Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

ProtView: A Versatile Tool for In Silico Protease Evaluation and Selection in a Proteomic and Proteogenomic Context.

Many tools have been created to generate in silico proteome digests with different protease enzymes and provide useful information for selecting optimal digest schemes for specific needs. This can save on time and resources and generate insights on the observable proteome. However, there remains a need for a tool that evaluates digest schemes beyond protein and amino acid coverages in the proteomic domain. Here, we present ProtView, a versatile in silico protease combination digest evaluation workflow that maps in silico-digested peptides to both protein and genome references, so that the potential observable portions of the proteome, transcriptome, and genome can be identified. The proteomic identification and quantification of evidence for transcriptional, co-transcriptional, post-transcriptional, translational, and post-translational regulation can all be examined in silico with ProtView prior to an experiment. Benchmarking against biological data comparing multiple proteases shows that ProtView can correctly estimate performances among the digest schemes. ProtView provides this information in a way that is easy to interpret, allowing for digest schemes to be evaluated before carrying out an experiment, in context that can optimize both proteomic and proteogenomic experiments. ProtView is available at https://github.com/SSPuliasis/ProtView.

S-acylation in plants: an expanding field.

S-acylation is a common yet poorly understood fatty acid-based post-translational modification of proteins in all eukaryotes, including plants. While exact roles for S-acylation in protein function are largely unknown the reversibility of S-acylation indicates that it is likely able to play a regulatory role. As more studies reveal the roles of S-acylation within the cell it is becoming apparent that how S-acylation affects proteins is conceptually different from other reversible modifications such as phosphorylation or ubiquitination; a new mind-set is therefore required to fully integrate these data into our knowledge of plant biology. This review aims to highlight recent advances made in the function and enzymology of S-acylation in plants, highlights current and emerging technologies for its study and suggests future avenues for investigation.

Variable Effects of C-Terminal Fusions on FLS2 Function: Not All Epitope Tags Are Created Equal.

Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli. Thus, RLKs function in diverse processes, including sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signals, and monitoring cell wall status. However, despite their fundamental role in plant biology, very few antibodies are available against RLKs, which necessitates the use of epitope tags and fluorescent protein fusions in biochemical analyses such as immunoblot analysis and intracellular visualization. Epitope tags are widely used and are typically assumed to be benign, with no influence on protein function. FLAGELLIN SENSITIVE2 (FLS2) is the receptor for bacterial flagellin and often is used as a model for RLK function. Previous work implies that carboxyl-terminal epitope fusions to FLS2 maintain protein function. Here, a detailed complementation analysis of Arabidopsis (Arabidopsis thaliana) fls2 mutant plants expressing various FLS2 C-terminal epitope fusions revealed highly variable and unpredictable FLS2-mediated signaling outputs. In addition, only one out of four FLS2 epitope fusions maintained the ability to inhibit plant growth in response to flg22 treatment comparable to that in the wild type or control untagged transgenic lines. These results raise concerns over the widespread use of RLK epitope tag fusions for functional studies. Many of the subtleties of FLS2 function, and by extension those of other RLKs, may have been overlooked or inappropriately interpreted through the use of RLK epitope tag fusions.

Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.

An outlook on protein S-acylation in plants: what are the next steps?

S-acylation, also known as palmitoylation, is the reversible post-translational addition of fatty acids to proteins. Historically thought primarily to be a means for anchoring otherwise soluble proteins to membranes, evidence now suggests that reversible S-acylation may be an important dynamic regulatory mechanism. Importantly S-acylation affects the function of many integral membrane proteins, making it an important factor to consider in understanding processes such as cell wall synthesis, membrane trafficking, signalling across membranes and regulating ion, hormone and metabolite transport through membranes. This review summarises the latest thoughts, ideas and findings in the field as well discussing future research directions to gain a better understanding of the role of this enigmatic regulatory protein modification.

The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

The importance of lipid modified proteins in plants.

Membranes have long been known to act as more than physical barriers within and between plant cells. Trafficking of membrane proteins, signalling from and across membranes, organisation of membranes and transport through membranes are all essential processes for plant cellular function. These processes rely on a myriad array of proteins regulated in a variety of manners and are frequently required to be directly associated with membranes. For integral membrane proteins, the mode of membrane association is readily apparent, but many peripherally associated membrane proteins are outwardly soluble proteins. In these cases the proteins are frequently modified by the addition of lipids allowing direct interaction with the hydrophobic core of membranes. These modifications include N-myristoylation, S-acylation (palmitoylation), prenylation and GPI anchors but until recently little was truly known about their function in plants. New data suggest that these modifications are able to act as more than just membrane anchors, and dynamic S-acylation in particular is emerging as a means of regulating protein function in a similar manner to phosphorylation. This review discusses how these modifications occur, their impact on protein function, how they are regulated, recent advances in the field and technical approaches for studying these modifications.

Current perspective on protein S-acylation in plants: more than just a fatty anchor?

Membranes are an important signalling platform in plants. The plasma membrane is the point where information about the external environment must be converted into intracellular signals, while endomembranes are important sites of protein trafficking, organization, compartmentalization, and intracellular signalling. This requires co-ordinating the spatial distribution of proteins, their activation state, and their interacting partners. This regulation frequently occurs through post-translational modification of proteins. Proteins that associate with the cell membrane do so through transmembrane domains, protein-protein interactions, lipid binding motifs/domains or use the post-translational addition of lipid groups as prosthetic membrane anchors. S-acylation is one such lipid modification capable of anchoring proteins to the membrane. Our current knowledge of S-acylation function in plants is fairly limited compared with other post-translational modifications and S-acylation in other organisms. However, it is becoming increasingly clear that S-acylation can act as more than just a simple membrane anchor: it can also act as a regulatory mechanism in signalling pathways in plants. S-acylation is, therefore, an ideal mechanism for regulating protein function at membranes. This review discusses our current knowledge of S-acylated proteins in plants, the interaction of different lipid modifications, and the general effects of S-acylation on cellular function.

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

A proteomic approach identifies many novel palmitoylated proteins in Arabidopsis.

S-acylation (palmitoylation) is a poorly understood post-translational modification of proteins involving the addition of acyl lipids to cysteine residues. S-acylation promotes the association of proteins with membranes and influences protein stability, microdomain partitioning, membrane targeting and activation state. No consensus motif for S-acylation exists and it therefore requires empirical identification. Here, we describe a biotin switch isobaric tagging for relative and absolute quantification (iTRAQ)-based method to identify S-acylated proteins from Arabidopsis. We use these data to predict and confirm S-acylation of proteins not in our dataset. We identified c. 600 putative S-acylated proteins affecting diverse cellular processes. These included proteins involved in pathogen perception and response, mitogen-activated protein kinases (MAPKs), leucine-rich repeat receptor-like kinases (LRR-RLKs) and RLK superfamily members, integral membrane transporters, ATPases, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs) and heterotrimeric G-proteins. The prediction of S-acylation of related proteins was demonstrated by the identification and confirmation of S-acylation sites within the SNARE and LRR-RLK families. We showed that S-acylation of the LRR-RLK FLS2 is required for a full response to elicitation by the flagellin derived peptide flg22, but is not required for localization to the plasma membrane. Arabidopsis contains many more S-acylated proteins than previously thought. These data can be used to identify S-acylation sites in related proteins. We also demonstrated that S-acylation is required for full LRR-RLK function.

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signaling.

Plant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signaling molecules. Receptor kinases are regulated by numerous post-translational modifications.1,2,3 Here, using the immune receptor kinases FLS24 and EFR,5 we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biochemical properties and behavior within the membrane environment.6 We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following the perception of its ligand, flg22, in a BAK1 co-receptor and PUB12/13 ubiquitin ligase-dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signaling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR suppressed elf18-triggered signaling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents, and native membrane DIBMA nanodiscs indicates that S-acylation stabilizes, and promotes retention of, activated receptor kinase complexes at the plasma membrane to increase signaling efficiency.

The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

• Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only.

Acetyl-CoA-carboxylase 1 (ACC1) plays a critical role in glucagon secretion.

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

Protein S-acylation in plants (Review).

Membrane resident proteins are a common feature of biology yet many of these proteins are not integral to the membrane. These peripheral membrane proteins are often bound to the membrane by the addition of fatty acyl chains to the protein. This modification, known as S-acylation or palmitoylation, promotes very strong membrane association but is also reversible allowing for a high degree of control over membrane association. Many S-acylated proteins are resident in sterol, sphingolipid and saturated-lipid enriched microdomains indicating an important role for S-acylation in protein partitioning within membranes. This review summarises the current knowledge of S-acylation in plants. S-acylated proteins play a wide variety of roles in plants and affect Ca(2+) signalling, K(+) movement, stress signalling, small and heterotrimeric G-protein membrane association and partitioning, tubulin function as well as pathogenesis. Although the study of S-acylation is in its infancy in plants this review illustrates that S-acylation is extremely important for plant function and that there are many unexplored aspects of S-acylation in plants. A full summary of the techniques and methods available to study S-acylation in plants is also presented.

Multiple roles for protein palmitoylation in plants.

Palmitoylation, more correctly known as S-acylation, aids in the regulation of cellular functions including stress response, disease resistance, hormone signalling, cell polarisation, cell expansion and cytoskeletal organization. S-acylation is the reversible addition of fatty acids to proteins, which increases their membrane affinity. Membrane-protein interactions are important for signalling complex formation and signal propagation, protein sequestration and segregation, protein stability, and maintaining polarity within the cell. S-acylation is a dynamic modification that modulates the activity and membrane association of many signalling molecules, including ROP GTPases, heterotrimeric G-proteins and calcium-sensing kinases. Recent advances in methods to study S-acylation are permitting an in-depth examination of its function in plants.

Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods.

S-Acylation is increasingly being recognized as an important dynamic posttranslational modification of cysteine residues in proteins. Various approaches have been described for assaying protein S-acylation with acyl-switch approaches being the most common and accessible. However, these approaches can be time-consuming with low reproducibility as a result of multiple protein precipitation/resuspension cleanup steps. Here we present a faster, cleaner, and more sensitive acyl-switch approach for detecting the S-acylation state of any protein, from any cell or tissue type, that can be detected by western blotting. In the case of acyl-RAC, the procedure is now performed without protein precipitation, greatly increasing speed and improving sample handling in the assay. This also allows for more samples to be processed simultaneously and opens the way for medium-throughput assays. Overall, maleimide scavenging improves the reliability of determination and quantification of protein S-acylation state by acyl-switch methods.

Juxta-membrane S-acylation of plant receptor-like kinases is likely fortuitous and does not necessarily impact upon function.

S-acylation is a common post-translational modification of membrane protein cysteine residues with many regulatory roles. S-acylation adjacent to transmembrane domains has been described in the literature as affecting diverse protein properties including turnover, trafficking and microdomain partitioning. However, all of these data are derived from mammalian and yeast systems. Here we examine the role of S-acylation adjacent to the transmembrane domain of the plant pathogen perceiving receptor-like kinase FLS2. Surprisingly, S-acylation of FLS2 adjacent to the transmembrane domain is not required for either FLS2 trafficking or signalling function. Expanding this analysis to the wider plant receptor-like kinase family we find that S-acylation adjacent to receptor-like kinase domains is common, affecting ~25% of Arabidopsis receptor-like kinases, but poorly conserved between orthologues through evolution. This suggests that S-acylation of receptor-like kinases at this site is likely the result of chance mutation leading to cysteine occurrence. As transmembrane domains followed by cysteine residues are common motifs for S-acylation to occur, and many S-acyl transferases appear to have lax substrate specificity, we propose that many receptor-like kinases are fortuitously S-acylated once chance mutation has introduced a cysteine at this site. Interestingly some receptor-like kinases show conservation of S-acylation sites between orthologues suggesting that S-acylation has come to play a role and has been positively selected for during evolution. The most notable example of this is in the ERECTA-like family where S-acylation of ERECTA adjacent to the transmembrane domain occurs in all ERECTA orthologues but not in the parental ERECTA-like clade. This suggests that ERECTA S-acylation occurred when ERECTA emerged during the evolution of angiosperms and may have contributed to the neo-functionalisation of ERECTA from ERECTA-like proteins.

Fats and function: protein lipid modifications in plant cell signalling.

The post-translational lipid modifications N-myristoylation, prenylation and S-acylation are traditionally associated with increasing protein membrane affinity and localisation. However this is an over-simplification, with evidence now implicating these modifications in a variety of roles such as membrane microdomain partitioning, protein trafficking, protein complex assembly and polarity maintenance. Evidence for a regulatory role is also emerging, with changes or manipulation of lipid modifications offering a means of directly controlling various aspects of protein function. Proteomics advances have revealed an enrichment of signalling proteins in the lipid-modified proteome, potentially indicating an important role for these modifications in responding to stimuli. This review highlights some of the key themes and possible functions of lipid modification during signalling processes in plants.