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

SummaryPlant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signalling molecules. Receptor kinases are regulated by numerous post-translational modifications. Here, using the immune receptor kinases FLS2 and EFR, 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 biophysical properties and behaviour within the membrane environment. We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following perception of its ligand flg22, in a BAK1 co-receptor dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signalling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR supressed elf18 triggered signalling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents and native membrane DIBMA nanodiscs indicates that S-acylation stabilises and promotes retention of activated receptor kinase complexes at the plasma membrane to increase signalling efficiency

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

Isolierung und Charakterisierung des ERP1-Gens aus Arabidopsis thaliana im Rahmen von Untersuchungen zur Nichtwirtsresistenz gegenüber Phytophthora infestans

Die Nichtwirtsresistenz von Arabidopsis thaliana gegen Phytophthora infestans basiert auf mehreren Ebenen der Abwehr. Die Myrosinase PEN2 und der ABC-Transporter PEN3 tragen effektiv zur Prä-invasiven Abwehr bei. Um weitere Gene zu identifizieren, die in diese Nichtwirts-Interaktion involviert sind, wurde ein genetischer screen mit re-mutagenisierten pen2-Pflanzen durchgeführt. Es wurden 14 unabhängige Mutanten isoliert, die entsprechend ihrer Phänotypen als enhanced response to Phytophthora (erp) bezeichnet wurden. Die Abwehrreaktion der erp1-Mutante gegenüber P. infestans und anderen filamentösen Pathogenen ging mit vermehrtem Zelltod und massiven PMR4-unabhängigen Kallose-Ablagerungen im Mesophyll einher. ERP1 kodiert die Phospholipid:Sterol-Acyltransferase PSAT1. erp1-Mutanten weisen stark reduzierte Sterolester-Mengen und eine veränderte Sterolhomöostase im Blatt auf. Dies legt eine Bedeutung von Sterolkonjugaten für die Abwehrreaktion gegen invasive filamentöse Pathogene nahe.The non-host resistance of Arabidopsis thaliana against Phytophthora infestans is based on a multi-layered defense response. The myrosinase PEN2 and the ABC-transporter PEN3 were shown to be required for an efficient pre-invasive resistance against P. infestans. To identify further genes involved in this non-host interaction, a genetic screen with re-mutagenized pen2 plants was performed. 14 independent mutants were isolated exhibiting an enhanced response to Phytophthora (erp) phenotype. The enhanced defense reaction of the erp1 mutant upon challenge with P. infestans and other filamentous pathogens was accompanied by an enhanced mesophyll cell death and excessive callose deposits in mesophyll cells, independent of PMR4. ERP1 encodes the phospholipid:sterol acyltransferase PSAT1. erp1 mutants showed highly reduced sterol ester levels and an altered sterol homeostasis in leaves. This suggests a role for sterol conjugates in defense responses against invasive filamentous pathogens.von Michaela Kopischk

NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies