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      Phosphorylation-Dependent Differential Regulation of Plant Growth, Cell Death, and Innate Immunity by the Regulatory Receptor-Like Kinase BAK1

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          Abstract

          Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.

          Author Summary

          Plants need to adapt to their ever-changing environment for survival. Transmembrane receptor kinases are essential to translate extracellular stimuli into intracellular responses. A key question is how plants maintain signaling specificity in response to multiple stresses and endogenous hormones. Growth responses induced by steroid hormones and innate immunity triggered by recognition of conserved microbial molecules depend on the common regulatory receptor-like kinase BAK1, which is also involved in cell death control. It is still unclear if BAK1 provides signaling specificity or if it is a mere signaling enhancer. Here, we describe the novel protein variant BAK1-5 that specifically blocks innate immune responses without affecting steroid responses or cell death. This unambiguously demonstrates that the role of BAK1 in plant signaling can be mechanistically separated. Importantly, the impairment of immune signaling is not caused by a loss of interaction of BAK1-5 with immune receptors but is due to an altered kinase activity. Thus, BAK1-dependent signaling pathways are under a differential phosphorylation-dependent regulation. The examination of this novel mutant version of BAK1 will enable detailed studies into the mechanistic role of BAK1 in plant innate immunity, but also more generally will provide invaluable insights into transmembrane receptor signaling specificity in plants.

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          Most cited references85

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          Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana

          The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
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            Cell signaling by receptor tyrosine kinases.

            Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses.
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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 Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                April 2011
                April 2011
                28 April 2011
                02 May 2011
                : 7
                : 4
                : e1002046
                Affiliations
                [1]The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom
                University of Toronto, Canada
                Author notes

                Conceived and designed the experiments: BS MR CZ. Performed the experiments: BS MR YK VN JS. Analyzed the data: BS MR VN JS AJ CZ. Wrote the paper: BS CZ.

                Article
                PGENETICS-D-10-00483
                10.1371/journal.pgen.1002046
                3085482
                21593986
                d0580de9-e155-4feb-9d25-18f943640844
                Schwessinger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 3 December 2010
                : 21 February 2011
                Page count
                Pages: 17
                Categories
                Research Article
                Biology
                Developmental Biology
                Plant Growth and Development
                Genetics
                Genetic Screens
                Immunology
                Immunity
                Innate Immunity
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Protein Kinase Signaling Cascade
                Signaling in Selected Disciplines
                Plant Signaling
                Mechanisms of Signal Transduction
                Membrane Receptor Signaling
                Cell Death
                Plant Science
                Plant Genetics
                Plant Growth and Development

                Genetics
                Genetics

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