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The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci . Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis - a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many biotrophic fungal plant pathogens develop feeding structures, haustoria, inside living plant cells, which are essential for their success. Extrahaustorial membranes (EHMs) surround haustoria and delimit the extrahaustorial matrices (EHMxs). Little is known about transport mechanisms across EHMs and what properties proteins and nutrients need in order to cross these membranes. To investigate this further, we expressed fluorescent proteins in the cytosol of infected barley leaf epidermal cells after particle bombardment and investigated properties that influenced their localisation in the powdery mildew EHMx. We showed that this translocation is favoured by a neutral isoelectric point (pI) between 6.0 and 8.4. However, for proteins larger than 50 kDa, pI alone does not explain their localisation, hinting towards a more complex interplay between pI, size, and sequence properties. We discuss the possibility that an EHM translocon is involved in protein uptake into the EHMx., (© 2019 John Wiley & Sons Ltd.)

Successful pathogens must efficiently defeat or delay host immune responses, including those triggered by release or exposure of microbe-associated molecular patterns (MAMPs). Knowledge of the molecular details leading to this phenomenon in genuine plant-pathogen interactions is still scarce. We took advantage of the well-established Arabidopsis thaliana - Pseudomonas syringae pv. tomato DC3000 pathosystem to explore the molecular prerequisites for the suppression of MAMP-triggered host defense by the bacterial invader. Using a transgenic Arabidopsis line expressing the calcium sensor apoaequorin, we discovered that strain DC3000 colonization results in a complete inhibition of MAMP-induced cytosolic calcium influx, a key event of immediate-early host immune signaling. A range of further plant-associated bacterial species is also able to prevent, either partially or fully, the MAMP-triggered cytosolic calcium pattern. Genetic analysis revealed that this suppressive effect partially relies on the bacterial type III secretion system (T3SS) but cannot be attributed to individual members of the currently known arsenal of strain DC3000 effector proteins. Although the phytotoxin coronatine and bacterial flagellin individually are dispensable for the effective inhibition of MAMP-induced calcium signatures, they contribute to the attenuation of calcium influx in the absence of the T3SS. Our findings suggest that the capacity to interfere with early plant immune responses is a widespread ability among plant-associated bacteria that, at least in strain DC3000, requires the combinatorial effect of multiple virulence determinants. This may also include the desensitization of host pattern recognition receptors by the prolonged exposure to MAMPs during bacterial pathogenesis.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus., Competing Interests: The authors have declared that no competing interests exist.

Attachment of the small ubiquitin-like modifier (SUMO) to substrate proteins modulates their turnover, activity, or interaction partners. However, how this SUMO conjugation activity concentrates the proteins involved and the substrates into uncharacterized nuclear bodies (NBs) remains poorly understood. Here, we characterized the requirements for SUMO NB formation and for their subsequent colocalization with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a master regulator of plant growth. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by darkness and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity alone was sufficient to target the SUMO machinery into NBs. Colocalization of these bodies with COP1 required, in addition to SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SAP and Miz 1 (SIZ1). We found that SIZ1 docks in the substrate-binding pocket of COP1 via two valine-proline peptide motifs, which represent a known interaction motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRYPTOCHROME 1 and CRYPTOCHROME 2. Our findings thus suggest that sumoylation stimulates COP1 activity within NBs. Moreover, the presence of SIZ1 and SUMO in these NBs explains how both the timing and amplitude of the high-temperature growth response is controlled. The strong colocalization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Many filamentous plant pathogens place specialized feeding structures, called haustoria, inside living host cells. As haustoria grow, they are believed to manipulate plant cells to generate a specialized, still enigmatic extrahaustorial membrane (EHM) around them. Here, we focused on revealing properties of the EHM. With the help of membrane-specific dyes and transient expression of membrane-associated proteins fused to fluorescent tags, we studied the nature of the EHM generated by barley leaf epidermal cells around powdery mildew haustoria. Observations suggesting that endoplasmic reticulum (ER) membrane-specific dyes labelled the EHM led us to find that Sar1 and RabD2a GTPases bind this membrane. These proteins are usually associated with the ER and the ER/cis-Golgi membrane, respectively. In contrast, transmembrane and luminal ER and Golgi markers failed to label the EHM, suggesting that it is not a continuum of the ER. Furthermore, GDP-locked Sar1 and a nucleotide-free RabD2a, which block ER to Golgi exit, did not hamper haustorium formation. These results indicated that the EHM shares features with the plant ER membrane, but that the EHM membrane is not dependent on conventional secretion. This raises the prospect that an unconventional secretory pathway from the ER may provide this membrane's material. Understanding these processes will assist future approaches to providing resistance by preventing EHM generation., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Loss-of-function of barley mildew locus o ( Mlo ) confers durable broad-spectrum penetration resistance to the barley powdery mildew pathogen, Blumeria graminis f. sp. hordei ( Bgh ). Given the importance of mlo mutants in agriculture, surprisingly few molecular components have been identified to be required for this type of resistance in barley. With the aim to identify novel cellular factors contributing to mlo -based resistance, we devised a pharmacological inhibitor screen. Of the 41 rationally chosen compounds tested, five caused a partial suppression of mlo resistance in barley, indicated by increased levels of Bgh host cell entry. These chemicals comprise brefeldin A (BFA), 2',3'-dideoxyadenosine (DDA), 2-deoxy-d-glucose, spermidine, and 1-aminobenzotriazole. Further inhibitor analysis corroborated a key role for both anterograde and retrograde endomembrane trafficking in mlo resistance. In addition, all four ribonucleosides, some ribonucleoside derivatives, two of the five nucleobases (guanine and uracil), some guanine derivatives as well as various polyamines partially suppress mlo resistance in barley via yet unknown mechanisms. Most of the chemicals identified to be effective in partially relieving mlo resistance in barley also to some extent compromised powdery mildew resistance in an Arabidopsis mlo2 mlo6 double mutant. In summary, our study identified novel suppressors of mlo resistance that may serve as valuable probes to unravel further the molecular processes underlying this unusual type of disease resistance., (© 2017 The Author(s).)

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O ( MLO ) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.

It is generally accepted in plant-microbe interactions research that disease is the exception rather than a common outcome of pathogen attack. However, in nature, plants with symptoms that signify colonization by obligate biotrophic powdery mildew fungi are omnipresent. The pervasiveness of the disease and the fact that many economically important plants are prone to infection by powdery mildew fungi drives research on this interaction. The competence of powdery mildew fungi to establish and maintain true biotrophic relationships renders the interaction a paramount example of a pathogenic plant-microbe biotrophy. However, molecular details underlying the interaction are in many respects still a mystery. Since its introduction in 1990, the Arabidopsis-powdery mildew pathosystem has become a popular model to study molecular processes governing powdery mildew infection. Due to the many advantages that the host Arabidopsis offers in terms of molecular and genetic tools this pathosystem has great capacity to answer some of the questions of how biotrophic pathogens overcome plant defense and establish a persistent interaction that nourishes the invader while in parallel maintaining viability of the plant host.

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)

Plants have evolved a complex array of defensive responses against pathogenic microorganisms. Recognition of microbes initiates signaling cascades that activate plant defenses. The membrane lipid phosphatidic acid, produced by phospholipase D (PLD), has been shown to take part in both abiotic and biotic stress signaling. In this study, the involvement of PLD in the interaction between Arabidopsis (Arabidopsis thaliana) and the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was investigated. This nonadapted pathogen is normally resisted by a cell wall-based defense, which stops the fungal hyphae from penetrating the epidermal cell wall. Chemical inhibition of phosphatidic acid production by PLD increased the penetration rate of Bgh spores on wild-type leaves. The analysis of transfer DNA knockout lines for all Arabidopsis PLD genes revealed that PLDδ is involved in penetration resistance against Bgh, and chemical inhibition of PLDs in plants mutated in PLDδ indicated that this isoform alone is involved in Bgh resistance. In addition, we confirmed the involvement of PLDδ in penetration resistance against another nonadapted pea powdery mildew fungus, Erysiphe pisi. A green fluorescent protein fusion of PLDδ localized to the plasma membrane at the Bgh attack site, where it surrounded the cell wall reinforcement. Furthermore, in the pldδ mutant, transcriptional up-regulation of early microbe-associated molecular pattern response genes was delayed after chitin stimulation. In conclusion, we propose that PLD is involved in defense signaling in nonhost resistance against powdery mildew fungi and put PLDδ forward as the main isoform participating in this process.

Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle-associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.

Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER) translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery. We provide support for a model suggesting that the retrotranslocon function of HvSec61βa is required for successful powdery mildew fungal infection. HvSec61βa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61βa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol.

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candidate, CSEP0055, contributes to the aggressiveness of the fungus. This result suggests that CSEP0055 is involved in the suppression of plant defence. A yeast two-hybrid screen indicated that CSEP0055 interacts with members of the barley pathogenesis-related protein families, PR1 and PR17. Interaction with PR17c was confirmed by bimolecular fluorescence complementation analyses. Down-regulation and over-expression of PR17c in epidermal cells of barley confirmed that this protein is important for penetration resistance against the powdery mildew fungus. In line with this, PR17c was found to be apoplastic, localizing to the papillae formed in response to this fungus. The CSEP0055 transcript did not start to accumulate until 24 h after inoculation. This suggests that this gene is expressed too late to influence primary penetration events, but rather sustains the fungus at sites of secondary penetration, where PR17c appears to be able to accumulate., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

The generation of intracellular microbe-associated molecular pattern (MAMP)-triggered Ca²⁺ transients was recently demonstrated to involve ionotropic Glutamate Receptor (iGluR)-like channels in Arabidopsis and tobacco. Here we elaborate on our previous findings and refine our insights in the putative agonist binding profile and potential mode of desensitization of MAMP-activated plant iGluRs. Based on results from pharmacological inhibition and desensitization experiments, we propose that plant iGluR complexes responsible for the MAMP-triggered Ca²⁺ signature have a binding profile that combines the specificities of mammalian NMDA-and non-NMDA types of iGluRs, possibly reflecting the evolutionary history of plant and animal iGluRs. We further hypothesize that, analogous to the mammalian NMDA-NR1 receptor, desensitization of plant iGluR-like channels might involve binding of the ubiquitous Ca²⁺ sensor calmodulin to a cytoplasmic C-terminal domain.

Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes plant immunity. Despite its relevance, the molecular components regulating FLS2-mediated signaling remain largely unknown. We show that plasma membrane ARABIDOPSIS-AUTOINHIBITED Ca(2+)-ATPase (ACA8) forms a complex with FLS2 in planta. ACA8 and its closest homolog ACA10 are required for limiting the growth of virulent bacteria. One of the earliest flg22 responses is the transient increase of cytosolic Ca(2+) ions, which is crucial for many of the well-described downstream responses (e.g. generation of reactive oxygen species and the transcriptional activation of defense-associated genes). Mutant aca8 aca10 plants show decreased flg22-induced Ca(2+) and reactive oxygen species bursts and exhibit altered transcriptional reprogramming. In particular, mitogen-activated protein kinase-dependent flg22-induced gene expression is elevated, whereas calcium-dependent protein kinase-dependent flg22-induced gene expression is reduced. These results demonstrate that the fine regulation of Ca(2+) fluxes across the plasma membrane is critical for the coordination of the downstream microbe-associated molecular pattern responses and suggest a mechanistic link between the FLS2 receptor complex and signaling kinases via the secondary messenger Ca(2+). ACA8 also interacts with other RKs such as BRI1 and CLV1 known to regulate plant development, and both aca8 and aca10 mutants show morphological phenotypes, suggesting additional roles for ACA8 and ACA10 in developmental processes. Thus, Ca(2+) ATPases appear to represent general regulatory components of RK-mediated signaling pathways.

Binding of specific microbial epitopes [MAMPs (microbe-associated molecular patterns)] to PRRs (pattern recognition receptors) and subsequent receptor kinase activation are key steps in plant innate immunity. One of the earliest detectable events after MAMP perception is a rapid and transient rise in cytosolic Ca2+ levels. In plants, knowledge about the signalling events leading to Ca2+ influx and on the molecular identity of the channels involved is scarce. We used a transgenic Arabidopsis thaliana line stably expressing the luminescent aequorin Ca2+ biosensor to monitor pharmacological interference with Ca2+ signatures following treatment with the bacterial peptide MAMPs flg22 and elf18, and the fungal carbohydrate MAMP chitin. Using a comprehensive set of compounds known to impede Ca2+-transport processes in plants and animals we found strong evidence for a prominent role of amino acid-controlled Ca2+ fluxes, probably through iGluR (ionotropic glutamate receptor)-like channels. Interference with amino acid-mediated Ca2+ fluxes modulates MAMP-triggered MAPK (mitogen-activated protein kinase) activity and affects MAMP-induced accumulation of defence gene transcripts. We conclude that the initiation of innate immune responses upon flg22, elf18 and chitin recognition involves apoplastic Ca2+ influx via iGluR-like channels.

Plasma-membrane-localized receptor kinases are essential for cell-cell communication and as sensors for the extracellular environment. Receptor function is dependent on their distribution in the membrane and interaction with other proteins that are either membrane-localized, present in the cytoplasm, or in the extracellular space. The organized distribution and mobility of receptor kinases is, therefore, thought to regulate the efficiency of downstream signaling. This chapter describes two methods to study receptor mobility in the plasma membrane. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). Especially, the combination of FRAP and FCS provides a better insight into plasma membrane receptor mobility.

Various fluorophore-based microscopic methods, comprising Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.

Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane-localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gbeta subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

The Arabidopsis thaliana somatic embryogenesis receptor-like kinase (SERK) family consists of five leucine-rich repeat receptor-like kinases (LRR-RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)-mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC-MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C-terminally located residue Ser-562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr-462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP-tagged SERK1 from plant extracts followed by MS/MS identified Ser-303, Thr-337, Thr-459, Thr-462, Thr-463, Thr-468, and Ser-612 or Thr-613 or Tyr-614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser-299 and Thr-462. This suggests both intra- and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser-887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.

The Arabidopsis (Arabidopsis thaliana) SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes belong to a small family of five plant receptor kinases that are involved in at least five different signaling pathways. One member of this family, BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1), also known as SERK3, is the coreceptor of the brassinolide (BR)-perceiving receptor BRI1, a function that is BR dependent and partially redundant with SERK1. BAK1 (SERK3) alone controls plant innate immunity, is also the coreceptor of the flagellin receptor FLS2, and, together with SERK4, can mediate cell death control, all three in a BR-independent fashion. SERK1 and SERK2 are essential for male microsporogenesis, again independent from BR. SERK5 does not appear to have any function under the conditions tested. Here, we show that the different SERK members are only redundant in pairs, whereas higher order mutant combinations only show additive phenotypes. Surprisingly, SERK members that are redundant within one are not redundant in another pathway. We also show that this evolution of functional pairs occurred by a change in protein function and not by differences in spatial expression. We propose that, in plants, closely related receptor kinases have a minimal homo- or heterodimeric configuration to achieve specificity.

Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 [AtSERK3]). Genetic, biochemical, and yeast (Saccharomyces cerevisiae) interaction studies suggested that the BRI1-BAK1 receptor complex initiates BR signaling, but the role of the BAK1 receptor is still not clear. Using transient expression in protoplasts of BRI1 and AtSERK3 fused to cyan and yellow fluorescent green fluorescent protein variants allowed us to localize each receptor independently in vivo. We show that BRI1, but not AtSERK3, homodimerizes in the plasma membrane, whereas BRI1 and AtSERK3 preferentially heterodimerize in the endosomes. Coexpression of BRI1 and AtSERK3 results in a change of the steady state distribution of both receptors because of accelerated endocytosis. Endocytic vesicles contain either BRI1 or AtSERK3 alone or both. We propose that the AtSERK3 protein is involved in changing the equilibrium between plasma membrane-located BRI1 homodimers and endocytosed BRI1-AtSERK3 heterodimers.