Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity.

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator.

The Cell Wall-Derived Xyloglucan Is a New DAMP Triggering Plant Immunity in Vitis vinifera and Arabidopsis thaliana.

Damage-associated molecular patterns (DAMPs) are endogenous molecules that can activate the plant innate immunity. DAMPs can derive from the plant cell wall, which is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides. Fragments of pectin, called oligogalacturonides (OG), can be released after wounding or by pathogen-encoded cell wall degrading enzymes (CWDEs) such as polygalacturonases (PGs). OG are known to induce innate immune responses, including the activation of mitogen-activated protein kinases (MAPKs), production of H2O2, defense gene activation, and callose deposition. Thus, we hypothesized that xyloglucans (Xh), derived from the plant cell wall hemicellulose, could also act as an endogenous elicitor and trigger a signaling cascade similar to OG. Our results indicate that purified Xh elicit MAPK activation and immune gene expression in grapevine (Vitis vinifera) and Arabidopsis (Arabidopsis thaliana) to trigger induced resistance against necrotrophic (Botrytis cinerea) or biotrophic (Hyaloperonospora arabidopsidis) pathogens. Xh also induce resveratrol production in grapevine cell suspension and callose deposition in Arabidopsis which depends on the callose synthase PMR4. In addition, we characterized some signaling components of Xh-induced immunity using Arabidopsis mutants. Our data suggest that Xh-induced resistance against B. cinerea is dependent on the phytoalexin, salicylate, jasmonate, and ethylene pathways.

Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Effects of potassium channel modulators on human detrusor smooth muscle myogenic phasic contractile activity: potential therapeutic targets for overactive bladder.

OBJECTIVES: Increased urinary bladder detrusor smooth muscle phasic contractility has been suggested to be associated with idiopathic bladder overactivity (OAB). We examined the role of voltage-dependent L-type calcium channels, adenosine triphosphate-sensitive potassium (K(ATP)) channels, and calcium-activated potassium (BK(Ca) and SK(Ca)) channels in the regulation of human detrusor phasic contractile activity. METHODS: Isolated human bladder strip phasic contractions were measured and quantified as the mean area under the force-time curve, amplitude, and frequency of phasic contractions in 22 bladder samples. RESULTS: Human detrusor strips displayed myogenic phasic contractions in the presence of atropine (10(-6) M), phentolamine (10(-6) M), propranolol (10(-6) M), suramin (10(-5) M), and tetrodotoxin (10(-6) M). The L-type calcium channel inhibitor nifedipine (300 nM) abolished the contractile activity. Blockade of K(ATP) channels by glibenclamide (1 and 10 microM) did not alter myogenic contractions. In contrast, the K(ATP) channel opener pinacidil (10 microM) markedly inhibited phasic contractility. Iberiotoxin (100 nM) and apamin (100 nM), potent and selective inhibitors of BK(Ca) and SK(Ca) channels, respectively, significantly increased the area under the force-time curve and the amplitude of contractions. CONCLUSIONS: Phasic contractions of human detrusor are dependent on calcium entry through L-type calcium channels. BK(Ca) and SK(Ca) channels play a key role in the modulation of human detrusor smooth muscle phasic contractility. Furthermore, these observations support the concept that increasing conductance through K(ATP), BK(Ca), and SK(Ca) channels may represent attractive pharmacologic targets for decreasing phasic contractions of detrusor smooth muscle in OAB.

Erectile dysfunction in hypercholesterolemic atherosclerotic apolipoprotein E knockout mice.

INTRODUCTION: Erectile dysfunction (ED) and cardiovascular diseases share the same risk factors. Although the use of hypercholesterolemic rabbit models has proven to be useful to illustrate the link between ED and hypercholesterolemia, the cost of daily maintenance of the animals and necessity for important amounts of drug have limited their use. AIM: We aimed to develop a new model of atherosclerosis-associated ED in a well-known experimental model of atherosclerosis, the apolipoprotein E knockout (ApoE KO) mouse. METHODS: Erectile function was evaluated by recording frequency-dependent increases in intracavernous pressure following electrical stimulation of the cavernous nerve in 26-, 32-, and 38-week-old ApoE KO mice fed a Western-type diet and age-matched C57BL6/J anesthetized mice. Atherosclerotic lesions were evaluated by planimetry in oil red O-stained aortas. RESULTS: We found that in contrast to C57BL6/J mice, ApoE mice displayed atherosclerotic lesions covering 22% of the aortic luminal surface at 26 weeks of age and increasing to 27% and 35% at 32 weeks and 38 weeks of age, respectively. The amplitude of erectile responses to electrical stimulation of the cavernous nerve was markedly impaired in 26-week-old ApoE KO mice as compared with age-matched C57BL6/J mice. Impairment in erectile function persisted in ApoE KO mice 32 and 38 weeks of age. CONCLUSIONS: The ApoE KO mouse, a well-characterized model to study disorders associated with hypercholesterolemia and atherosclerosis in cardiovascular research, could therefore be suitable for investigation of disease-modifying effects of new therapeutic strategies aiming to target both atherosclerosis and ED.