In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots

Long, Yuchen; Stahl, Yvonne; Weidtkamp‐Peters, Stefanie; Postma, Marten; Zhou, Wenkun; Goedhart, Joachim; Sánchez-Pérez, María Isabel; Gadella, Theodorus W. J.; Simon, Rüdiger; Scheres, Ben; Blilou, Ikram

doi:10.1038/nature23317

Cited by 135 publications

(143 citation statements)

References 41 publications

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“…Note that our cooperative combinatorial model does not exclude the existence of either additional shared targets or many unshared PLT and SCR target genes. Unshared targets can serve broader roles in root development, such as the progression of cell division and differentiation in the root PLT gradient (Mähönen et al 2014) and the regulation of division and differentiation of the cortical-endodermal lineage by SCR and its binding partner, SHR (Di Laurenzio et al 1996;Helariutta et al 2000;Sabatini et al 2003;Cui et al 2007;Cruz-Ramírez et al 2012;Clark et al 2016;Long et al 2017). The many additional nonoverlapping functions are consistent with the observation that the published regulated targets of the PLT and SCR pathways are quite distinct (Levesque et al 2006;Moreno-Risueno et al 2015;Santuari et al 2016).…”

Section: Discussionsupporting

confidence: 68%

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules

et al. 2018

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Continuous formation of somatic tissues in plants requires functional stem cell niches where undifferentiated cells are maintained. In Arabidopsis thaliana, PLETHORA (PLT) and SCARECROW (SCR) genes are outputs of apicalbasal and radial patterning systems, and both are required for root stem cell specification and maintenance. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene is specifically expressed in and required for functions of a small group of root stem cell organizer cells, also called the quiescent center (QC). PLT and SCR are required for QC function, and their expression overlaps in the QC; however, how they specify the organizer has remained unknown. We show that PLT and SCR genetically and physically interact with plant-specific teosinte-branched cycloidea PCNA (TCP) transcription factors to specify the stem cell niche during embryogenesis and maintain organizer cells post-embryonically. PLT-TCP-SCR complexes converge on PLT-binding sites in the WOX5 promoter to induce expression.

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Section: Discussionsupporting

confidence: 68%

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules

et al. 2018

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“…For instance, FRET–FLIM in combination with multiparameter fluorescence image spectroscopy revealed that CLAVATA1 and ARABIDOPSIS CRINKLY4, involved in root stemness control, can form homo‐ and heteromeric complexes that differ in their composition, according to the subcellular localizations at the plasma membrane and plasmodesmata (Stahl et al, ). Another study with in vivo FRET–FLIM used to resolve cell‐type‐specific complex formation between the transcription factors SHORT‐ROOT, SCARECROW, and JACKDAW that were stably expressed under the control of their endogenous promoters in the Arabidopsis root uncovered the cell fate specification‐depending spatial separation of protein interactions (Long et al, ). In addition, FRET has a good time resolution; hence, it can be applied to examine transient interactions in real‐time in the relevant physiological contexts.…”

Section: Choosing the Right Validation Techniquementioning

confidence: 99%

Exploring the protein–protein interaction landscape in plants

Struk

Jacobs

Martín-Fontecha

et al. 2018

Plant Cell & Environment

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Protein-protein interactions (PPIs) represent an essential aspect of plant systems biology. Identification of key protein players and their interaction networks provide crucial insights into the regulation of plant developmental processes and into interactions of plants with their environment. Despite the great advance in the methods for the discovery and validation of PPIs, still several challenges remain. First, the PPI networks are usually highly dynamic, and the in vivo interactions are often transient and difficult to detect. Therefore, the properties of the PPIs under study need to be considered to select the most suitable technique, because each has its own advantages and limitations. Second, besides knowledge on the interacting partners of a protein of interest, characteristics of the interaction, such as the spatial or temporal dynamics, are highly important. Hence, multiple approaches have to be combined to obtain a comprehensive view on the PPI network present in a cell. Here, we present the progress in commonly used methods to detect and validate PPIs in plants with a special emphasis on the PPI features assessed in each approach and how they were or can be used for the study of plant interactions with their environment.

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“…However, advances in imaging techniques now allow to study colocalization and interactions of RLKs in living plant cells (Bücherl et al, ; Somssich et al, ). In particular, Förster Resonance Energy Transfer (FRET) is an attractive technique as it allows to resolve protein–protein interactions in live plants in a cell‐specific and dynamic manner (Lampugnani, Wink, Persson, & Somssich, ; Long et al, ; Somssich et al, ; Weidtkamp‐Peters & Stahl, ).…”

Section: Introductionmentioning

confidence: 99%

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments

et al. 2019

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Receptor‐like kinases (RLK) and receptor‐like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo‐ and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is, however, limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Förster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (a) The emission and excitation spectra of the donor and acceptor, respectively, must overlap; (b) they should not interfere with proper folding, activity, or localization of the fusion proteins; (c) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near‐endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET‐based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.

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In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots

Cited by 135 publications

References 41 publications

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules

Exploring the protein–protein interaction landscape in plants

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments

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