SCHENGEN receptor module drives localized ROS production and lignification in plant roots
2020; Springer Nature; Volume: 39; Issue: 9 Linguagem: Inglês
10.15252/embj.2019103894
ISSN1460-2075
AutoresSatoshi Fujita, Damien De Bellis, Kai H. Edel, Philipp Köster, Tonni Grube Andersen, Emanuel Schmid‐Siegert, Valérie Dénervaud Tendon, Alexandre Pfister, Peter Marhavý, Robertas Ursache, Verónica G. Doblas, Marie Barberon, Jean Daraspe, Audrey Creff, Gwyneth Ingram, Jörg Kudla, Niko Geldner,
Tópico(s)Plant-Microbe Interactions and Immunity
ResumoArticle18 March 2020Open Access Transparent process SCHENGEN receptor module drives localized ROS production and lignification in plant roots Satoshi Fujita Corresponding Author Satoshi Fujita [email protected] orcid.org/0000-0002-3514-3349 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Damien De Bellis Damien De Bellis orcid.org/0000-0001-9480-7208 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Kai H Edel Kai H Edel Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Philipp Köster Philipp Köster Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Tonni Grube Andersen Tonni Grube Andersen Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Emanuel Schmid-Siegert Emanuel Schmid-Siegert Vital-IT Competence Center, Swiss Institute of Bioinformatics, Lausanne, Switzerland Search for more papers by this author Valérie Dénervaud Tendon Valérie Dénervaud Tendon Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Alexandre Pfister Alexandre Pfister Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Peter Marhavý Peter Marhavý orcid.org/0000-0002-0178-2230 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Robertas Ursache Robertas Ursache Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Verónica G Doblas Verónica G Doblas orcid.org/0000-0002-5476-3228 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Marie Barberon Marie Barberon Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Jean Daraspe Jean Daraspe Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Audrey Creff Audrey Creff Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France Search for more papers by this author Gwyneth Ingram Gwyneth Ingram Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France Search for more papers by this author Jörg Kudla Jörg Kudla Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Niko Geldner Corresponding Author Niko Geldner [email protected] orcid.org/0000-0002-2300-9644 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Satoshi Fujita Corresponding Author Satoshi Fujita [email protected] orcid.org/0000-0002-3514-3349 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Damien De Bellis Damien De Bellis orcid.org/0000-0001-9480-7208 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Kai H Edel Kai H Edel Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Philipp Köster Philipp Köster Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Tonni Grube Andersen Tonni Grube Andersen Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Emanuel Schmid-Siegert Emanuel Schmid-Siegert Vital-IT Competence Center, Swiss Institute of Bioinformatics, Lausanne, Switzerland Search for more papers by this author Valérie Dénervaud Tendon Valérie Dénervaud Tendon Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Alexandre Pfister Alexandre Pfister Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Peter Marhavý Peter Marhavý orcid.org/0000-0002-0178-2230 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Robertas Ursache Robertas Ursache Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Verónica G Doblas Verónica G Doblas orcid.org/0000-0002-5476-3228 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Marie Barberon Marie Barberon Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Jean Daraspe Jean Daraspe Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Audrey Creff Audrey Creff Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France Search for more papers by this author Gwyneth Ingram Gwyneth Ingram Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France Search for more papers by this author Jörg Kudla Jörg Kudla Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany Search for more papers by this author Niko Geldner Corresponding Author Niko Geldner [email protected] orcid.org/0000-0002-2300-9644 Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Author Information Satoshi Fujita *,1,6, Damien De Bellis1,2, Kai H Edel3, Philipp Köster3,7, Tonni Grube Andersen1,8, Emanuel Schmid-Siegert4, Valérie Dénervaud Tendon1, Alexandre Pfister1, Peter Marhavý1,9, Robertas Ursache1, Verónica G Doblas1,10, Marie Barberon1,11, Jean Daraspe2, Audrey Creff5, Gwyneth Ingram5, Jörg Kudla3 and Niko Geldner *,1 1Department of Plant Molecular Biology, Biophore, University of Lausanne, Lausanne, Switzerland 2Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland 3Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany 4Vital-IT Competence Center, Swiss Institute of Bioinformatics, Lausanne, Switzerland 5Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France 6Present address: National Institute of Genetics, Mishima, Shizuoka, Japan 7Present address: Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland 8Present address: Max Planck Institute for Plant Breeding Research, Cologne, Germany 9Present address: Umeå Plant Science Centre (UPSC), Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden 10Present address: Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles, France 11Present address: Department of Botany and Plant Biology, Quai Ernest-Ansermet, Geneva, Switzerland *Corresponding author. Tel: +81 55 981 6799; E-mail: [email protected] *Corresponding author. Tel: +41 21 692 4192; E-mail: [email protected] The EMBO Journal (2020)39:e103894https://doi.org/10.15252/embj.2019103894 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Production of reactive oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants, and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and immediate molecular action of ROS. A well-understood ROS action in plants is to provide the co-substrate for lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here, we establish a kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicate that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate. Synopsis In plants, establishment of the Casparian strip diffusion barrier in the root endodermis is achieved by spatially precise deposition of lignin. Here, asymmetrically localized SCHENGEN1 (SGN1) kinase is shown to regulate Casparian strip formation via local induction of ROS-dependent lignification. Polar localization of SGN1, a cytoplasmic kinase anchored to the plasma membrane, determines localized lignin accumulation in the endodermis. SGN1 transduces activation of the SGN3 receptor by CIF2 secreted peptide. SGN1 locally activates ROS production at the Casparian strip via direct phosphorylation of NADPH oxidases. CIF2-induced transcriptional changes regulate Casparian strip scaffold establishment and compensatory repair upon barrier defects. Introduction As in animals, NADPH oxidase-produced ROS in plants is important for a multitude of processes and the number of NADPH oxidase genes (10 in Arabidopsis, called RESPIRATORY BURST OXIDASE HOMOLOGs, RBOHs, A-J) suggests a high complexity of regulation of ROS production in plants. Among its many roles, ROS-dependent regulation of plant cell wall structure and function is considered to be among its most important (Kärkönen & Kuchitsu, 2015). The cell wall is the nano-structured, sugar-based, pressure-resisting extracellular matrix of plants, and NOXs are thought to be the predominant ROS source in this compartment (also termed apoplast; Kärkönen & Kuchitsu, 2015). A staggering number of kinases have been shown to regulate plant NOXs and the activation mechanism of NOX-dependent ROS production is well established, especially in response to microbial pattern recognition by immune receptors (Zipfel, 2014). However, the specific role and direct molecular targets of ROS during microbial pattern recognition have remained elusive (Qi et al, 2017). The same applies to the central role of ROS in tip growing cells, such as root hairs or pollen tubes, where ROS is thought to be part of an intricate oscillation of cell wall stiffening and loosening, aimed at allowing cell wall expansion without catastrophic collapse (Monshausen et al, 2007; Boisson-Dernier et al, 2013). In this case, ROS is proposed to be important for counteracting cell wall loosening pH decreases, but it is again unclear what direct targets of ROS would mediate cell wall stiffening. Cell wall lignification by apoplastic peroxidases can therefore be considered as the most well-established role of ROS, where the peroxidases themselves are the direct "ROS targets", using it as a co-substrate for the oxidation of mono-lignols (Liu, 2012; Barbosa et al, 2019). In the case of lignification, however, a molecularly defined signaling pathway that induces ROS production during lignification has not been defined. A few years ago, our group identified a specific NADPH oxidase, RBOHF, to be required for the localized formation of lignin in the root endodermis (Lee et al, 2013). Lignin is a poly-phenolic polymer that is generated by the radical coupling of mono-lignols, oxidized through the action of ROS-dependent peroxidases, as well as laccases (Liu, 2012). The hydrophobic lignin polymer impregnates the cellulosic cell wall of plants, rendering it unextendible and highly resistant to degradation. Lignin in the root endodermis is deposited in a central, longitudinal band around every endodermal cell. Named Casparian strips (CS), these ring-like lignin structures fuse into a supracellular network, establishing a tissue-wide, extracellular diffusion barrier (Fig 1A), analogous to epithelial tight junctions in animals (Geldner, 2013; Barberon & Geldner, 2014). Functionality of this barrier can be easily visualized by a block of penetration of a fluorescent cell wall dye, propidium iodide, into the vasculature (Alassimone et al, 2010; Naseer et al, 2012). CS localization occurs through the action of CASPARIAN STRIP DOMAIN PROTEINS (CASPs), 4-TM proteins, which form a highly scaffolded transmembrane protein platform, assembling RBOHF and cell wall peroxidases and other proteins at the Casparian strip domain (CSD; Roppolo et al, 2011; Hosmani et al, 2013; Lee et al, 2013). CSD formation and lignin deposition are coordinated such that the aligned rings of endodermal neighbors' fuse, leading to a supracellular network that seals the extracellular space between endodermal cells, generating a tissue-wide diffusion barrier. Figure 1. Apolar SGN1 leads to ectopic lignin accumulation in endodermal cells A. Schematic of Casparian strip development (magenta). Casparian strips start to appear as centrally aligned discontinuous dots in the endodermal cell layer, progressing into a network of fused rings functioning as a root apoplastic barrier. B. Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white, respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex-facing (outer) side. Scale Bar = 5 μm. C, D. Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcofluor White) are shown in magenta and white, respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 μm. E. Schematic illustrating how signal activation can be governed by SGN1 localization and peptide ligand diffusion from the stele. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface. Download figure Download PowerPoint More recently, we identified a pair of peptide ligands, a leucine-rich repeat receptor-like kinase (LRR-RLK) and a cytoplasmic kinase, whose phenotypes, genetic interaction, and specific subcellular localizations led us to propose that they combine into a barrier surveillance pathway. Previous reports had shown that the CIF1/2 (CASPARIAN STRIP INTEGRITY FACTORs 1/2) peptides are SCHENGEN3 (SGN3) (also called GASSHO1 (GSO1)) ligands and that the SGN1 and SGN3 kinases govern Casparian strip integrity (Pfister et al, 2014; Alassimone et al, 2016; Doblas et al, 2017; Nakayama et al, 2017). cif1 cif2 and sgn1 and sgn3 mutants have similar, discontinuous CS, caused by a discontinuous CSD, as well as a conspicuous absence, or strong attenuation, of compensatory lignification and suberization observed in other CS mutants (Hosmani et al, 2013; Pfister et al, 2014; Kamiya et al, 2015; Doblas et al, 2017; Kalmbach et al, 2017; Li et al, 2017). Their phenotypic similarities suggested that these factors act in one pathway. CIF1 and 2 peptides do not express in the endodermis, where the CS is formed, but in the stele. In contrast, both SGN1 and SGN3 present specific localization on the endodermal plasma membrane; SGN3 receptor-like kinase resides along both sides of the CS, while palmitoylated SGN1 polarly localizes on cortex-facing (outer) plasma membranes (Pfister et al, 2014; Alassimone et al, 2016). Remarkably, their localization overlaps only in a small region next to the cortex-facing side of the CS (Alassimone et al, 2016). This would require peptides from the stele to diffuse across the CS in order to access the signaling complex. This is only possible while the CS is still permeable (Doblas et al, 2017; Fig 1E). This pathway would therefore provide a mechanism that allows to probe the tissue-wide integrity of an extracellular diffusion barrier and to respond to barrier defects through compensatory overlignification (Doblas et al, 2017). Here, we demonstrate that the receptor, cytoplasmic kinase, and NADPH oxidase molecularly connect into one pathway, with great resemblance to plant immune signaling pathways, but whose direct action is to locally produce ROS for spatially restricted lignification. We establish the crucial importance of the restricted subcellular localization of its components and demonstrate that stimulation of this signaling pathway additionally leads to strong transcriptional activation of target genes that further drive and sustain endodermal lignification, as well as suberization and endodermal sub-domain formation and differentiation. We thus provide a molecular circuitry in which an endogenous peptide from the stele stimulates localized signaling kinases and NADPH oxidases in the endodermis, causing extracellular ROS production at micrometer-scale precision and a precisely localized lignification of the plant cell wall. Results We generated a new cif1-2 cif2-2 double mutant allele by CRISPR-Cas9 in a pure Col background, because the previous cif1-1 cif2-1 double mutant allele (Nakayama et al, 2017) was a mixture of Ws and Col alleles. The new CRISPR allele was complemented by CIF1 or CIF2 application, visualized by PI (propidium iodide) uptake assays or reconstitution of CASP1 membrane domain connectivity (Fig EV1D and E, and Appendix Fig S1A). Click here to expand this figure. Figure EV1. Apolar SGN1 leads to ectopic lignin accumulation in endodermal cells PI penetration assay. Scoring of number of cells after the onset of cell elongation until PI signal is excluded from the inner side of the endodermis (10 roots in total were tested in each condition during two independent assays). In the box plot, boxes are showing ranges from the first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters indicate significant statistical differences (P < 0.01, one-way ANOVA and Tukey test). Localization of CASP1-mCherry, driven by CASP1 promoter, in pCASP1::SGN1-Citrine or pCASP1::myrpalmSGN1-Citrine transgenic lines. For each crossed line, more than 10 roots were observed and showed similar localization patterns. Scale bar = 10 μm. Localization patterns of SGN1 (WT, kinase dead (KD)), myrpalm-SGN1 (WT and KD), and lignin deposition patterns in each indicated transgenic line. Arrowheads indicate excess lignification. For this experiment, two independent lines were tested. From each transgenic line, 2 spots from 12 roots were observed and representative pictures are shown. Scale bars are 10 μm in SGN1-Cit, 5 μm in lignin and cell wall pictures, 20 μm in overview of lignin deposition. One-base pair insertion sites of cif1-2 and cif2-2. Red letters indicate inserted bases in each locus. PI penetration phenotype of the cif1 cif2 double mutant with or without 100 nM peptide treatment. Seedlings were germinated on the medium with or without peptides. At least five roots were observed in each condition. Asterisks indicate the stele. Scale bar = 40 μm. Whole root views of suberin deposition patterns in polar- or apolar-SGN1 transgenic lines. esb1 (enhanced suberin 1) is shown as a representative oversuberized mutant. Scale bar = 500 μm. Quantification of the ratio of suberized zones and root lengths in each mutant or transgenic line. esb1 is shown as a representative oversuberized mutant. In the box plot, boxes are showing ranges from the first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters are indicating statistically significant differences (n = 16–36 roots, P < 0.01, ANOVA and Tukey test). Download figure Download PowerPoint Apolar SGN1 kinase leads to constitutive barrier defect signaling Central to the barrier surveillance model is the polar localization of SGN1, which is thought to limit the potential for signal activation to the cortex side of the endodermis, requiring passage of CIF peptides across the CS region (Fig 1A and E). Consistently, application of peptide ligand to the media, leading to stimulation from the outside, causes overlignification at the cortex-facing endodermal edges (Fig 1B). In an attempt to falsify the model we had proposed and to interrogate the importance of SGN1 polar localization, we generated a SGN1 variant that localized in an apolar fashion, by adding a myristoylation (myr) and palmitoylation (palm) motifs on the N-terminus (Vermeer et al, 2004). This myrpalm SGN1-mCitrine (Citrine) variant was expressed under the control of the endodermis-specific CASP1 promoter, which is strongly active during Casparian strip formation and complemented the sgn1 barrier phenotype (Fig EV1A). In planta, the wild-type SGN1-Citrine variant resides polarly on the cortex-facing side of endodermal cells, while myrpalm SGN1-Citrine was observed at both sides of the endodermal plasma membranes, even though preferentially accumulation at the cortex side could still be observed (Fig 1C and D). Both variants were excluded from the central position where the Casparian strip domain is formed (Fig EV1B), and the localization patterns of the two variants did not change when introgressed into sgn1, sgn3, or cif1 cif2 mutants (Fig 1C and D). An apolar SGN1 localization would allow SGN3 to encounter SGN1 also on the stele-facing side, not only on the cortex side (Fig 1E). This should lead to constitutive signal activation in the absence of barrier defects, because the CIF peptides would now be able to access a SGN3/SGN1 signaling module on the stele-facing side without crossing the barrier. Indeed, we found that the apolar SGN1 variant caused both, ectopic lignin deposition and precocious suberization in endodermal cells (Figs 1D and EV1F and G), as previously described for endodermal barrier mutants. Yet, no barrier defect was observed in the lines complemented with apolar SGN1 (Fig EV1A) and, consistently, we found CASP1-mCherry distribution to be normal in these lines, forming a continuous band in the central position of the endodermal cells, indistinguishable from wild type (Fig EV1B). This indicates that the presence of SGN1 at the plasma membrane to the inside of the CS leads to signaling in the absence of barrier defects. CASP1 promoter-driven wild-type SGN1 lines as a control complemented the mutant (Fig EV1A) and did not cause any changes in lignin accumulation pattern (Fig 1C). Interestingly, the apolar SGN1 lines accumulated lignin mainly on the stele-facing edges of the endodermal cell walls, as opposed to the cortical lignin deposition observed by ectopic ligand treatment (compare Fig 1B with 1D, arrows). The ectopic lignin deposition in apolar SGN1 lines was fully dependent on the presence of receptor and ligand; as in both sgn3 and cif1 cif2 mutants, no excess lignification at the stele-facing side could be observed in apolar SGN1 lines (Fig 1D). This strongly suggests that mislocalized SGN1 does not become constitutively active, but leads to continuous, ectopic transduction of CIF1/2 signals through SGN3. An apolar, but kinase-dead variant of SGN1 was also unable to induce ectopic lignification (Fig EV1C), suggesting that a phosphorylation relay downstream of SGN1 is necessary for lignification. SGN1 is a downstream component of the CIF/SGN3 pathway Previous data showed that sgn1 is less sensitive to high doses of externally applied CIF peptide, consistent with a role of SGN1 downstream of the SGN3 receptor (Doblas et al, 2017), at least with respect to CIF-induced excess lignification. In order to address whether SGN1 is indeed a generally required downstream component of the CIF/SGN3 pathway during CS formation, we evaluated whether the sgn1 mutant is resistant to complementation by CIF2 peptide treatment. In contrast to the full complementation of the discontinuous CS domain of cif1 cif2 double mutants, the sgn1 mutant did not show full restoration of domain integrity, neither on 10 nM nor on 100 nM CIF2 medium (Fig 2A, Appendix Fig S1A). While some degree of rescue occurred, only about 50% of discontinuities were rescued. This was corroborated by testing CS functionality using the propidium iodide (PI) assay. Only a weak complementation of barrier formation was observed even when grown on 100 nM CIF2 medium (Fig 2B). Our results indicate that SGN1 functions downstream of CIFs/SGN3, but suggest that additional factors can partially compensate for its absence, most probably homologs of the extended RLCKVII family to which SGN1 belongs. Figure 2. SGN1 acts as a transducer of CIF2 signaling and is phosphorylated by the SGN3 receptor Quantification of defects in CSD formation as number of holes per 100 μm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences (P < 0.05, one-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays). Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences (P < 0.05, one-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition). [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie-stained gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid autophosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown. Download figure Download PowerPoint We then tested for direct connectivity between SGN3 receptor kinase and SGN1 by carrying out an in vitro kinase assay. Some RLCKVII members are reported to be phosphorylated and activated by LRR receptor kinases (Lu et al, 2010; Kim et al, 2011). A glutathione S-transferase (GST)-fused SGN3 kinase domain was incubated with a kinase-dead form of a trigger factor (TF)-SGN1 fusion protein—which does not have autophosphorylation activity—in the presence of radioactive ATP. Kinase-dead TF-SGN1 was efficiently phosphorylated by SGN3 kinase domain, but not by a kinase-dead form of SGN3 in vitro (Fig 2C, Appendix Fig S1B). This further supports our model that SGN1 is a direct downstream component of the SGN3/CIF pathway. Two NADPH oxidases are absolutely required for CIF-induced lignification in the endodermis Our generation of an apolar SGN1 thus appears to have reconstituted a functional CIF/SGN3 pathway at the stele-facing (inner) endodermal surface, causing ectopic lignification in the absence of barrier defects. Yet, the second intriguing aspect of this manipulation is the observation that lignification occurs almost exclusively at the inner endodermal edges (Fig 1D), the side where endogenous CIF peptide must be assumed to be present. External treatment, by contrast, leads to predominant lignification at the outer endodermal edges (Fig 1B). This surprising spatial correlation between the site of sign
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