Biology subjects

Holzwart, E.

Publications and source records attributed to Holzwart, E..

3 recordsLinked to original sources

bioRxiv2019

Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells

Protein-protein interaction studies provide valuable insights into cellular signaling. The well-studied brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Forster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized in other nanodomains. In general, our three-fluorophore FRET approaches provide a feasible basis for studying the interaction and sub-compartmentalization of proteins in great detail.

plant biology↗

bioRxiv2019

A novel mutant allele uncouples brassinosteroid-dependent and independent functions of BRI1

Plants depend on an array of cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1), which upon binding of its hormone ligands forms a complex with shape-complimentary co-receptors and initiates a signal transduction cascade leading to a wide range of responses. BR biosynthetic and receptor mutants have similar growth defects on the macroscopic level, which had initially led to the assumption of a largely linear signalling pathway. However, recent evidence suggests that BR signalling is interconnected with a number of other pathways through a variety of different mechanisms. We recently described that feedback information from the cell wall is integrated at the level of the receptor complex through interaction with RLP44. Moreover, BRI1 is required for a second function of RLP44, the control of procambial cell fate. Here, we report on a BRI1 mutant, bri1cnu4, which differentially affects canonical BR signalling and RLP44 function in the vasculature. While BR signalling is only mildly impaired, bri1cnu4 mutants show ectopic xylem in the position of procambium. Mechanistically, this is explained by an increased association of RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semi-dominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signalling-related roles of BRI1 from its non-canonical functions.\n\nOne sentence summaryA novel mutant allows to dissect brassinosteroid signalling related and non-canonical functions of the receptor-like kinase BRI1.

plant biology↗

bioRxiv2018

Integration of Brassinosteroid and Phytosulfokine Signalling Controls Vascular Cell Fate in the Arabidopsis Root

Multicellularity arose independently in plants and animals, but invariably requires robust determination and maintenance of cell fate. This is exemplified by the highly specialized water-and nutrient-conducting cells of the plant vasculature, which are specified long before their commitment to terminal differentiation. Here, we show that the hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is required for root vascular cell fate maintenance, as BRI1 mutants show ectopic xylem in procambial position. However, this phenotype is unrelated to classical brassinosteroid signalling outputs. Instead, BRI1 is required for the expression and function of its interaction partner RECEPTOR-LIKE PROTEIN 44 (RLP44), which, in turn, associates with the receptor for the peptide hormone phytosulfokine (PSK). We show that PSK signalling is required for the maintenance of procambial cell identity and is quantitatively controlled by RLP44, which promotes complex formation between the receptor for PSK and its co-receptor. Mimicking the loss of RLP44, PSK-related mutants show ectopic xylem in the position of procambium, whereas rlp44 can be rescued by exogenous PSK. Based on these findings, we propose that RLP44 controls cell fate by connecting BRI1 and PSK signalling, providing a mechanistic framework for the integration of signalling mediated by the plethora of plant receptor-like kinases at the plasma membrane.

plant biology↗