the mechanism of activation of the associated non-arginine-aspartate (non-RD) kinases is unknown. The rice PRR XA21 recognizes the pathogen-associated molecular pattern, Ax21 (activator of XA21-mediated immunity). Here we show that the XA21 juxtamembrane (JM) domain is required for kinase autophosphorylation. Threonine 705 in the XA21 JM domain is essential for XA21 autophosphorylation in vitro and XA21-mediated innate immunity in vivo. The replacement of Thr(705) by an alanine or glutamic acid abolishes XA21 autophosphorylation and eliminates interactions between XA21 and four XA21-binding proteins in yeast and rice. Although threonine residues analogous to Thr(705) of XA21 are present in the JM domains of most RD and non-RD plant receptor-like kinases, this residue is not required for autophosphorylation of the Arabidopsis RD RLK BRI1 (brassinosteroid insensitive 1). The threonine 705 of XA21 is conserved only in the JM domains of plant RLKs but not in those of fly, human, or mouse suggesting distinct regulatory mechanisms. These results contribute to growing knowledge regarding the mechanism by which non-RD RLKs function in plant.

Enhanced disease resistance and hypersensitivity to BTH by introduction of an NH1/OsNPR1 paralog.

Non-expresser of pathogenesis-related genes 1 (NPR1) is the master regulator of salicylic acid-mediated systemic acquired resistance. Over-expression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While over-expression of NH3 using the maize ubiquitin-1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT-NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo-induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10-fold in nNT-NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to benzothiadiazole (BTH) and 2,6-dichloroisonicotinic acid (INA) treatment as measured by increased cell death. Expression analysis of pathogenesis-related (PR) genes showed that nNT-NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defence. This approach avoids using undesirable constitutive, high-level expression and may prove to be more practical for engineering resistance.

The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors.

We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21-mediated innate immunity.

Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Kinases that are associated with the receptors or are part of the receptors themselves modulate signaling through phosphorylation events. The rice (Oryza sativa L.) XA21 receptor kinase is a key recognition and signaling determinant in the innate immune response. A yeast two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as bait, identified a protein phosphatase 2C (PP2C), called XA21 binding protein 15 (XB15). The interaction of XA21 and XB15 was confirmed in vitro and in vivo by glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, respectively. XB15 fusion proteins purified from Escherichia coli and from transgenic rice carry PP2C activity. Autophosphorylated XA21 can be dephosphorylated by XB15 in a temporal- and dosage-dependent manner. A serine residue in the XA21 JM domain is required for XB15 binding. Xb15 mutants display a severe cell death phenotype, induction of pathogenesis-related genes, and enhanced XA21-mediated resistance. Overexpression of Xb15 in an XA21 rice line compromises resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. These results demonstrate that Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response.

Silencing of the Rice Gene LRR1 Compromises Rice Xa21 Transcript Accumulation and XA21-Mediated Immunity.

BACKGROUND: The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. We previously demonstrated that an auxilin-like protein, XA21 BINDING PROTEIN 21 (XB21), positively regulates resistance to Xoo. RESULTS: To further investigate the function of XB21, we performed a yeast two-hybrid screen. We identified 22 unique XB21 interacting proteins, including LEUCINE-RICH REPEAT PROTEIN 1 (LRR1), which we selected for further analysis. Silencing of LRR1 in the XA21 genetic background (XA21-LRR1Ri) compromises resistance to Xoo compared with control XA21 plants. XA21-LRR1Ri plants have reduced Xa21 transcript levels and reduced expression of genes that serve as markers of XA21-mediated activation. Overexpression of LRR1 is insufficient to alter resistance to Xoo in rice lines lacking XA21. CONCLUSIONS: Taken together, our results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity.

Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light.

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Previous reports indicate that rice has a disease-resistance pathway similar to the Arabidopsis SAR pathway. Here we report the isolation and characterization of a rice NPR1 homologue (NH1). Transgenic rice plants overexpressing NH1 (NH1ox) acquire high levels of resistance to Xanthomonas oryzae pv. oryzae. The resistance phenotype is heritable and correlates with the presence of the transgene and reduced bacterial growth. Northern analysis shows that NH1ox rice spontaneously activates defense genes, contrasting with NPR1-overexpressing Arabidopsis, where defense genes are not activated until induction. Wild-type NH1, but not a point mutant corresponding to npr1-1, interacts strongly with the rice transcription factor rTGA2.2 in yeast two-hybrid. Greenhouse-grown NH1ox plants develop lesion-mimic spots on leaves at preflowering stage although no other developmental effects are observed. However, when grown in growth chambers (GCs) under low light, NH1ox plants are dwarfed, indicating elevated sensitivity to light. The GC-grown NH1ox plants show much higher salicylic acid (SA) levels than the wild type, whereas greenhouse-grown NH1ox plants contain lower SA. These results indicate that NH1 may be involved in the regulation of SA in response to environmental changes.

An XA21-associated kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors.

The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR.

Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice.

Glycolate oxidase (GLO) is a key enzyme in photorespiration, catalyzing the oxidation of glycolate to glyoxylate. Arabidopsis GLO is required for nonhost defense responses to Pseudomonas syringae and for tobacco Pto/AvrPto-mediated defense responses. We previously described identification of rice GLO1 that interacts with a glutaredoxin protein, which in turn interacts with TGA transcription factors. TGA transcription factors are well known to participate in NPR1/NH1-mediated defense signaling, which is crucial to systemic acquired resistance in plants. Here we demonstrate that reduction of rice GLO1 expression leads to enhanced resistance to Xanthomonas oryzae pv oryzae (Xoo). Constitutive silencing of GLO1 leads to programmed cell death, resulting in a lesion-mimic phenotype and lethality or reduced plant growth and development, consistent with previous reports. Inducible silencing of GLO1, employing a dexamethasone-GVG (Gal4 DNA binding domain-VP16 activation domain-glucocorticoid receptor fusion) inducible system, alleviates these detrimental effects. Silencing of GLO1 results in enhanced resistance to Xoo, increased expression of defense regulators NH1, NH3, and WRKY45, and activation of PR1 expression.

The endoplasmic reticulum-quality control component SDF2 is essential for XA21-mediated immunity in rice.

Plant genomes contain large number of plasma membrane (PM)-localized immune receptors, also called pattern recognition receptors (PRRs). PRRs are synthesized in the endoplasmic reticulum (ER) and then translocated to the PM, where they recognize conserved pathogen-associated molecular patterns (PAMPs) and activate innate immune response. The rice XA21 immune receptor confers resistance to the Gram-negative bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). To identify components that mediate XA21-mediated signaling, we performed co-purification experiments using C-terminal GFP tagged XA21 protein. Several endoplasmic reticulum-quality control (ER-QC) proteins including stromal-derived factor 2 (SDF2) co-purified with XA21. Silencing of the SDF2 genes in the XA21 rice genetic background compromises resistance to Xoo but does not affect plant growth and development.

A rice transient assay system identifies a novel domain in NRR required for interaction with NH1/OsNPR1 and inhibition of NH1-mediated transcriptional activation.

BACKGROUND: Arabidopsis NPR1 is a master regulator of systemic acquired resistance. NPR1 binds to TGA transcription factors and functions as a transcriptional co-activator. In rice, NH1/OsNPR1 functions to enhance innate immunity. NRR disrupts NH1 function, when over-expressed. RESULTS: We have established a rice transient protoplast assay to demonstrate that NH1 is a transcriptional co-activator and that NRR represses NH1-mediated activation. We identified three NRR homologues (RH1, RH2, and RH3). RH1 and RH3, but not RH2, also effectively repress NH1-mediated transcriptional activation. NRR, RH1, RH2, and RH3 share sequence similarity in a region beyond the previously identified NPR1-interacting domain. This region is required for strong interaction with NH1. A double point mutation, W66A/F70A, in this novel NH1-interacting domain severely reduces interaction with NH1. Mutation W66A/F70A also greatly reduces the ability of NRR to repress NH1-mediated activation. RH2 carries a deviation (amino acids AV) in this region as compared to consensus sequences (amino acids ED) among NRR, RH1, and RH3. A substitution (AV to ED) in RH2 results in strong binding of mutant RH2ED to NH1 and effective repression of NH1-mediated activation. CONCLUSIONS: The protoplast-based transient system can be used to dissect protein domains associated with their functions. Our results demonstrate that the ability of NRR and its homologues to repress NH1-mediated transcriptional activation is tightly correlated with their ability to bind to NH1. Furthermore, a sequence is identified as a novel NH1-interacting domain. Importantly, this novel sequence is widely present in plant species, from cereals to castor bean plants, to poplar trees, to Arabidopsis, indicating its significance in plants.

Overexpression of the endoplasmic reticulum chaperone BiP3 regulates XA21-mediated innate immunity in rice.

Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the innate immune response. Although PRR-mediated signaling events are critical to the survival of plants and animals, secretion and localization of PRRs have not yet been clearly elucidated. Here we report the in vivo interaction of the endoplasmic reticulum (ER) chaperone BiP3 with the rice XA21 PRR, which confers resistance to the Gram negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). We show that XA21 is glycosylated and is primarily localized to the ER and also to the plasma membrane (PM). In BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. BiP3 overexpression does not affect the general rice defense response, cell death or brassinolide-induced responses. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo.

Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv. oryzae.

Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) activates the innate immune response. The rice PRR, XA21, confers robust resistance at adult stages to most strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Seedlings are still easily infected by Xoo, causing severe yield losses. Here we report that Xa21 is induced by Xoo infection and that ectopic expression of Xa21 confers resistance at three leaf stage (three-week-old), overcoming the developmental limitation of XA21-mediated resistance. Ectopic expression of Xa21 also up-regulates a larger set of defense-related genes as compared to Xa21 driven by the native promoter. These results indicate that altered regulation of Xa21 expression is useful for developing enhanced resistance to Xoo at multiple developmental stages.