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Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif., (© 2021. The Author(s).)

Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which - epsilon - is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development.

To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.

Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.