RESUMEN
Grapevine (Vitis vinifera) is one of the major economic fruit crops but suffers many diseases, causing damage to the quality of grapes. Strain G166 was isolated from the rhizosphere of grapevine and was found to exhibited broad-spectrum antagonistic activities against fungal pathogens on grapes in vitro, such as Coniella diplodiella, Botrytis cinerea, and Colletotrichum gloeosporioides. Whole-genome sequencing revealed that G166 contained a 6,613,582 bp circular chromosome with 5749 predicted coding DNA sequences and an average GC content of 60.57%. TYGS analysis revealed that G166 belongs to Pseudomonas viciae. Phenotype analysis indicated that P. viciae G166 remarkably reduced the severity of grape white rot disease in the grapevine. After inoculation with C. diplodiella, more H2O2 and MDA accumulated in the leaves and resulted in decreases in the Pn and chlorophyll content. Conversely, G166-treated grapevine displayed less oxidative damage with lower H2O2 levels and MDA contents under the pathogen treatments. Subsequently, G166-treated grapevine could sustain a normal Pn and chlorophyll content. Moreover, the application of P. viciae G166 inhibited the growth of mycelia on detached leaves and berries, while more disease symptoms occurred in non-bacterized leaves and berries. Therefore, P. viciae G166 served as a powerful bioagent against grape white rot disease. Using antiSMASH prediction and genome comparisons, a relationship between non-ribosomal peptide synthase clusters and antifungal activity was found in the genome of P. viciae G166. Taken together, P. viciae G166 shows promising antifungal potential to improve fruit quality and yield in ecological agriculture.
RESUMEN
Water deficit caused by osmotic stress and drought limits crop yield and tree growth worldwide. Screening and identifying candidate genes from stress-resistant species are a genetic engineering strategy to increase drought resistance. In this study, an increased concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in the nonsecretor mangrove species Kandelia candel. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localizes to chloroplasts. Enzymatic activity characterization revealed that KcTrxf recombinant protein possesses the disulfide reductase function. KcTrxf overexpression contributes to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) production. KcTrxf was shown to reduce the stomatal aperture by enhancing K+ efflux in guard cells, which increased the water-retaining capacity in leaves under drought conditions. Notably, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits plants exposed to drought by reducing water loss by promoting stomatal closure. KcTrxf-transgenic plants limited drought-induced H2O2 in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Additionally, glutathione (GSH) contributing to reactive oxygen species (ROS) scavenging and transgenic plants are more efficient at regenerating GSH from oxidized glutathione (GSSG) under conditions of drought stress. Notably, KcTrxf-transgenic plants had increased glucose and fructose contents under drought stress conditions, presumably resulting from KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributes to drought tolerance by increasing the water status, by enhancing osmotic adjustment, and by maintaining ROS homeostasis in transgene plants.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Tiorredoxinas en Cloroplasto/genética , Tiorredoxinas en Cloroplasto/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Nicotiana/metabolismo , Rhizophoraceae/química , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Sequías , Fructosa/metabolismo , Glucosa/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Malondialdehído/metabolismo , Manitol/toxicidad , NADH NADPH Oxidorreductasas/metabolismo , Presión Osmótica , Filogenia , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Estomas de Plantas/citología , Estomas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Análisis de Secuencia , Nicotiana/efectos de los fármacos , Regulación hacia Arriba , Agua/metabolismoRESUMEN
In agricultural production, sustainability is currently one of the most significant concerns. The genetic modification of plant growth-promoting rhizobacteria may provide a novel way to use natural bacteria as microbial inoculants. In this study, the root-colonizing strain Pseudomonas protegens Pf-5 was genetically modified to act as a biocontrol agent and biofertilizer with biological nitrogen fixation activity. Genetic inactivation of retS enhanced the production of 2,4-diacetylphloroglucinol, which contributed for the enhanced antifungal activity. Then, the entire nitrogenase island with native promoter from Pseudomonas stutzeri DSM4166 was introduced into a retS mutant strain for expression. Root colonization patterns assessed via confocal laser scanning microscopy confirmed that GFP-tagged bacterial were mainly located on root surfaces and at the junctions between epidermal root cells. Moreover, under pathogen and N-limited double treatment conditions, the fresh weights of seedlings inoculated with the recombinant retS mutant-nif strain were increased compared with those of the control. In conclusion, this study has innovatively developed an eco-friendly alternative to the agrochemicals that will benefit global plant production significantly.
Asunto(s)
Proteínas Bacterianas , Agentes de Control Biológico , Fijación del Nitrógeno , Ingeniería de Proteínas , Pseudomonas stutzeri , Nitrogenasa/metabolismo , Raíces de Plantas , Pseudomonas , Pseudomonas stutzeri/metabolismoRESUMEN
As a versatile therapeutic modality, peptides attract much attention because of their great binding affinity, low toxicity, and the capability of targeting traditionally "undruggable" protein surfaces. However, the deficiency of cell permeability and metabolic stability always limits the success of in vitro bioactive peptides as drug candidates. Peptide macrocyclization is one of the most established strategies to overcome these limitations. Over the past decades, more than 40 cyclic peptide drugs have been clinically approved, the vast majority of which are derived from natural products. The de novo discovered cyclic peptides on the basis of rational design and in vitro evolution, have also enabled the binding with targets for which nature provides no solutions. The current review summarizes different classes of cyclic peptides with diverse biological activities, and presents an overview of various approaches to develop cyclic peptide-based drug candidates, drawing upon series of examples to illustrate each strategy.
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Descubrimiento de Drogas , Péptidos Cíclicos/uso terapéutico , Secuencia de Aminoácidos , Ensayos Clínicos como Asunto , Ciclización , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacologíaRESUMEN
Multiple sclerosis (MS) is an inflammatory disease in central nervous system (CNS) with limited therapeutic drugs. In the present study, we explored the anti-inflammatory/neuroprotective properties of Acteoside (AC), an active compound from medicinal herb Radix Rehmanniae (RR), and neuroprotective effects of AC on MS pathology by using an experimental autoimmune encephalomyelitis (EAE) model. We tested the hypothesis that AC could alleviate EAE pathogenesis through inhibiting inflammation and ONOO--mediated mitophagy activation in vivo and in vitro. The results showed that AC treatment effectively ameliorated neurological deficit score and postponed disease onset in the EAE mice. AC treatment inhibited inflammation/demyelination, alleviated peripheral activation and CNS infiltration of encephalitogenic CD4+ T cells and CD11b+ activated microglia/macrophages in the spinal cord of EAE mice. Meanwhile, AC treatment reduced ONOO- production, down-regulated the expression of iNOS and NADPH oxidases, and inhibited neuronal apoptotic cell death and mitochondrial damage in the spinal cords of the EAE mice. Furthermore, AC treatment decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited the translocation of Drp1 to the mitochondria. In vitro studies further proved that AC possessed strong ONOO- scavenging capability and protected the neuronal cells from nitrative cytotoxicity via suppressing ONOO--mediated excessive mitophagy. Taken together, Acteoside could be a potential therapeutic agent for multiple sclerosis treatment. The suppression of ONOO--induced excessive mitophagy activation could be one of the critical mechanisms contributing to its anti-inflammatory and anti-demyelinating properties.
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Encefalomielitis Autoinmune Experimental , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Glucósidos , Ratones , Ratones Endogámicos C57BL , Mitofagia , Ácido Peroxinitroso , FenolesRESUMEN
Pseudomonas protegens CHA0 is a well-characterized, root-colonizing bacterium with broad-spectrum biocontrol ability. Therefore, it has a great potential to curb plant diseases and to partly replace synthetic chemical pesticides that are harmful to humans. Here, we obtained the multifunctional mutant CHA0-ΔretS-Nif via Red/ET recombineering technology. After deletion of the retS gene and integration of the nitrogen-fixing gene island (Nif) into the CHA0 genome, the resulting mutant, CHA0-ΔretS-Nif, manifested improved both bactericidal activity and biological nitrogen-fixation function. A pot experiment of Arabidopsis thaliana indicated that the strain CHA0-ΔretS-Nif promoted plant growth via expressing several secondary factors, such as the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and nitrogenase. In order to grow this biocontrol strain at an industrial level, the growth conditions in a 1 L continuous-flow fermenter were optimized to 28 °C, pH of 7.0, and 600 rpm. Moreover, growth experiments in a 5 L fermenter with these optimal growth conditions yielded the maximum cell density, providing vital insights for the industrialization and large-scale fermentation of P. protegens CHA0 for further applications. CHA0-ΔretS-Nif possesses both bactericidal and nitrogen-fixation activities and thus could be used as a biological agent to enhance crop production.
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Arabidopsis/crecimiento & desarrollo , Agentes de Control Biológico/metabolismo , Fijación del Nitrógeno/genética , Enfermedades de las Plantas/prevención & control , Pseudomonas/genética , Pseudomonas/metabolismo , Arabidopsis/microbiología , Proteínas Bacterianas/genética , Producción de Cultivos/métodos , Eliminación de Gen , Fijación del Nitrógeno/fisiología , Nitrogenasa/metabolismo , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Simbiosis/genética , Factores de Virulencia/genéticaRESUMEN
Bacterial genomes encode numerous cryptic biosynthetic gene clusters (BGCs) that represent a largely untapped source of drugs or pesticides. Mining of the cryptic products is limited by the unavailability of streamlined genetic tools in native producers. Precise genome engineering using bacteriophage recombinases is particularly useful for genome mining. However, recombinases are usually host-specific. The genome-guided discovery of novel recombinases and their transient expression could boost cryptic BGC mining. Herein, we reported a genetic system employing Red recombinases from Burkholderiales strain DSM 7029 for efficient genome engineering in several Burkholderiales species that currently lack effective genetic tools. Using specialized recombinases-assisted in situ insertion of functional promoters, we successfully mined five cryptic nonribosomal peptide synthetase/polyketide synthase BGCs, two of which were silent. Two classes of lipopeptides, glidopeptins and rhizomides, were identified through extensive spectroscopic characterization. This recombinase expression strategy offers utility within other bacteria species, allowing bioprospecting for potentially scalable discovery of novel metabolites with attractive bioactivities.
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Bacteriófagos/enzimología , Burkholderia/genética , Genoma Bacteriano , Familia de Multigenes , Recombinasas/química , Proteínas Virales/químicaRESUMEN
Ca(2+)-dependent protein kinases (CDPKs) play vital roles in plant adaptations to environmental challenges. The precise regulatory mechanism of CDPKs in mediating salt stress still remains unclear, although several CDPK members have been identified to be involved in salt stress accumulation in various plants, such as Arabidopsis thaliana and Oryza sativa. Here, we investigated the function of an Arabidopsis CDPK, CPK27, in salt stress-signaling. CPK27 is a membrane-localized protein kinase; its expression was induced by NaCl. cpk27-1, a T-DNA insertion mutant of CPK27, was much more sensitive to salt stress than wild-type plants in terms of seed germination and post-germination seedling growth. In ion-flux assay, cpk27-1 mutants exhibited a lower capacity than wild-type plants to extrude Na(+) and import H(+) after a long-term salt treatment (110mM NaCl for 10days). Moreover, the content of Na(+) was higher and K(+) was lower in cpk27-1 mutants than in wild-type plants under salt stress. In addition, the level of salt-elicited H2O2 production was higher in cpk27-1 mutants than in wild-type plants Col after a short-term NaCl shock and long-term salt treatment. Collectively, our results suggest that CPK27 is required for plant adaptation to salt stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Calcio/metabolismo , Proteínas Quinasas/metabolismo , Cloruro de Sodio/metabolismo , Estrés Fisiológico/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Germinación/genética , Germinación/fisiología , Oryza/genética , Oryza/metabolismo , Oryza/fisiología , Plantones/genética , Plantones/metabolismo , Plantones/fisiología , Sodio/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Na(+) uptake and transport in Kandelia candel and antioxidative defense were investigated under rising NaCl stress from 100 to 300 mM. Salinized K. candel roots had a net Na(+) efflux with a declined flux rate during an extended NaCl exposure. Na(+) buildup in leaves enhanced H2O2 levels, superoxide dismutase (SOD) activity, and increased transcription of CSD gene encoding a Cu/Zn SOD. Sequence and subcellular localization analyses have revealed that KcCSD is a typical Cu/Zn SOD in chloroplast. The transgenic tobacco experimental system was used as a functional genetics model to test the effect of KcCSD on salinity tolerance. KcCSD-transgenic lines were more Na(+) tolerant than wild-type (WT) tobacco in terms of lipid peroxidation, root growth, and survival rate. In the latter, 100 mM NaCl led to a remarkable reduction in chlorophyll content and a/b ratio, decreased maximal chlorophyll a fluorescence, and photochemical efficiency of photosystem II. NaCl stress in WT resulted from H2O2 burst in chloroplast. Na(+) injury to chloroplast was less pronounced in KcCSD-transgenic plants due to upregulated antioxidant defense. KcCSD-transgenic tobacco enhanced SOD activity by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 day. Catalase activity rose in KcCSD overexpressing tobacco plants. KcCSD-transgenic plants better scavenged NaCl-elicited reactive oxygen species (ROS) compared to WT ones. In conclusion, K. candel effectively excluded Na(+) in roots during a short exposure; and increased CSD expression to reduce ROS in chloroplast in a long-term and high saline environment.
RESUMEN
Using 3-month-old seedlings of Bruguiera gymnorrhiza (L.) Savigny and Kandelia candel (L.) Druce, we compared species differences in ionic homeostasis control between the two non-secretor mangrove species. A high salinity (400 mM NaCl, 4 weeks) resulted in a decline of the K(+)/Na(+) ratio in root and leaf tissues, and the reduction was more pronounced in K. candel (41-66%) as compared with B. gymnorrhiza (5-36%). Salt-altered flux profiles of Na(+), K(+), H(+) and Ca(2+) in roots and effects of exogenous hydrogen peroxide (H(2)O(2)), nitric oxide (NO) and Ca(2+) on root ion ï¬uxes were examined in seedlings that were hydroponically treated short term with 100 mM NaCl (ST, 24 h) and long term with 200 mM NaCl (LT, 7 days). Short term and LT salinity resulted in Na(+) efflux and a correspondingly increased H(+) influx in roots of both species, although a more pronounced effect was observed in B. gymnorrhiza. The salt-enhanced exchange of Na(+) with H(+) was obviously inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or sodium orthovanadate (a plasma membrane H(+)-ATPase inhibitor), indicating that the Na(+) efflux resulted from active Na(+) exclusion across the plasma membrane. Short term and LT salinity accelerated K(+) efflux in the two species, but K. candel exhibited a higher flux rate. The salt-induced K(+) efflux was markedly restricted by the K(+) channel blocker, tetraethylammonium chloride, indicating that the K(+) efflux is mediated by depolarization-activated channels, e.g., KORCs (outward rectifying K(+) channels) and NSCCs (non-selective cation channels). Exogenous H(2)O(2) application (10 mM) markedly increased the apparent Na(+) efflux and limited K(+) efflux in ST-treated roots, although H(2)O(2) caused a higher Na(+) efflux in B. gymnorrhiza roots. CaCl(2) (10 mM) reduced the efflux of K(+) in salinized roots of the two mangroves, but its enhancement of Na(+) efflux was found only in B. gymnorrhiza. Under ST treatment, sodium nitroprusside (SNP) (100 âM, an NO donor) increased Na(+) efflux at the root apex of the two species; however, its inhibition of K(+) loss was seen only in K. candel. Of note, NaCl caused an obvious influx of Ca(2+) in B. gymnorrhiza roots, which was enhanced by H(2)O(2) (10 mM). Therefore, the salt-induced Ca(2+) benefits B. gymnorrhiza in maintaining K(+)/Na(+) homeostasis under high external salinity.