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      Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses

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          Abstract

          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.

          Author Summary

          Plants possess multi-layered immune recognition systems. Early in the infection process, plants use receptor proteins to recognize pathogen molecules. Some of these receptors are present in only in a subset of plant species. Transfer of these taxonomically restricted immune receptors between plant species by genetic engineering is a promising approach for boosting the plant immune system. Here we show the successful transfer of an immune receptor from a species in the mustard family, called EFR, to rice. Rice plants expressing EFR are able to sense the bacterial ligand of EFR and elicit an immune response. We show that the EFR receptor is able to use components of the rice immune signaling pathway for its function. Under laboratory conditions, this leads to an enhanced resistance response to two weakly virulent isolates of an economically important bacterial disease of rice.

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          Most cited references56

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          Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

          A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.
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            Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

            Higher eukaryotes sense microbes through the perception of pathogen-associated molecular patterns (PAMPs). Arabidopsis plants detect a variety of PAMPs including conserved domains of bacterial flagellin and of bacterial EF-Tu. Here, we show that flagellin and EF-Tu activate a common set of signaling events and defense responses but without clear synergistic effects. Treatment with either PAMP results in increased binding sites for both PAMPs. We used this finding in a targeted reverse-genetic approach to identify a receptor kinase essential for EF-Tu perception, which we called EFR. Nicotiana benthamiana, a plant unable to perceive EF-Tu, acquires EF-Tu binding sites and responsiveness upon transient expression of EFR. Arabidopsis efr mutants show enhanced susceptibility to the bacterium Agrobacterium tumefaciens, as revealed by a higher efficiency of T-DNA transformation. These results demonstrate that EFR is the EF-Tu receptor and that plant defense responses induced by PAMPs such as EF-Tu reduce transformation by Agrobacterium.
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              A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence.

              Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                30 March 2015
                March 2015
                : 11
                : 3
                : e1004809
                Affiliations
                [1 ]Department of Plant Pathology and the Genome Center, University of California, Davis, Davis, California, United States of America
                [2 ]Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
                [3 ]The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom
                [4 ]Department of Energy Joint Genome Institute, Walnut Creek, California, United States of America
                University of California Riverside, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Performed the experiments: BS OB NT NH VN DR PEC AD CJP VRS RK MC CD JLH. Wrote the paper: BS OB NT PCR. Designed Experiments and Analyzed Data: BS OB NT NH CJP VRS JLH PCR. Provided Material: VN CZ.

                [¤a]

                Current address: Department of Plant Pathology and Weed Research, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel

                [¤b]

                Current address: bio-protocol, Palo Alto, California, United States of America

                Article
                PPATHOGENS-D-14-01786
                10.1371/journal.ppat.1004809
                4379099
                25821973
                6d4b1cee-5700-4ae4-a8e3-7522aeeaa6fc

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

                History
                : 25 July 2014
                : 12 March 2015
                Page count
                Figures: 6, Tables: 2, Pages: 34
                Funding
                This work was supported by an EMBO long-term post-doctoral fellowship (ALTF 1290-2011) and a Human Frontier Science Program long-term post-doctoral fellowship (LT000674/2012) to BS. This work was funded by the Gatsby Charitable Foundation (CZ). The work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This work was supported by the NIH GM59962 to PCR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files except for the raw sequence file for the RNAseq experiment of Kitaake and Ubi::EFR::XA21::GFP plants which is available from JGI and can be found here http://genome.jgi.doe.gov/pages/dynamicOrganismDownload.jsf?organism=OrysatspilotREDO"

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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