Enhancement of MAMP signaling by chimeric receptors improves disease resistance in plants

Plants activate defense responses through the recognition of microbe-associated molecular patterns (MAMPs). Recently, several pattern-recognition receptors (PRRs) have been identified in plants, paving the way for manipulating MAMP signaling. CEBiP is a receptor for the chitin elicitor (CE) identified in the rice plasma membrane and XA21 is a member of the receptor-like protein kinase (RLK) family that confers disease resistance to rice bacterial leaf blight expressing the sulfated protein Ax21. To improve resistance to rice blast, the most serious fungal disease of rice, we aimed to create a defense system that combines high affinity of CEBiP for CE and the ability of XA21 to confer disease resistance. Cultured rice cells expressing the chimeric receptor CRXA, which consists of CEBiP and the intracellular region of XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after exposure to CE. Rice plants expressing the chimeric receptor exhibited more resistance to rice blast. Engineering PRRs may be a new strategy in molecular breeding for achieving disease resistance.Key words: chimeric receptor, chitin signal, disease resistance, HR cell death, MAMP-induced resistance, rice blast fungus     

CEBiP is the major chitin oligomer-binding protein in rice and plays a main role in the perception of chitin oligomers

CEBiP, a plasma membrane-localized glycoprotein of rice, directly binds with chitin elicitors (CE), and has been identified as a receptor for CE by using CEBiP-RNAi rice cells. To further clarify the function of CEBiP, we produced CEBiP-disrupted rice plants by applying an efficient Agrobacterium-mediated gene-targeting system based on homologous recombination, which has recently been developed for rice. Homologous recombination occurred at the CEBiP locus in ~0.5 % of the positive/negative selected calli. In the self-pollinated next generation, it was confirmed that the first exon of CEBiP was replaced with the hygromycin selection cassette as designed, and that the expression of CEBiP was completely deficient in homozygous cebip lines. Affinity-labeling analysis using biotinylated N-acetylchitooctaose demonstrated that CEBiP is the major CE-binding protein in rice cultured cells and leaves, which was consistent with the result that the response to CE in cebip cells was greatly diminished. Nevertheless, we observed a significant decrease in disease resistance against Magnaporthe oryzae, the causal agent of rice blast disease, only when the cebip leaf sheaths were inoculated with a weakly virulent strain, suggesting that CE perception during the infection process of M. oryzae is limited. The response to peptidoglycan and lipopolysaccharides in cebip cells was not affected, strongly suggesting that CEBiP is a CE-specific receptor.     

Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.     

Dissection of the factors affecting development-controlled and race-specific disease resistance conferred by leucine-rich repeat receptor kinase-type <Emphasis Type="Italic">R</Emphasis> genes in rice

Development-controlled resistance and resistance specificity frequently restrict the application of a disease resistance (R) gene in crop breeding programs. Xa3/Xa26 and Xa21, encoding leucine-rich repeat (LRR)-kinase type plasma membrane proteins, mediate race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, one of the most devastating rice diseases. Plants carrying Xa3/Xa26 and plants carrying Xa21 have different resistance spectra and the functions of the two R genes are regulated by developmental stage. Four chimeric genes encoding proteins consisting of different parts of XA3/XA26 and XA21 were constructed by domain swapping and transformed into a susceptible rice variety. The resistance spectra and development-regulated resistance of the transgenic plants carrying Xa3/Xa26, Xa21, or chimeric gene to different Xoo strains were analyzed in the same genetic background. The results suggest that the gradually increased expression of Xa3/Xa26 and Xa21 plays an important role in the progressively enhanced Xoo resistance during rice development. In addition, the LRR domains of XA3/XA26 and XA21 are important determinants of race-specific recognition during rice–Xoo interaction, but juxtamembrane regions of the two R proteins also appear to contribute to resistance specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Suppression of bacterial infection in rice by treatment with a sulfated peptide

The rice XA21 receptor kinase confers robust resistance to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). A tyrosine‐sulfated peptide from Xoo, called RaxX, triggers XA21‐mediated immune responses, including the production of ethylene and reactive oxygen species and the induction of defence gene expression. It has not been tested previously whether these responses confer effective resistance to Xoo. Here, we describe a newly established post‐inoculation treatment assay that facilitates investigations into the effect of the sulfated RaxX peptide in planta. In this assay, rice plants were inoculated with a virulent strain of Xoo and then treated with the RaxX peptide 2 days after inoculation. We found that post‐inoculation treatment of XA21 plants with the sulfated RaxX peptide suppresses the development of Xoo infection in XA21 rice plants. The treated plants display restricted lesion development and reduced bacterial growth. Our findings demonstrate that exogenous application of sulfated RaxX activates XA21‐mediated immunity in planta, and provides a potential strategy for the control of bacterial disease in the field.     

Four receptor‐like cytoplasmic kinases regulate development and immunity in rice

Receptor‐like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs – OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21‐mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.     

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 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.     

Rice BiP3 regulates immunity mediated by the PRRs XA3 and XA21 but not immunity mediated by the NB-LRR protein,Pi5

Plant innate immunity is mediated by pattern recognition receptors (PRRs) and intracellular NB-LRR (nucleotide-binding domain and leucine-rich repeat) proteins. Overexpression of the endoplasmic reticulum (ER) chaperone, luminal-binding protein 3 (BiP3) compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) mediated by the rice PRR XA21 [12]. Here we show that BiP3 overexpression also compromises resistance mediated by rice XA3, a PRR that provides broad-spectrum resistance to Xoo. In contrast, BiP3 overexpression has no effect on resistance mediated by rice Pi5, an NB-LRR protein that confers resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae). Our results suggest that rice BiP3 regulates membrane-resident PRR-mediated immunity.     

<Emphasis Type="Italic">HvCEBiP</Emphasis>, a gene homologous to rice chitin receptor <Emphasis Type="Italic">CEBiP</Emphasis>, contributes to basal resistance of barley to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background  

Rice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses.     

The XA21 binding protein XB25 is required for maintaining XA21‐mediated disease resistance

Plant genomes encode a large number of proteins that potentially function as immune receptors in the defense against pathogen invasion. As a well‐characterized receptor kinase consisting of 23 tandem leucine‐rich repeats, a transmembrane domain and a serine/threonine kinase, the rice (Oryza sativa) protein XA21 confers resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae (Xoo) races that cause bacterial blight disease. We report here that XA21 binding protein 25 (XB25) belongs to the plant‐specific ankyrin‐repeat (PANK) family. XB25 physically interacts, in vitro, with the transmembrane domain of XA21 through its N–terminal binding to transmembrane and positively charged domain (BTMP) repeats. In addition, XB25 associates with XA21 in planta. The downregulation of Xb25 results in reduced levels of XA21 and compromised XA21‐mediated disease resistance at the adult stage. Moreover, the accumulation of XB25 is induced by Xoo infection. Taken together, these results indicate that XB25 is required for maintaining XA21‐mediated disease resistance.     

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.     

Quantitative Measurements of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> Distribution in Rice Using Fluorescent-Labeling

The rice host sensor, XA21, confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight disease. Using in planta fluorescence imaging of Xoo strain PXO99Az expressing a green fluorescent protein (Xoo-gfp) we show that XA21 restricts Xoo spread at the point of infection. This noninvasive and quantitative method to measure spatial distribution of Xoo populations in planta facilitates detailed assessment of plant disease resistance.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

The Receptor-Like Cytoplasmic Kinase OsRLCK102 Regulates XA21-Mediated Immunity and Plant Development in Rice

Receptor-like cytoplasmic kinases (RLCKs) belong to a large subgroup of kinases that play pivotal roles in plant development and in protecting plants from various stresses. Here, we report the isolation and characterization of rice OsRLCK102, from the OsRLCK VII subgroup. Silencing of OsRLCK102 compromised receptor kinase XA21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) but did not affect plant basal resistance to Xoo or Magnaporthe oryzae (M. oryzae). Plants with silenced OsRLCK102 exhibit architecture alterations, including reduced plant height, enlarged angle of the lamina joint, decreased rates of seed setting and enhanced sensitivity to hormone brassinolide (BR). Collectively, our study reveals that OsRLCK102 positively regulates XA21-mediated immunity and negatively regulates rice development through BR signaling in rice.     

Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.     

Induction of systemic resistance in rice to bacterial blight by 1,2,3-benzothiadiazole 7-carbothioic acid-S-methyl ester (BTH) treatments

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. There was very little or marginal effect of BTH on the induction of resistance to BB in IR24 rice plants. When the same concentrations of BTH were applied to IRBB21 (Xa-21) rice plants, they showed pronounced triggering of systemic resistance to BB pathogen even at 0.1 mM concentration of BTH applied either as seed treatment or as foliar spry. Disease severity index was reduced to 5 (against a score of 9 in untreated) and there was 85–86% reduction in BB incidence in plants that received 0.1 mM BTH. These results provide evidence that BTH-induced systemic resistance complements the R-gene resistance in IRBB21 plants but not in IR24 rice plants.