Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

Oligogalacturonide application increases resistance to Fusarium head blight in durum wheat.

Fusariosis causes substantial yield losses in the wheat crop worldwide and compromises food safety because of the presence of toxins associated with the fungal disease. Among the current approaches to crop protection, the use of elicitors able to activate natural defense mechanisms in plants is a strategy gaining increasing attention. Several studies indicate that applications of plant cell-wall-derived elicitors, such as oligogalacturonides (OGs) derived from partial degradation of pectin, induce local and systemic resistance against plant pathogens. The aim of this study was to establish the efficacy of OGs in protecting durum wheat (Triticum turgidum subsp. durum), which is characterized by an extreme susceptibility to Fusarium graminearum. To evaluate the functionality of OGs, spikes and seedlings of cv. Svevo were inoculated with OGs, F. graminearum spores, and a co-treatment of both. Results demonstrated that OGs are active elicitors of wheat defenses, triggering typical immune marker genes and determining regulation of fungal genes. Moreover, bioassays on spikes and transcriptomic analyses on seedlings showed that OGs can regulate relevant physiological processes in Svevo with dose-dependent specificity. Thus, the OG sensing system plays an important role in fine tuning immune signaling pathways in durum wheat.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

ROS and RNS production, subcellular localization and signaling triggered by immunogenic danger signals.

Plants continuously monitor the environment to detect changing conditions and to properly respond, avoiding deleterious effects on their fitness and survival. An enormous number of cell-surface and intracellular immune receptors are deployed to perceive danger signals associated with microbial infections. Ligand binding by cognate receptors represents the first essential event in triggering plant immunity and determining the tissue invasion attempt outcome. Reactive oxygen and nitrogen species (ROS/RNS) are secondary messengers rapidly produced in different subcellular localizations upon the perception of immunogenic signals. Danger signal transduction inside the plant cells involves cytoskeletal rearrangements as well as several organelles and interactions between them to activate key immune signaling modules. Such immune processes depend on ROS and RNS accumulation, highlighting their role as key regulators in the execution of the immune cellular programme. In fact, ROS and RNS are synergic and inter-dependent intracellular signals required for decoding danger signals and for the modulation of defense-related responses. Here we summarize the current knowledge on ROS/RNS production, compartmentalization and signaling in plant cells that have perceived immunogenic danger signals.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens.

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

Proteolytic Processing of SERK3/BAK1 Regulates Plant Immunity, Development, and Cell Death.

Plants have evolved many receptor-like kinases (RLKs) to sense extrinsic and intrinsic cues. The signaling pathways mediated by multiple Leucine-rich repeat (LRR) RLK (LRR-RLK) receptors require ligand-induced receptor-coreceptor heterodimerization and transphosphorylation with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR KINASES family LRR-RLKs. Here we reveal an additional layer of regulation of BAK1 via a Ca2+-dependent proteolytic cleavage process that is conserved in Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and Saccharomyces cerevisiae The proteolytic cleavage of BAK1 is intrinsically regulated in response to developmental cues and immune stimulation. The surface-exposed Asp (D287) residue of BAK1 is critical for its proteolytic cleavage and plays an essential role in BAK1-regulated plant immunity, growth hormone brassinosteroid-mediated responses, and cell death containment. BAK1D287A mutation impairs BAK1 phosphorylation on its substrate BOTRYTIS-INDUCED KINASE1 (BIK1), and its plasma membrane localization. Intriguingly, it aggravates BAK1 overexpression-triggered cell death independent of BIK1, suggesting that maintaining homeostasis of BAK1 through a proteolytic process is crucial to control plant growth and immunity. Our data reveal that in addition to layered transphosphorylation in the receptor complexes, the proteolytic cleavage is an important regulatory process for the proper functions of the shared coreceptor BAK1 in diverse cellular signaling pathways.

Danger-associated peptide signaling in Arabidopsis requires clathrin.

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

The crossroads of receptor-mediated signaling and endocytosis in plants.

Plants deploy numerous plasma membrane receptors to sense and rapidly react to environmental changes. Correct localization and adequate protein levels of the cell-surface receptors are critical for signaling activation and modulation of plant development and defense against pathogens. After ligand binding, receptors are internalized for degradation and signaling attenuation. However, one emerging notion is that the ligand-induced endocytosis of receptor complexes is important for the signal duration, amplitude, and specificity. Recently, mutants of major endocytosis players, including clathrin and dynamin, have been shown to display defects in activation of a subset of signal transduction pathways, implying that signaling in plants might not be solely restricted to the plasma membrane. Here, we summarize the up-to-date knowledge of receptor complex endocytosis and its effect on the signaling outcome, in the context of plant development and immunity.

GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding.

Conserved microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) act as danger signals to activate the plant immune response. These molecules are recognized by surface receptors that are referred to as pattern recognition receptors. Oligogalacturonides (OGs), DAMPs released from the plant cell wall homogalacturonan, have also been proposed to act as local signals in the response to wounding. The Arabidopsis Wall-Associated Kinase 1 (WAK1), a receptor of OGs, has been described to form a complex with a cytoplasmic plasma membrane-localized kinase-associated protein phosphatase (KAPP) and a glycine-rich protein (GRP-3) that we find localized mainly in the cell wall and, in a small part, on the plasma membrane. By using Arabidopsis plants overexpressing WAK1, and both grp-3 and kapp null insertional mutant and overexpressing plants, we demonstrate a positive function of WAK1 and a negative function of GRP-3 and KAPP in the OG-triggered expression of defence genes and the production of an oxidative burst. The three proteins also affect the local response to wounding and the basal resistance against the necrotrophic pathogen Botrytis cinerea. GRP-3 and KAPP are likely to function in the phasing out of the plant immune response.

Tomato pomace food waste from different variants as a high antioxidant potential resource.

Pomaces obtained from three San Marzano tomato genotypes including the wild type (WT), Sun Black (SB), and colorless fruit epidermis (CL) were dried at 50 °C and analyzed for nutritional composition, total polyphenol (TPC), flavonoid (TFC) content, polyphenol qualitative profile, total antioxidant capacity (TAC), and antimicrobial activity. Commercial dried tomato powder (CTRP) was included as a control. No differences were detected nutritionally, in TPC and antimicrobial activity, but significant changes were observed for TFC and TAC, underlying variation in the phenolic profile. SB pomace (SBP) had the highest TFC and TAC. LC-HRMS analysis showed a flavonoid-enriched profile in SBP besides the exclusive presence of anthocyanins, with petanin and negretein as the most abundant. Among flavonoids, quercetin-hexose-deoxyhexose-pentose, naringenin, and rutin were the major. Overall, we showed the potential of dried tomato pomace, especially SBP, as an extremely valuable waste product to be transformed into a functional ingredient, reducing the food industry waste.

Oligogalacturonide-auxin antagonism does not require posttranscriptional gene silencing or stabilization of auxin response repressors in Arabidopsis.

α-1-4-Linked oligogalacturonides (OGs) derived from plant cell walls are a class of damage-associated molecular patterns and well-known elicitors of the plant immune response. Early transcript changes induced by OGs largely overlap those induced by flg22, a peptide derived from bacterial flagellin, a well-characterized microbe-associated molecular pattern, although responses diverge over time. OGs also regulate growth and development of plant cells and organs, due to an auxin-antagonistic activity. The molecular basis of this antagonism is still unknown. Here we show that, in Arabidopsis (Arabidopsis thaliana), OGs inhibit adventitious root formation induced by auxin in leaf explants as well as the expression of several auxin-responsive genes. Genetic, biochemical, and pharmacological experiments indicate that inhibition of auxin responses by OGs does not require ethylene, jasmonic acid, and salicylic acid signaling and is independent of RESPIRATORY BURST OXIDASE HOMOLOGUE D-mediated reactive oxygen species production. Free indole-3-acetic acid levels are not noticeably altered by OGs. Notably, OG- as well as flg22-auxin antagonism does not involve any of the following mechanisms: (1) stabilization of auxin-response repressors; (2) decreased levels of auxin receptor transcripts through the action of microRNAs. Our results suggest that OGs and flg22 antagonize auxin responses independently of Aux/Indole-3-Acetic Acid repressor stabilization and of posttranscriptional gene silencing.

Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

Biotechnological Resources to Increase Disease-Resistance by Improving Plant Immunity: A Sustainable Approach to Save Cereal Crop Production.

Plant diseases are globally causing substantial losses in staple crop production, undermining the urgent goal of a 60% increase needed to meet the food demand, a task made more challenging by the climate changes. Main consequences concern the reduction of food amount and quality. Crop diseases also compromise food safety due to the presence of pesticides and/or toxins. Nowadays, biotechnology represents our best resource both for protecting crop yield and for a science-based increased sustainability in agriculture. Over the last decades, agricultural biotechnologies have made important progress based on the diffusion of new, fast and efficient technologies, offering a broad spectrum of options for understanding plant molecular mechanisms and breeding. This knowledge is accelerating the identification of key resistance traits to be rapidly and efficiently transferred and applied in crop breeding programs. This review gathers examples of how disease resistance may be implemented in cereals by exploiting a combination of basic research derived knowledge with fast and precise genetic engineering techniques. Priming and/or boosting the immune system in crops represent a sustainable, rapid and effective way to save part of the global harvest currently lost to diseases and to prevent food contamination.

Corrigendum: Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome.

[This corrects the article DOI: 10.3389/fpls.2017.02234.].

Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome.

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional ß-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance. Highlights: The loss of ANP function affects cell wall composition and leads to typical cell wall damage-induced phenotypes, such as ectopic lignification and jasmonic acid accumulation.

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea.

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2'-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography-mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.

Wounding in the plant tissue: the defense of a dangerous passage.

Plants are continuously exposed to agents such as herbivores and environmental mechanical stresses that cause wounding and open the way to the invasion by microbial pathogens. Wounding provides nutrients to pathogens and facilitates their entry into the tissue and subsequent infection. Plants have evolved constitutive and induced defense mechanisms to properly respond to wounding and prevent infection. The constitutive defenses are represented by physical barriers, i.e., the presence of cuticle or lignin, or by metabolites that act as toxins or deterrents for herbivores. Plants are also able to sense the injured tissue as an altered self and induce responses similar to those activated by pathogen infection. Endogenous molecules released from wounded tissue may act as Damage-Associated Molecular Patterns (DAMPs) that activate the plant innate immunity. Wound-induced responses are both rapid, such as the oxidative burst and the expression of defense-related genes, and late, such as the callose deposition, the accumulation of proteinase inhibitors and of hydrolytic enzymes (i.e., chitinases and gluganases). Typical examples of DAMPs involved in the response to wounding are the peptide systemin, and the oligogalacturonides, which are oligosaccharides released from the pectic component of the cell wall. Responses to wounding take place both at the site of damage (local response) and systemically (systemic response) and are mediated by hormones such as jasmonic acid, ethylene, salicylic acid, and abscisic acid.

Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development.

Oligogalacturonides (OGs) are oligomers of alpha-1,4-linked galacturonosyl residues released from plant cell walls upon partial degradation of homogalacturonan. OGs are able to elicit defense responses, including accumulation of reactive oxygen species and pathogenesis-related proteins, and protect plants against pathogen infections. Recent studies demonstrated that OGs are perceived by wall-associated kinases and share signaling components with microbe-associated molecular patterns. For this reason OGs are now considered true damage-associated molecular patterns that activate the plant innate immunity and may also be involved in the activation of responses to mechanical wounding. Furthermore, OGs appear to modulate developmental processes, likely through their ability to antagonize auxin responses. Here we review our current knowledge on the role and mode of action of this class of oligosaccharides in plant defense and development.