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.     

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.     

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.     

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.     

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.     

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.     

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.     

The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots

During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots.     

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.     

Variation and inheritance of the Xanthomonas raxX-raxSTAB gene cluster required for activation of XA21-mediated immunity

The rice XA21-mediated immune response is activated on recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The 60-residue RaxX precursor is post-translationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The 5-kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type I secretion system. To assess raxX-raxSTAB gene cluster evolution and to determine its phylogenetic distribution, we first identified rax gene homologues in other genomes. We detected the complete raxX-raxSTAB gene cluster only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae. The phylogenetic distribution of the raxX-raxSTAB gene cluster is consistent with the occurrence of multiple lateral (horizontal) gene transfer events during Xanthomonas speciation. RaxX natural variants contain a restricted set of missense substitutions, as expected if selection acts to maintain peptide hormone-like function. Indeed, eight RaxX variants tested all failed to activate the XA21-mediated immune response, yet retained peptide hormone activity. Together, these observations support the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX.     

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     

Small protein-mediated quorum sensing in a Gram-negative bacterium

The rice XA21 pattern recognition receptor binds a type I secreted sulfated peptide, called axY^S22, derived from the Ax21 (activator of XA21-mediated immunity) protein. The conservation of Ax21 in all sequenced Xanthomonas spp. and closely related genera suggests that Ax21 serves a key biological function. Here we show that the predicted N-terminal sequence of Ax21 is cleaved prior to secretion outside the cell and that mature Ax21 serves as a quorum sensing (QS) factor in Xanthomonas oryzae pv. oryzae. Ax21-mediated QS controls motility, biofilm formation and virulence. We provide genetic evidence that the Xoo RaxH histidine kinase serves as the bacterial receptor for Ax21. This work establishes a critical role for small protein-mediated QS in a Gram-negative bacterium.     

Salicylic acid-induced accumulation of biochemical components associated with resistance against Xanthomonas oryzae pv. oryzae in rice

Seed treatment and foliar sprays of salicylic acid (SA) provided protection in rice against bacterial leaf blight (BLB) caused by bacterial Xanthomonas oryzae pv. oryzae (Xoo). Treatment of rice with exogenous SA reduced disease severity by more than 38%. Superoxide anion production and hypersensitive response increased approximately 28% and 110% at 6 and 48?h after Xoo inoculation, respectively, for plants treated with SA. Moreover, the Xoo in treated rice plants grew more slowly, resulting in a population that was half of that observed in the control. Fourier transform infrared spectroscopy analysis revealed that the higher ratios of 1233/1517, 1467/1517, and 1735/1517?cm^?1 observed in treated rice suggested alteration of monomer composition of lignin and pectin in the rice cell wall. Exogenous SA-treated rice had more amide I β-sheet structure and lipids as shown by the peaks at 1629, 2851, and 1735?cm^?1. These biochemical changes of rice treated with SA and inoculated with Xoo were related to primed resistance of the rice plants to BLB disease.