Signal Peptide Peptidase and PI4Kß1/2 play opposite roles in plant ER stress response and immunity.

The Arabidopsis pi4kß1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) ß1 and PI4Kß2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kß1,2 plants display autoimmunity and shorter roots. Though the pi4kß1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kß1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kß1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kß and SPP in ER stress and plant immunity.

A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

Puncta-localized TRAF domain protein TC1b contributes to the autoimmunity of snc1.

Immune receptors play important roles in the perception of pathogens and initiation of immune responses in both plants and animals. Intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors constitute a major class of receptors in vascular plants. In the Arabidopsis thaliana mutant suppressor of npr1-1, constitutive 1 (snc1), a gain-of-function mutation in the NLR gene SNC1 leads to SNC1 overaccumulation and constitutive activation of defense responses. From a CRISPR/Cas9-based reverse genetics screen in the snc1 autoimmune background, we identified that mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains, can suppress snc1 phenotypes. TC1b does not appear to be a general immune regulator as it is not required for defense mediated by other tested immune receptors. TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented by ACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b impacts snc1 autoimmunity in a unique way. TC1b can form oligomers and localizes to punctate structures of unknown function. The puncta localization of TC1b strictly requires its coiled-coil (CC) domain, whereas the functionality of TC1b requires the four TRAF domains in addition to the CC. Overall, we uncovered the TRAF domain protein TC1b as a novel positive contributor to plant immunity.

EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gß, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gß1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).

Mixed Linkage ß-1,3/1,4-Glucan Oligosaccharides Induce Defense Responses in Hordeum vulgare and Arabidopsis thaliana.

Plants detect conserved microbe-associated molecular patterns (MAMPs) and modified "self" molecules produced during pathogen infection [danger associated molecular patterns (DAMPs)] with plasma membrane-resident pattern recognition receptors (PRRs). PRR-mediated MAMP and/or DAMP perception activates signal transduction cascades, transcriptional reprogramming and plant immune responses collectively referred to as pattern-triggered immunity (PTI). Potential sources for MAMPs and DAMPs are microbial and plant cell walls, which are complex extracellular matrices composed of different carbohydrates and glycoproteins. Mixed linkage ß-1,3/1,4-glucan (ß-1,3/1,4-MLG) oligosaccharides are abundant components of monocot plant cell walls and are present in symbiotic, pathogenic and apathogenic fungi, oomycetes and bacteria, but have not been detected in the cell walls of dicot plant species so far. Here, we provide evidence that the monocot crop plant H. vulgare and the dicot A. thaliana can perceive ß-1,3/1,4-MLG oligosaccharides and react with prototypical PTI responses. A collection of Arabidopsis innate immunity signaling mutants and >100 Arabidopsis ecotypes showed unaltered responses upon treatment with ß-1,3/1,4-MLG oligosaccharides suggesting the employment of a so far unknown and highly conserved perception machinery. In conclusion, we postulate that ß-1,3/1,4-MLG oligosaccharides have the dual capacity to act as immune-active DAMPs and/or MAMPs in monocot and dicot plant species.

SCFSNIPER7 controls protein turnover of unfoldase CDC48A to promote plant immunity.

The unfoldase CDC48 (Cell Division Cycle 48) is highly conserved in eukaryotes, serving as an AAA + ATPase to extract ubiquitinated proteins from large protein complexes and membranes. Although its biochemical properties have been studied extensively in yeast and animal systems, the biological roles and regulations of the plant CDC48s have been explored only recently. Here we describe the identification of a novel E3 ligase from the SNIPER (snc1-influencing plant E3 ligase reverse genetic) screen, which contributes to plant defense regulation by targeting CDC48A for degradation. SNIPER7 encodes an F-box protein and its overexpression leads to autoimmunity. We identified CDC48s as interactors of SNIPER7 through immunoprecipitation followed by mass spectrometry proteomic analysis. SNIPER7 overexpression lines phenocopy the autoimmune mutant Atcdc48a-4. Furthermore, CDC48A protein levels are reduced or stabilized when SNIPER7 is overexpressed or inhibited, respectively, suggesting that CDC48A is the ubiquitination substrate of SCFSNIPER7 . Taken together, this study reveals a new mechanism where a SCFSNIPER7 complex regulates CDC48 unfoldase levels and modulates immune output.

Quantitative Hormone Signaling Output Analyses of Arabidopsis thaliana Interactions With Virulent and Avirulent Hyaloperonospora arabidopsidis Isolates at Single-Cell Resolution.

The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are central regulators of biotic and abiotic stress responses in Arabidopsis thaliana. Here, we generated modular fluorescent protein-based reporter lines termed COLORFUL-PR1pro, -VSP2pro, and -PDF1.2apro. These feature hormone-controlled nucleus-targeted transcriptional output sensors and the simultaneous constitutive expression of spectrally separated nuclear reference and plasma membrane-localized reporters. This set-up allowed the study of cell-type specific hormone activities, cellular viability and microbial invasion. Moreover, we developed a software-supported high-throughput confocal microscopy imaging protocol for output quantification to resolve the spatio-temporal dynamics of respective hormonal signaling activities at single-cell resolution. Proof-of-principle analyses in A. thaliana leaves revealed distinguished hormone sensitivities in mesophyll, epidermal pavement and stomatal guard cells, suggesting cell type-specific regulatory protein activities. In plant-microbe interaction studies, we found that virulent and avirulent Hyaloperonospora arabidopsidis (Hpa) isolates exhibit different invasion dynamics and induce spatio-temporally distinct hormonal activity signatures. On the cellular level, these hormone-controlled reporter signatures demarcate the nascent sites of Hpa entry and progression, and highlight initiation, transduction and local containment of immune signals.

Live-cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana.

Subdiffraction super-resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live-cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule-associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time-lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live-cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew.

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid.

The phytohormone salicylic acid (SA) controls biotic and abiotic plant stress responses. Plastid-produced chorismate is a branch-point metabolite for SA biosynthesis. Most pathogen-induced SA derives from isochorismate, which is generated from chorismate by the catalytic activity of ISOCHORISMATE SYNTHASE1. Here, we ask how and in which cellular compartment isochorismate is converted to SA. We show that in Arabidopsis, the pathway downstream of isochorismate requires only two additional proteins: ENHANCED DISEASE SUSCEPTIBILITY5, which exports isochorismate from the plastid to the cytosol, and the cytosolic amidotransferase avrPphB SUSCEPTIBLE3 (PBS3). PBS3 catalyzes the conjugation of glutamate to isochorismate to produce isochorismate-9-glutamate, which spontaneously decomposes into SA and 2-hydroxy-acryloyl-N-glutamate. The minimal requirement of three compartmentalized proteins controlling unidirectional forward flux may protect the pathway against evolutionary forces and pathogen perturbations.

A Poly(A) Ribonuclease Controls the Cellotriose-Based Interaction between Piriformospora indica and Its Host Arabidopsis.

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.

Chitin-induced and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) phosphorylation-dependent endocytosis of Arabidopsis thaliana LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5 (LYK5).

To detect potential pathogens, plants perceive the fungal polysaccharide chitin through receptor complexes containing lysin motif receptor-like kinases (LysM-RLKs). To investigate the ligand-induced spatial dynamics of chitin receptor components, we studied the subcellular behaviour of two Arabidopsis thaliana LysM-RLKs involved in chitin signalling, CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) and LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5. We performed standard and quantitative confocal laser scanning microscopy on stably transformed A. thaliana plants expressing fluorescently tagged CERK1 and LYK5 from their native promoters. Microscopy approaches were complemented by biochemical analyses in plants and in vitro. Both CERK1 and LYK5 localized to the plasma membrane and showed constitutive endomembrane trafficking. After chitin treatment, however, CERK1 remained at the plasma membrane while LYK5 relocalized into mobile intracellular vesicles. Detailed analyses revealed that chitin perception transiently induced the internalization of LYK5 into late endocytic compartments. Plants that lacked CERK1 or expressed an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. CERK1 could phosphorylate LYK5 in vitro and chitin treatment induced CERK1-dependent phosphorylation of LYK5 in planta. Our results suggest that chitin-induced phosphorylation by CERK1 triggers LYK5 internalization. Thus, our work identifies phosphorylation as a key regulatory step in endocytosis of plant RLKs and also provides evidence for receptor complex dissociation after ligand perception.

Nucleoporin NUP88/MOS7 is required for manifestation of phenotypes associated with the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 mutant cerk1-4.

Arabidopsis nucleoporin MOS7/NUP88 was identified in a forward-genetic screen for components that contribute to auto-immunity of the deregulated Resistance (R) gene mutant snc1, and is required for immunity to biotrophic and hemi-biotrophic pathogens. In a recent study, we showed that MOS7 is also essential to mount a full defense response against the necrotrophic fungal pathogen Botrytis cinerea, suggesting that MOS7 modulates plant defense responses to different types of pathogenic microbes. Here, we extend our analyses of MOS7-dependent plant immune responses and report the genetic requirement of MOS7 for manifestation of phenotypes associated with the CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) mutant cerk1-4.

The putative kinase substrate MUSE7 negatively impacts the accumulation of NLR proteins.

Stringent modulation of immune signaling in plants is necessary to enable a rapid response to pathogen attack without spurious defense activation. To identify genes involved in plant immunity, a forward genetic screen for enhancers of the autoimmune snc1 (suppressor of npr1, constitutive 1) mutant was conducted. The snc1 mutant contains a gain-of-function mutation in a gene encoding a NOD-like receptor (NLR) protein. The isolated muse7 (mutant, snc1-enhancing, 7) mutant was shown to confer a reversion to autoimmune phenotypes in the wild-type-like mos4 (modifier of snc1, 4) snc1 background. Positional cloning revealed that MUSE7 encodes an evolutionarily conserved putative kinase substrate of unknown function. The muse7 single mutants display enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. While transcription of SNC1 is not enhanced, elevated SNC1 protein accumulation is associated with mutations in muse7. Accumulation of two additional NLR proteins, RPS2 (RESISTANCE TO PSEUDOMONAS SYRINGAE 2) and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE pv. MACULICOLA 1), was also observed in muse7 plants. Although proteasome-mediated degradation of NLR proteins is a well studied event in plant immunity, no interactions were detected between MUSE7 and selected components of this pathway. This study has demonstrated a role for MUSE7 in modulating plant immune responses through negatively affecting NLR accumulation, and will benefit future studies of MUSE7 homologs in other species.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants.

Advancing basic and applied plant research requires the continuous innovative development of the available technology toolbox. Essential components of this toolbox are methods that simplify the assembly, delivery, and expression of multiple transgenes of interest. To allow simultaneous and directional multigene assembly on the same plant transformation vector, several strategies based on overlapping sequences or restriction enzymes have recently been developed. However, the assembly of homologous and repetitive DNA sequences can be inefficient and the frequent occurrence of target sequences recognized by commonly used restriction enzymes can be a limiting factor. Here, we noted that recognition sites for the restriction enzyme SfiI are rarely occurring in plant genomes. This fact was exploited to establish a multigene assembly system called "COLORFUL-Circuit." To this end, we developed a set of binary vectors which provide a flexible and cost efficient cloning platform. The gene expression cassettes in our system are flanked with unique SfiI sites, which allow simultaneous multi-gene cassette assembly in a hosting binary vector. We used COLORFUL-Circuit to transiently and stably express up to four fluorescent organelle markers in addition to a selectable marker and analyzed the impact of assembly design on coexpression efficiency. Finally, we demonstrate the utility of our optimized "COLORFUL-Circuit" system in an exemplary case study, in which we monitored simultaneously the subcellular behavior of multiple organelles in a biotrophic plant-microbe interaction by Confocal Laser Scanning Microscopy.

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis.

The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.

Knowing your friends and foes--plant receptor-like kinases as initiators of symbiosis or defence.

The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane-localized receptor complexes. A critical step in their activation is ligand-induced homo- or hetero-oligomerization of leucine-rich repeat (LRR)- and/or lysin motif (LysM) receptor-like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen-associated molecular patterns (PAMPs), including the bacterial flagellin-derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont-derived (lipo)-chitooligosaccharides. The structurally related chitin-oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM-RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin-like domain (MLD)-LRR-RLK Symbiosis Receptor-like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR- and LysM-mediated signalling, the involvement of MLD-LRR-RLKs in symbiosis and defence, and the role of endocytosis in RLK function.