AHL-Priming Protein 1 mediates N-3-oxo-tetradecanoyl-homoserine lactone priming in Arabidopsis.

BACKGROUND: N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) is one of the N-acyl homoserine lactones (AHL) that mediate quorum sensing in Gram-negative bacteria. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short-chain AHL are easily taken up by plants and transported over long distances. They promote root elongation and growth. Plants typically do not uptake hydrophobic long sidechain AHL such as oxo-C14-HSL, although they prime plants for enhanced resistance to biotic and abiotic stress. Many studies have focused on priming effects of oxo-C14-HSL for enhanced plant resistance to stress. However, specific plant factors mediating oxo-C14-HSL responses in plants remain unexplored. Here, we identify the Arabidopsis protein ALI1 as a mediator of oxo-C14-HSL-induced priming in plants. RESULTS: We compared oxo-C14-HSL-induced priming between wild-type Arabidopsis Col-0 and an oxo-C14-HSL insensitive mutant ali1. The function of the candidate protein ALI1 was assessed through biochemical, genetic, and physiological approaches to investigate if the loss of the ALI1 gene resulted in subsequent loss of AHL priming. Through different assays, including MAP kinase activity assay, gene expression and transcriptome analysis, and pathogenicity assays, we revealed a loss of AHL priming in ali1. This phenomenon was reverted by the reintroduction of ALI1 into ali1. We also investigated the interaction between ALI1 protein and oxo-C14-HSL using biochemical and biophysical assays. Although biophysical assays did not reveal an interaction between oxo-C14-HSL and ALI1, a pull-down assay and an indirect method employing biosensor E. coli LuxCDABE support such interaction. We expressed fluorescently tagged ALI1 in tobacco leaves to assess the localization of ALI1 and demonstrate that ALI1 colocalizes with the plasma membrane, tonoplast, and endoplasmic reticulum. CONCLUSIONS: These results suggest that the candidate protein ALI1 is indispensable for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis and that the ALI1 protein may interact with oxo-C14-HSL. Furthermore, ALI1 protein is localized in the cell periphery. Our findings advance the understanding of interactions between plants and bacteria and provide an avenue to explore desired outcomes such as enhanced stress resistance, which is useful for sustainable crop protection.

Biocontrol of Plant Diseases Using Glycyrrhiza glabra Leaf Extract.

The growing concern regarding the potential risks of pesticides and their impact on nontarget organisms stimulates the development and application of alternative, environmentally friendly products. It seems necessary to develop alternatives for conventional products and for those already widely used in organic agriculture, e.g., copper. Very importantly, such alternative products should not limit the productivity and profitability of agriculture. In this study, we examined the efficacy of licorice (Glycyrrhiza glabra) leaf extract as such an alternative. We tested its impact on the virulence of Pseudomonas syringae toward the model plant Arabidopsis thaliana and the crop plant tomato (Solanum lycopersicum) as well as of Clavibacter michiganensis, Xanthomonas campestris, and Phytophthora infestans toward tomato, at multiple levels. We demonstrate that licorice leaf extract acts as a direct fungicide and bactericide. Moreover, it acts against a metalaxyl-resistant P. infestans strain. In addition, the extract from licorice leaves influences the plant immune system, modulating the plant responses to the challenge with pathogen(s); this involves both salicylic acid and ethylene-based responses. Our results show that in addition to the well-known use of licorice root extract in medicine, the leaf extract can be an effective alternative in organic and integrated farming, contributing to copper reduction and resistance management.[Formula: see text] Copyright © 2022 The Author(s). This is an open-access article distributed under the CC BY 4.0 International license.

Detection of Bacterial Quorum Sensing Molecules.

Bacterial cells use the quorum sensing system to communicate with each other. The gram-negative species very often use N-acyl homoserine lactones for this purpose. One of the easiest ways to detect these molecules is the use of particular reporter strains, which possess different kinds of reporter genes under the control of AHL-responsive promoters. Here we present some of the possibilities available today, even for not specialized researchers.

N-Acyl-Homoserine Lactone Primes Plants for Cell Wall Reinforcement and Induces Resistance to Bacterial Pathogens via the Salicylic Acid/Oxylipin Pathway.

The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection.

RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-ß-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.

Modified N-acyl-homoserine lactones as chemical probes for the elucidation of plant-microbe interactions.

Gram-negative bacteria often use N-acyl-homoserine lactones (AHLs) as signal molecules to monitor their local population densities and to regulate gene-expression in a process called "Quorum Sensing" (QS). This cell-to-cell communication allows bacteria to adapt to environmental changes and to behave as multicellular communities. QS plays a key role in both bacterial virulence towards the host and symbiotic interactions with other organisms. Plants also perceive AHLs and respond to them with changes in gene expression or modifications in development. Herein, we report the synthesis of new AHL-derivatives for the investigation and identification of AHL-interacting proteins. We show that our new compounds are still recognised by different bacteria and that a novel biotin-tagged-AHL derivative interacts with a bacterial AHL receptor.

Homoserine lactones influence the reaction of plants to rhizobia.

Bacterial quorum sensing molecules not only grant the communication within bacterial communities, but also influence eukaryotic hosts. N-acyl-homoserine lactones (AHLs) produced by pathogenic or beneficial bacteria were shown to induce diverse reactions in animals and plants. In plants, the reaction to AHLs depends on the length of the lipid side chain. Here we investigated the impact of two bacteria on Arabidopsis thaliana, which usually enter a close symbiosis with plants from the Fabaceae (legumes) family and produce a long-chain AHL (Sinorhizobium meliloti) or a short-chain AHL (Rhizobium etli). We demonstrate that, similarly to the reaction to pure AHL molecules, the impact, which the inoculation with rhizosphere bacteria has on plants, depends on the type of the produced AHL. The inoculation with oxo-C14-HSL-producing S. meliloti strains enhanced plant resistance towards pathogenic bacteria, whereas the inoculation with an AttM lactonase-expressing S. meliloti strain did not. Inoculation with the oxo-C8-HSL-producing R. etli had no impact on the resistance, which is in agreement with our previous hypothesis. In addition, plants seem to influence the availability of AHLs in the rhizosphere. Taken together, this report provides new insights in the role of N-acyl-homoserine lactones in the inter-kingdom communication at the root surface.

DSCAM contributes to dendrite arborization and spine formation in the developing cerebral cortex.

Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental processes including axon guidance, dendrite arborization, and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM(del17)) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization, volume, and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness, but these reductions could not be attributed to changes in neuron production or cell death. DSCAM(del17) mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM(del17) dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42, mutant mice displayed a decrease in the percentage of large, stable spines and an increase in the percentage of small, immature spines. Together, our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development.

Arabidopsis growth and defense are modulated by bacterial quorum sensing molecules.

N-acyl-homoserine lactones (AHLs) play an important role in the communication within the rhizosphere; they serve as a chemical base for interactions within and between different species of Gram-negative bacteria. Not only bacteria, also plants perceive and react to AHLs with diverse responses. Here we describe a negative correlation between the length of AHLs' lipid chains and the observed growth promotion in Arabidopsis thaliana. Moreover, we speculate on a positive correlation between the reinforcement of defense mechanisms and the length of the lipid moieties. Observation presented here may be of great importance for understanding of the complex interplay between plants and their environment, as well as for agronomic applications.

N-acyl-homoserine lactone confers resistance toward biotrophic and hemibiotrophic pathogens via altered activation of AtMPK6.

Pathogenic and symbiotic bacteria rely on quorum sensing to coordinate the collective behavior during the interactions with their eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signals in such communication. Here we show that plants have evolved means to perceive AHLs and that the length of acyl moiety and the functional group at the γ position specify the plant's response. Root treatment with the N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) reinforced the systemic resistance to the obligate biotrophic fungi Golovinomyces orontii in Arabidopsis (Arabidopsis thaliana) and Blumeria graminis f. sp. hordei in barley (Hordeum vulgare) plants. In addition, oxo-C14-HSL-treated Arabidopsis plants were more resistant toward the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000. Oxo-C14-HSL promoted a stronger activation of mitogen-activated protein kinases AtMPK3 and AtMPK6 when challenged with flg22, followed by a higher expression of the defense-related transcription factors WRKY22 and WRKY29, as well as the PATHOGENESIS-RELATED1 gene. In contrast to wild-type Arabidopsis and mpk3 mutant, the mpk6 mutant is compromised in the AHL effect, suggesting that AtMPK6 is required for AHL-induced resistance. Results of this study show that AHLs commonly produced in the rhizosphere are crucial factors in plant pathology and could be an agronomic issue whose full impact has to be elucidated in future analyses.

Presenilin-dependent receptor processing is required for axon guidance.

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-ß precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.

Systemic resistance in Arabidopsis conferred by the mycorrhizal fungus Piriformospora indica requires jasmonic acid signaling and the cytoplasmic function of NPR1.

We analyzed the requirement of specific defense pathways for powdery mildew (Golovinomyces orontii) resistance induced by the basidiomycete Piriformospora indica in Arabidopsis. Piriformospora indica root colonization reduced G. orontii conidia in wild-type (Col-0), npr1-3 (nonexpressor of PR genes 1-3) and NahG plants, but not in the npr1-1 null mutant. Therefore, cytoplasmic but not nuclear localization of NPR1 is required for P. indica-induced resistance. Two jasmonate signaling mutants were non-responsive to P. indica, and jasmonic acid-responsive vegetative storage protein expression was primed and thus elevated in response to powdery mildew, suggesting that P. indica confers resistance reminiscent of induced systemic resistance (ISR).

DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.

During nervous system development, spinal commissural axons project toward and across the ventral midline. They are guided in part by netrin-1, made by midline cells, which attracts the axons by activating the netrin receptor DCC. However, previous studies suggest that additional receptor components are required. Here, we report that the Down's syndrome Cell Adhesion Molecule (DSCAM), a candidate gene implicated in the mental retardation phenotype of Down's syndrome, is expressed on spinal commissural axons, binds netrin-1, and is necessary for commissural axons to grow toward and across the midline. DSCAM and DCC can each mediate a turning response of these neurons to netrin-1. Similarly, Xenopus spinal neurons exogenously expressing DSCAM can be attracted by netrin-1 independently of DCC. These results show that DSCAM is a receptor that can mediate turning responses to netrin-1 and support a key role for netrin/DSCAM signaling in commissural axon guidance in vertebrates.

Netrin signaling leading to directed growth cone steering.

In the developing nervous system, nerve cells and axons respond to various attractive and repulsive guidance cues while traveling to their final destination. Netrins are bifunctional guidance cues that attract several classes of axons but repel others. The response of an axon to netrins is dictated by the composition of netrin receptors on the cell surface and the internal state of the growth cone. Recent analyses have identified several signal transduction pathways that contribute to netrin-mediated guidance. A model emerges in which tyrosine phosphorylation, phosphatidylinositol signaling and regulation by Rho GTPases act in concert to trigger extension of axons and turning of growth cones in response to Netrin1.

Expression of barley BAX Inhibitor-1 in carrots confers resistance to Botrytis cinerea.

SUMMARY BAX Inhibitor-1 (BI-1) is a protein that controls heterologous BAX-induced cell death, the hypersensitive reaction and abiotic stress-induced cell death in plants. When over-expressed in epidermal cells of barley, barley BI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. When we expressed barley BI-1 in carrot susceptible to the necrotrophic fungus Botrytis cinerea, we obtained BI-1-mediated resistance to fungus-induced leaf cell death and less fungal spreading on the leaves. Barley BI-1 also mediated resistance to Chalara elegans in carrot roots. The results support the idea that cell death inhibition is an applicable approach to control cell-death-inducing pathogens in crop plants.

Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis.

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. Here, by using a three-dimensional culture system, we provide evidence for a novel role for Netrins, best known as axonal guidance molecules, in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds.

The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.

Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation.

Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.

Regulation of cortical dendrite development by Slit-Robo interactions.

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.