Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 6 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.Eukaryotic plasma membranes (PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (Yetukuri et al., 2008;Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant PM contains an important number of sterols, among which b-sitosterol, stigmasterol, and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (SG) and acyl steryl glycosides (ASG) that represent up to approximately 15% of the tobacco (Nicotiana tabacum) PM (Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.DOI: http://dx.doi.org/10.7554/eLife.26404.001

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h.