Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Prunus dulcis response to novel defense elicitor peptides and control of Xylella fastidiosa infections.

KEY MESSAGE: New defense elicitor peptides have been identified which control Xylella fastidiosa infections in almond. Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), threatening the agricultural economy of relevant Mediterranean crops such as almond (Prunus dulcis). Plant defense elicitor peptides would be promising to manage diseases such as almond leaf scorch, but their effect on the host has not been fully studied. In this work, the response of almond plants to the defense elicitor peptide flg22-NH2 was studied in depth using RNA-seq, confirming the activation of the salicylic acid and abscisic acid pathways. Marker genes related to the response triggered by flg22-NH2 were used to study the effect of the application strategy of the peptide on almond plants and to depict its time course. The application of flg22-NH2 by endotherapy triggered the highest number of upregulated genes, especially at 6 h after the treatment. A library of peptides that includes BP100-flg15, HpaG23, FV7, RIJK2, PIP-1, Pep13, BP16-Pep13, flg15-BP100 and BP16 triggered a stronger defense response in almond plants than flg22-NH2. The best candidate, FV7, when applied by endotherapy on almond plants inoculated with X. fastidiosa, significantly reduced levels of the pathogen and decreased disease symptoms. Therefore, these novel plant defense elicitors are suitable candidates to manage diseases caused by X. fastidiosa, in particular almond leaf scorch.

Atopic Dermatitis: Disease Background and Risk Factors.

Multiple risk factors have been associated with the development of atopic dermatitis (AD). Recent advances in understanding the role of genetics in this disease have been made, with discovery of the filaggrin (FLG) gene as the most notable so far. In addition to FLG gene mutations as a risk factor for AD, a positive family history of atopic or allergic disease in either parent has been shown to confer a greater risk of developing AD. Atopic dermatitis usually presents early in life and is thought to represent the initial step in the "atopic march," which is characterized by the development of other atopic diseases later in life such as asthma, allergic rhinitis, and/or rhinoconjunctivitis, food allergies, and hay fever. Other comorbid diseases that have been associated with AD include increase risk of viral and bacterial skin infections, neuropsychiatric diseases such as attention-deficit hyperactivity disorders (ADHD), and autistic spectrum disorder (ASD). Patients with AD have also been found to have worse sleep quality overall compared to patients without AD. In this chapter, we will discuss the risk factors associated with development of atopic dermatitis as well as the most commonly reported comorbidities in patients with this disease.

Specific modulation of the root immune system by a community of commensal bacteria.

Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.

A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.

BACKGROUND: The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. METHODS AND RESULTS: For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. CONCLUSIONS: Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders.

The LeEIX Locus Determines Pathogen Resistance in Tomato.

The mechanisms underlying the ability of plants to differentiate between pathogens and commensals in their environment are currently unresolved. It has been suggested that spatiotemporal regulation of pattern-recognition receptor (PRR) content could be one of the components providing plants with the ability to distinguish between pathogens and nonpathogenic microbes. The LeEIX PRRs recognize xylanases derived from beneficial or commensal plant colonizers of Trichoderma species, including the xylanase known as EIX. Here, we investigated possible general roles of PRRs from the LeEIX locus in immunity and pathogen resistance in tomato. Mutating the inhibitory PRR LeEIX1, or overexpressing the activating PRR LeEIX2, resulted in resistance to a wide range of pathogens and increased basal and elicited immunity. LeEIX1 knockout caused increases in the expression level of several tested PRRs, including FLS2, as well as bacterial pathogen resistance coupled with an increase in flg22-mediated immunity. The wild tomato relative Solanum pennellii contains inactive LeEIX PRR variants. S. pennellii does not respond to elicitation with the LeEIX PRR ligand EIX. Given that EIX is derived from a mostly nonpathogenic microbe, the connection of its PRRs to disease resistance has not previously been investigated directly. Here, we observed that compared with S. lycopersicum cultivar M82, S. pennellii was more sensitive to several fungal and bacterial pathogens. Our results suggest that the LeEIX locus might determine resistance to fungal necrotrophs, whereas the resistance to biotrophs is effected in combination with a gene/quantitative trait locus not within the LeEIX locus.

Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana.

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.

Filaggrin loss-of-function mutations are associated with persistence of egg and milk allergy.

BACKGROUND: A genetic defect in the epidermal barrier protein filaggrin (FLG) plays a major role in the etiology of eczema and associated allergic airways diseases. However, it is still controversial to what extend loss-of-function (LOF) mutations in FLG contribute to the development and persistence of food allergies. OBJECTIVES: This study tested association of FLG LOF mutations with allergic reactions to diverse foods and investigated their potential effect on the persistence of early food allergies. METHODS: This study recruited 890 children with challenge-proven food allergy for the German Genetics of Food Allergy Study (GOFA). Longitudinal data were available for 684 children. All children were clinically characterized, including their allergic responses to specific foods, and genotyped for the 4 most common LOF mutations in FLG; R501X, 2282del4, R2447X, and S3247X. Associations between FLG mutations and food allergies were analyzed by logistic regression using the German Multicenter Allergy Study cohort as the control population. RESULTS: FLG mutations were associated with allergies to diverse foods including hen's egg (HE), cow's milk (CM), peanut, hazelnut, fish, soy, cashew, walnut, and sesame with similar risk estimates. Effects remained significant after adjusting for the eczema status. Interestingly, FLG mutations increased the risk of a persistent course of HE and CM allergy. CONCLUSIONS: Using the gold standard for food allergy diagnosis, this study demonstrates that FLG LOF mutations confer a risk of any food allergy independent of eczema. These mutations predispose to the persistence of HE and CM allergy and should be considered in the assessment of tolerance development.

Root-specific CLE3 expression is required for WRKY33 activation in Arabidopsis shoots.

KEY MESSAGE: This study focused on the role of CLE1-7 peptides as defense mediators, and showed that root-expressed CLE3 functions as a systemic signal to regulate defense-related gene expression in shoots. In the natural environment, plants employ diverse signaling molecules including peptides to defend themselves against various pathogen attacks. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes (CLE1-7) respond to biotic stimuli. CLE3 showed significant up-regulation upon treatment with flg22, Pep2, and salicylic acid (SA). Quantitative real-time PCR (qRT-PCR) analysis revealed that CLE3 expression is regulated by the NON-EXPRESSOR OF PR GENES1 (NPR1)-dependent SA signaling and flg22-FLAGELLIN-SENSITIVE 2 (FLS2) signaling pathways. We demonstrated that SA-induced up-regulation of CLE3 in roots was required for activation of WRKY33, a gene involved in the regulation of systemic acquired resistance (SAR), in shoots, suggesting that CLE3 functions as a root-derived signal that regulates the expression of defense-related genes in shoots. Microarray analysis of transgenic Arabidopsis lines overexpressing CLE3 under the control of a ß-estradiol-inducible promoter revealed that root-confined CLE3 overexpression affected gene expression in both roots and shoots. Comparison of CLE2- and CLE3-induced genes indicated that CLE2 and CLE3 peptides target a few common but largely distinct downstream genes. These results suggest that root-derived CLE3 is involved in the regulation of systemic rather than local immune responses. Our study also sheds light on the potential role of CLE peptides in long-distance regulation of plant immunity.

The cell-type specific role of Arabidopsis bZIP59 transcription factor in plant immunity.

Stomatal movement participates in plant immunity by directly affecting the invasion of bacteria, but the genes that regulate stomatal immunity have not been well identified. Here, we characterised the function of the bZIP59 transcription factor from Arabidopsis thaliana, which is constitutively expressed in guard cells. The bzip59 mutant is partially impaired in stomatal closure induced by Pseudomonas syringae pv. tomato strain (Pst) DC3000 and is more susceptible to Pst DC3000 infection. By contrast, the line overexpressing bZIP59 enhances resistance to Pst DC3000 infection. Furthermore, the bzip59 mutant is also partially impaired in stomatal closure induced by flagellin flg22 derived from Pst DC3000, and epistasis analysis revealed that bZIP59 acts upstream of reactive oxygen species (ROS) and nitric oxide (NO) and downstream of salicylic acid signalling in flg22-induced stomatal closure. In addition, the bzip59 mutant showed resistance and sensitivity to Sclerotinia sclerotiorum and Tobacco mosaic virus that do not invade through stomata, respectively. Collectively, our results demonstrate that bZIP59 plays an important role in the stomatal immunity and reveal that the same transcription factor can positively and negatively regulate disease resistance against different pathogens.

Associations between child filaggrin mutations and maternal diet with the development of allergic diseases in children.

BACKGROUND: Filaggrin (FLG) loss-of-function mutations in children and maternal diet in pregnancy have been implicated in child allergy outcomes. This paper studies the questions: "do FLG mutations modify the effect of maternal diet on the odds of development of allergic diseases?" and "which factor leads to the highest rate of diagnosis allergic diseases over time, maternal diet, or FLG mutations?". METHODS: Exact logistic regressions studied effect modification. Cox proportional hazard models compared the rate of allergic disease development in three groups (N = 624): (1) children with FLG mutation, (2) children without FLG mutation whose mothers did not eat an allergy preventive diet, and (3) children without FLG mutation whose mothers ate an allergy preventive diet. Maternal diet was classified using a validated index. RESULTS: Cox models showed the development of atopic dermatitis, asthma, and wheeze was significantly higher for children in group 1 versus 3 (HR = 2.40 [1.32, 4.37], HR = 2.29 [1.05, 4.97], and HR 2.10 [1.004, 4.38], respectively), but not significantly higher for children in group 1 versus 2 (HR = 1.30 [0.74, 2.29], HR = 1.27 [0.61, 2.63], and HR = 1.29 [0.65, 2.58], respectively). Development of allergic rhinitis was significantly higher for group 1 versus 2 and 3 (1 vs. 2: HR = 2.29 [1.10, 4.76]; 1 vs. 3: HR = 3.21 [1.46, 7.08]). There was no significant effect modification for any outcome. CONCLUSION: Children with FLG mutation had similar risk of atopic dermatitis, asthma, and wheeze as children without an FLG mutation whose mothers did not eat an allergy preventive diet during pregnancy. Child FLG mutation did not modify the effect of maternal diet. The results suggest that maternal diet in pregnancy, a modifiable risk factor, could be a target for preventive interventions.

Distribution of flagellin CD2-1, flg22, and flgII-28 recognition systems in plant species and regulation of plant immune responses through these recognition systems.

The first layer of active plant immunity relies upon the recognition of pathogen-associated molecular patterns (PAMPs), and the induction of PTI. Flagellin is the major protein component of the bacterial flagellum. Flagellin-derived peptide fragments such as CD2-1, flg22, and flgII-28 function as PAMPs in most higher plants. To determine the distribution of CD2-1, flg22, and flgII-28 recognition systems within plant species, the inducibility of PTI by CD2-1, flg22, and flgII-28 in 8 plant species, including monocotyledonous and dicotyledonous plants, was investigated. CD2-1 caused PTI responses in Oryza sativa, Brachypodium distachyon, and Asparagus persicus; flg22 caused PTI responses in Phyllostachys nigra, A. persicus, Arabidopsis thaliana, Nicotiana tabacum, Solanum lycopersicum, and Lotus japonicus; and flgII-28 caused PTI responses only in S. lycopersicum. Furthermore, quantitative analysis of FLS2 receptor revealed that the responsiveness of flg22 in plants was dependent on the expression level of the receptor.

Nickel penetration into stratum corneum in FLG null carriers-A human experimental study.

BACKGROUND: The filaggrin gene (FLG) plays a role in skin diseases, with the skin barrier function being impaired in FLG null carriers. The role of FLG status in relation to nickel penetration into the skin remains unclear. OBJECTIVES: To elucidate the association between FLG status and nickel penetration into stratum corneum (SC) in individuals without self-reported history of nickel allergy. METHODS: Forty participants (23 FLG wt and 17 FLG null) were exposed to a nickel solution (80 µg/cm2 ) which was applied onto 2 × 2 cm on their left forearm. After 4 h, the area was tape-stripped with 10 consecutive tapes. Nickel in each tape was quantified using inductively coupled plasma mass spectrometry. RESULTS: The average recovered nickel dose was 35%-48%. A tendency towards lower recovery was seen in FLG null carriers compared to FLG wt carriers, and lower recovery in those with history of skin and/or respiratory symptoms compared to those without such history. This was however not statistically significant. CONCLUSION: FLG null carriers had less nickel recovered by tape strips compared with FLG wt carriers and, compared with individuals without a history of skin and/or respiratory symptoms, indicating higher nickel penetration into SC for FLG null carriers, but further studies are needed.

Current insights into the genetics of food allergy.

Food allergy (FA), a growing public health burden in the United States, and familial aggregation studies support strong roles for both genes and environment in FA risk. Deepening our understanding of the molecular and cellular mechanisms driving FAs is paramount to improving its prevention, diagnosis, and clinical management. In this review, we document lessons learned from the genetics of FA that have aided our understanding of these mechanisms. Although current genetic association studies suffer from low power, heterogeneity in definition of FA, and difficulty in our ability to truly disentangle FA from food sensitization (FS) and general atopy genetics, they reveal a set of genetic loci, genes, and variants that continue to implicate the importance of barrier and immune function genes across the atopic march, and FA in particular. The largest reported effects on FA are from MALT1 (odds ratio, 10.99), FLG (average odds ratio, ∼2.9), and HLA (average odds ratio, ∼2.03). The biggest challenge in the field of FA genetics is to elucidate the specific mechanism of action on FA risk and pathogenesis for these loci, and integrative approaches including genetics/genomics with transcriptomics, proteomics, and metabolomics will be critical next steps to translating these genetic insights into practice.

Screening and Research on Skin Barrier Damage Protective Efficacy of Different Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) may prevent skin barrier damage, although their protective mechanisms and active monomeric constituents remain unclear. Here, three MELs were extracted from Candida antarctica cultures containing fermented olive oil then purified using silica gel-based column chromatography and semipreparative HPLC. All three compounds (MEL-A, MEL-B, MEL-C) were well separated and stable, and reliable materials were used for NMR and HRESIMS chemical structure determinations and for assessing MELs' protective effects against skin damage. Notably, MEL-B and MEL-C effectively protected HaCaT cells from UVB-induced damage by upregulating the contents of filaggrin (FLG) and transglutaminase-1 (TGM1), as determined via ELISA. Moreover, MEL-B treatment (20 µg/mL) of UVB-irradiated HaCaT cells led to the upregulation of both the expression of mRNA genes and the key proteins FLG, LOR, and TGM1, which are known to be decreased in damaged skin cells. Additionally, histopathological analysis results revealed a markedly reduced intracellular vacuolation and cell damage, reflecting improved skin function after MEL-B treatment. Furthermore, immunofluorescence results revealed that MEL-B protected EpiKutis® three-dimensional cultured human skin cells from sodium dodecyl sulfate-induced damage by up-regulating FLG, LOR, and TGM1 expression. Accordingly, MELs' protection against skin barrier damage depended on MEL-B monomeric constituent activities, thus highlighting their promise as beneficial ingredients for use in skin-care products.

The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure.

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high-throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non-redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image-based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.

Proteobacteria Contain Diverse flg22 Epitopes That Elicit Varying Immune Responses in Arabidopsis thaliana.

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and ß-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. The flg22 peptides of γ- and ß-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and ß-Proteobacteria, a monophyletic group containing many common phytopathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca2+ and H+ reveals new insights into ion signaling in plants.

Whereas the role of calcium ions (Ca2+ ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+ - and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca2+ -dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+ . In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+ -increases and cytosolic acidification by c. 2 min, suggesting a Ca2+ /pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+ - and pH-responses. We propose close interrelation in Ca2+ - and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.

MAP4K4 associates with BIK1 to regulate plant innate immunity.

To perceive pathogens, plants employ pattern recognition receptor (PRR) complexes, which then transmit these signals via the receptor-like cytoplasmic kinase BIK1 to induce defense responses. How BIK1 activity and stability are controlled is still not completely understood. Here, we show that the Hippo/STE20 homolog MAP4K4 regulates BIK1-mediated immune responses. MAP4K4 associates and phosphorylates BIK1 at Ser233, Ser236, and Thr242 to ensure BIK1 stability and activity. Furthermore, MAP4K4 phosphorylates PP2C38 at Ser77 to enable flg22-induced BIK1 activation. Our results uncover that a Hippo/STE20 homolog, MAP4K4, maintains the homeostasis of the central immune component BIK1.