HSL1 and BAM1/2 impact epidermal cell development by sensing distinct signaling peptides.

The membrane receptor kinases HAESA and HSL2 recognize a family of IDA/IDL signaling peptides to control cell separation processes in different plant organs. The homologous HSL1 has been reported to regulate epidermal cell patterning by interacting with a different class of signaling peptides from the CLE family. Here we demonstrate that HSL1 binds IDA/IDL peptides with high, and CLE peptides with lower affinity, respectively. Ligand sensing capability and receptor activation of HSL1 require a SERK co-receptor kinase. Crystal structures with IDA/IDLs or with CLE9 reveal that HSL1-SERK1 complex recognizes the entire IDA/IDL signaling peptide, while only parts of CLE9 are bound to the receptor. In contrast, the receptor kinase BAM1 interacts with the entire CLE9 peptide with high affinity and specificity. Furthermore, the receptor tandem BAM1/BAM2 regulates epidermal cell division homeostasis. Consequently, HSL1-IDLs and BAM1/BAM2-CLEs independently regulate cell patterning in the leaf epidermal tissue.

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth.

Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2-RALF4 and LRX8-RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX-RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms.

BRI1 controls vascular cell fate in the Arabidopsis root through RLP44 and phytosulfokine signaling.

Multicellularity arose independently in plants and animals, but invariably requires a robust determination and maintenance of cell fate that is adaptive to the environment. This is exemplified by the highly specialized water- and nutrient-conducting cells of the plant vasculature, the organization of which is already prepatterned close to the stem-cell niche, but can be modified according to extrinsic cues. 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 seems unrelated to canonical brassinosteroid signaling outputs. Instead, BRI1 is required for the expression and function of its interacting partner RECEPTOR-LIKE PROTEIN 44 (RLP44), which, in turn, associates with the receptor for the peptide hormone phytosulfokine (PSK). We show that PSK signaling is required for the maintenance of procambial cell identity and quantitatively controlled by RLP44, which promotes complex formation between the PSK receptor and its coreceptor. Mimicking the loss of RLP44, PSK-related mutants show ectopic xylem in the position of the procambium, whereas rlp44 is rescued by exogenous PSK. Based on these findings, we propose that RLP44 controls cell fate by connecting BRI1 and PSK signaling, providing a mechanistic framework for the dynamic balancing of signaling mediated by the plethora of plant receptor-like kinases at the plasma membrane.

Crystal structures of two tandem malectin-like receptor kinases involved in plant reproduction.

Complex cell-to-cell communication between the male pollen tube and the female reproductive organs is required for plant fertilization. A family of Catharanthus roseus receptor kinase 1-like (CrRLK1L) membrane receptors has been genetically implicated in this process. Here, crystal structures of the CrRLK1Ls ANXUR1 and ANXUR2 are reported at 1.48 and 1.1 Šresolution, respectively. The structures reveal a novel arrangement of two malectin-like domains connected by a short ß-hairpin linker and stabilized by calcium ions. The canonical carbohydrate-interaction surfaces of related animal and bacterial carbohydrate-binding modules are not conserved in plant CrRLK1Ls. In line with this, the binding of chemically diverse oligosaccharides to ANXUR1 and HERCULES1 could not be detected. Instead, CrRLK1Ls have evolved a protein-protein interface between their malectin domains which forms a deep cleft lined by highly conserved aromatic and polar residues. Analysis of the glycosylation patterns of different CrRLK1Ls and their oligomeric states suggests that this cleft could resemble a binding site for a ligand required for receptor activation of CrRLK1Ls.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.

A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling.

The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.