Summary Remorins are well‐established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post‐translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S‐acylation on defined residues in these proteins. S‐acylation of cysteine residues in a C‐terminal hydrophobic core contributes to membrane association of most remorin proteins. While S‐acylation patterns differ between members of this multi‐gene family, initial membrane association is mediated by protein–protein or protein–lipid interactions. However, S‐acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane‐resident proteins may be involved in the recruitment of remorins into membrane domains. S‐acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S‐acylation is a reversible post‐translational modification, stimulus‐dependent intracellular trafficking of these proteins can be envisioned.

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants' innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in longdisordered regions ([50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein-protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.

Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs)Alike other RLKs the Medicago truncatula entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, molecular mechanisms underlying such dynamic transitions and their functional relevance remained poorly understood. Here, we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in symrem1 mutants upon rhizobial inoculation resulting in premature abortion of host cell infections.These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/179036 doi: bioRxiv preprint first posted online Dec. 31, 2017; 3 SIGNIFICANCE STATEMENT:Pattern recognition receptors control the cellular entry of pathogenic as well as symbiotic microbes. While ligand-induced changes in receptor mobility at the plasma membrane and their localization in membrane nanodomains appears as a general feature, the molecular mechanism and the biological relevance of this phenomenon remained unknown. Here, we show that immobilization of the symbiotic cell entry receptor LYK3 in nanodomains requires the presence of actin and the two molecular scaffold proteins FLOT4 and SYMREM1. While FLOT4 forms the initial core structure, infection-induced expression and subsequent physical interaction of SYMREM1 with LYK3 stabilizes the activated receptors in membrane nanodomains. This recruitment prevents its stimulusdependent endocytosis and ensures progression of the primary infection thread into root cortical cells.All rights reserved. No reuse allowed without permission.

Plant-specific remorin proteins reside in subdomains of plasma membranes, originally termed membrane rafts. They probably facilitate cellular signal transduction by direct interaction with signaling proteins such as receptor-like kinases and may dynamically modulate their lateral segregation within plasma membranes. Recent evidence suggests such functions of remorins during plant–microbe interactions and innate immune responses, where differential phosphorylation of some of these proteins has been described to be dependent on the perception of the microbe-associated molecular pattern (MAMP) flg22 and the presence of the NBS–LRR resistance protein RPM1. A number of specifically phosphorylated residues in their highly variable and intrinsically disordered N-terminal regions have been identified. Sequence diversity of these evolutionary distinct domains suggests that remorins may serve a wide range of biological functions. Here, we describe patterns and features of intrinsic disorder in remorin protein and discuss possible functional implications of phosphorylation within these rapidly evolving domains.

Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs). Like other RLKs, the entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, the molecular mechanisms underlying such dynamic transitions and their functional relevance have remained poorly understood. Here we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in mutants on rhizobial inoculation, resulting in premature abortion of host cell infections. These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.