Direct interaction of ligand-receptor pairs specifying stomatal patterning. Genes Dev

Department of Biology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
Genes & development (Impact Factor: 10.8). 01/2012; 26(2):126-36. DOI: 10.1101/gad.179895.111
Source: PubMed


Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2-ERECTA and EPF1-ERL1 as ligand-receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

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    • "For example, the kinase-deleted version of the receptor kinase ERECTA (ER: At2g26330) accumulates at much higher levels than the full-length, endogenous ERECTA protein (Shpak et al, 2003), while the removal of the kinase domain in the CLAVA- TA1 (CLV1: At1g75820) receptor improves its expression without affecting the ligand-binding affinity of its ectodomain (Ogawa et al, 2008). These truncated proteins have been successfully used in binding assays such as pull down, gel filtration, fluorescence anisotropy, and surface plasmon resonance (Pollard, 2010, Luoni et al, 2006, Lee et al 2012). However, interpretation of this binding data has to include a thorough analysis of the potential biological effects caused by removing the kinase domains and the changes in stoichiometry caused by the over-accumulation of stabilized receptors. "
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    ABSTRACT: The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane.
    The Arabidopsis Book 06/2015; 13:e0180. DOI:10.1199/tab.0180
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    • "Because RLPs lack a kinase domain, it has been hypothesized that these cell surface receptors recruit one or more RLKs to initiate cytoplasmic signalling. Indeed, in Arabidopsis thaliana (At), the RLP TOO MANY MOUTHS (TMM), which regulates stomatal patterning, interacts with the LRR-RLK ERECTA (Lee et al., 2012), whereas CLAVATA 2 (CLV2), which is involved in meristem maintenance, forms a complex with the transmembrane (TM) kinase CORYNE, as well as with the LRR- RLK CLV1, to activate downstream signalling (Bleckmann et al., 2010; Müller et al., 2008; Zhu et al., 2010). Recently, it has been reported that the LRR-RLK SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR, hereafter referred to as SOBIR1) (Gao et al., 2009; Leslie et al., 2010) constitutively interacts with Cf proteins and is required for their function (Liebrand et al., 2013, 2014b). "
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    ABSTRACT: Receptor-like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine-rich repeats (LRRs) and, in contrast to receptor-like kinases (RLKs), lack a cytoplasmic kinase required for initiating downstream signalling. Recent studies revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1-1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf-4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf-4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked-down by virus-induced gene silencing, showed that the LRR domain as well as kinase activity of SOBIR1 are required for the Cf-4/Avr4-triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitating Cf-4 accumulation. Together, these results suggest that in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for initiating downstream signalling through its kinase domain. This article is protected by copyright. All rights reserved.
    Molecular Plant Pathology 04/2015; DOI:10.1111/mpp.12266 · 4.72 Impact Factor
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    • "Conversely, CLEL6 and CLEL7 are small peptides that play a role in later asymmetric divisions within lateral root development, although their corresponding LRR-RLK has not been identified (Meng et al., 2012). The ERECTA LRR-RLK influences both ( pro)cambial and stomatal development, demonstrating one case where a single receptor can regulate asymmetric cell division in multiple cell types (Shpak et al., 2005; Lee et al., 2012; Etchells et al., 2013). Perhaps this conservation of function is due to the fact that these are examples of asymmetric amplifying divisions. "
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