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Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana . The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N -glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man ₃XylFucGlcNAc ₄, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The K d values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes.

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

Most plants have the ability to establish a symbiosis with arbuscular mycorrhizal (AM) fungi, which allows better plant nutrition. A plant signaling pathway, called the common symbiosis signaling pathway (CSSP), is essential for the establishment of both AM and root nodule symbioses. The CSSP is activated by microbial signals. Plant receptor(s) for AM fungal signals required for the activation of the CSSP and initial fungal penetration are currently unknown. We set up conditions to use virus‐induced gene silencing (VIGS) in Solanum lycopersicum to study the genes potentially involved in AM. We show that the lysin motif receptor‐like kinase SlLYK10, whose orthologs in legumes are essential for nodulation, but not for AM, and SlCCaMK, a component of the CSSP, are required for penetration of the AM fungus Rhizophagus irregularis into the roots of young tomato plants. Our results support the hypothesis that the SILYK10 ancestral gene originally played a role in AM and underwent duplication and neofunctionalization for a role in nodulation in legumes. Moreover, we conclude that VIGS is an efficient method for fast screening of genes playing major roles in AM.

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

Over the past few decades, we have witnessed a surge around the world in the emergence of antibiotic-resistant bacteria. This global health threat arose mainly due to the overuse and misuse of antibiotics as well as a relative lack of new drug classes in development pipelines. Innovative antibacterial therapeutics and strategies are, therefore, in grave need. For the last twenty years, antimicrobial enzymes encoded by bacteriophages, viruses that can lyse and kill bacteria, have gained tremendous interest. There are two classes of these phage-derived enzymes, referred to also as enzybiotics: peptidoglycan hydrolases (lysins), which degrade the bacterial peptidoglycan layer, and polysaccharide depolymerases, which target extracellular or surface polysaccharides, i.e., bacterial capsules, slime layers, biofilm matrix, or lipopolysaccharides. Their features include distinctive modes of action, high efficiency, pathogen specificity, diversity in structure and activity, low possibility of bacterial resistance development, and no observed cross-resistance with currently used antibiotics. Additionally, and unlike antibiotics, enzybiotics can target metabolically inactive persister cells. These phage-derived enzymes have been tested in various animal models to combat both Gram-positive and Gram-negative bacteria, and in recent years peptidoglycan hydrolases have entered clinical trials. Here, we review the testing and clinical use of these enzymes.

Globally, Food security main threaten by abiotic stress like salinity and levels amongst the majority serious environmental stressors which reduce crop yield mass production. Biochar application has received much attention in agricultural practices as it enhances crop quality and production. The present study was carried out to analyze the role of lysine zinc and biochar on growth enhancement of wheat ( Triticum aestivum L. cv. PU-2011) under saline stress (EC 7.17 dSm -1 ). Seeds were sown in pots containing saline soil with and without 2% biochar, and foliar application of Zn-lysine (0, 1.0, and 2.0 mM) was made at different time intervals during plant growth. A combined application of biochar and Zn-lysine 2.0 mM highly improved the physiological attributes such as chlorophyll a (37%), chlorophyll b (60%), total chlorophyll (37%), carotenoids (16%), photosynthesis rate ( Pn ) 45%, stomatal conductance ( gs ) 53%, transpiration rate ( Tr ) 56%, and water use efficiency ( WUE ) 55%. The levels of malondialdehyde (MDA) 38%, hydrogen peroxide (H 2 O 2 ) 62%, and electrolyte leakage (EL) 48% were decreased with the combined application of biochar and Zn-lysine 2.0 mM as compared with other treatments. The activities of catalase (CAT) 67%, superoxide dismutase (SOD) 70%, and ascorbate peroxidase (APX) 61% as well as catalase (CAT) 67% were regulated with the combined biochar and Zn-lysine 2.0 mM treatment. Similarly, the combined application of biochar and zinc-lysine (2.0 mM) enhanced the growth and yield attributes such as shoot length (79%), root fresh weight (62%), shoot fresh weight (36%), root dry weight (86%), shoot dry weight (39%), grain weight (57%), and spike length (43%) as compared with untreated control. The concentrations of sodium (Na) decreased whereas potassium (K), iron (Fe), and zinc (Zn) concentrations were enhanced in plants with the combined application of Zn-lysine and biochar. Overall, results showed that the combined application of Zn-lysine (2.0 mM) and biochar significantly inhibited the negative effect of salinity and improved the growth and physiological performance of wheat plants. The combined use of Zn-lysine and biochar might be a practical solution to tackle salt stress in plants, but field studies by growing various crops under varied environmental conditions are needed before any recommendation to farmers.

Lower respiratory tract infections and tuberculosis are responsible for the death of about 4.5 million people each year and are the main causes of mortality in children under 5 years of age. is the most common bacterial pathogen associated with severe pneumonia, although other Gram-positive and Gram-negative bacteria are involved in respiratory infections as well. The ability of these pathogens to persist and produce infection under the appropriate conditions is also associated with their capacity to form biofilms in the respiratory mucous membranes. Adding to the difficulty of treating biofilm-forming bacteria with antibiotics, many of these strains are becoming multidrug resistant, and thus the alternative therapeutics available for combating this kind of infections are rapidly depleting. Given these concerns, it is urgent to consider other unconventional strategies and, in this regard, phage lysins represent an attractive resource to circumvent some of the current issues in infection treatment. When added exogenously, lysins break specific bonds of the peptidoglycan and have potent bactericidal effects against susceptible bacteria. These enzymes possess interesting features, including that they do not trigger an adverse immune response and raise of resistance is very unlikely. Although Gram-negative bacteria had been considered refractory to these compounds, strategies to overcome this drawback have been developed recently. In this review we describe the most relevant and results obtained to date with lysins against bacterial respiratory pathogens.

Sow endometritis is usually caused by multiple species of pathogenic bacteria. Numerous isolates from endometritis patients have developed antimicrobial resistance. Thus, novel antibacterial agents and strategies to combat endometritis are needed. A total of 526 bacteria, including Staphylococcus spp. (26.3%), Streptococcus spp. (12.3%), E. coli (28.9%), Enterococcus spp. (20.1%), Proteus spp. (9.5%), and Corynebacterium spp. (2.8%), were isolated from sows with endometritis. We constructed a novel chimeric lysin, ClyL, which is composed of a cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain from the phage lysin LysGH15 and a cell wall-binding domain (CBD) from the prophage lysin Lys0859. The activities of ClyL and Lys0859 were most pronounced for the Staphylococcus and Streptococcus strains isolated from sow endometritis and bovine mastitis, respectively. ClyL and Lys0859 were combined to create a phage lysin cocktail, which demonstrated a synergistic effect against the coinfection of Staphylococcus and Streptococcus in vitro and in vivo. Furthermore, the combination of phage lysin cocktail and cefquinome had a synergistic bactericidal effect on boar semen that did not influence the activity of sperm. Remarkably, the incidence rate of sow endometritis was 0% (0/7) when the combination of phage lysin cocktail and cefquinome was used in semen via artificial insemination compared with 50% (3/6) when PBS was administered. Overall, the administration of a phage lysin cocktail and cefquinome in semen via artificial insemination is a promising novel strategy to prevent sow endometritis after artificial insemination.

Globally, Food security main threaten by abiotic stress like salinity and levels amongst the majority serious environmental stressors which reduce crop yield mass production. Biochar application has received much attention in agricultural practices as it enhances crop quality and production. The present study was carried out to analyze the role of lysine zinc and biochar on growth enhancement of wheat ( L. cv. PU-2011) under saline stress (EC 7.17 dSm ). Seeds were sown in pots containing saline soil with and without 2% biochar, and foliar application of Zn-lysine (0, 1.0, and 2.0 mM) was made at different time intervals during plant growth. A combined application of biochar and Zn-lysine 2.0 mM highly improved the physiological attributes such as chlorophyll a (37%), chlorophyll b (60%), total chlorophyll (37%), carotenoids (16%), photosynthesis rate ( ) 45%, stomatal conductance ( ) 53%, transpiration rate ( ) 56%, and water use efficiency ( ) 55%. The levels of malondialdehyde (MDA) 38%, hydrogen peroxide (H O ) 62%, and electrolyte leakage (EL) 48% were decreased with the combined application of biochar and Zn-lysine 2.0 mM as compared with other treatments. The activities of catalase (CAT) 67%, superoxide dismutase (SOD) 70%, and ascorbate peroxidase (APX) 61% as well as catalase (CAT) 67% were regulated with the combined biochar and Zn-lysine 2.0 mM treatment. Similarly, the combined application of biochar and zinc-lysine (2.0 mM) enhanced the growth and yield attributes such as shoot length (79%), root fresh weight (62%), shoot fresh weight (36%), root dry weight (86%), shoot dry weight (39%), grain weight (57%), and spike length (43%) as compared with untreated control. The concentrations of sodium (Na) decreased whereas potassium (K), iron (Fe), and zinc (Zn) concentrations were enhanced in plants with the combined application of Zn-lysine and biochar. Overall, results showed that the combined application of Zn-lysine (2.0 mM) and biochar significantly inhibited the negative effect of salinity and improved the growth and physiological performance of wheat plants. The combined use of Zn-lysine and biochar might be a practical solution to tackle salt stress in plants, but field studies by growing various crops under varied environmental conditions are needed before any recommendation to farmers.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.