RESUMEN
With recent large-scale applications and validations, the relative binding free energy (RBFE) calculated using alchemical free energy methods has been proven to be an accurate measure to probe the binding of small-molecule drug candidates. On the other hand, given the flexibility of peptides, it is of great interest to find out whether sufficient sampling could be achieved within the typical time scale of such calculation, and a similar level of accuracy could be reached for peptide drugs. However, the systematic evaluation of such calculations on protein-peptide systems has been less reported. Most reported studies of peptides were restricted to a limited number of data points or lacking experimental support. To demonstrate the applicability of the alchemical free energy method for protein-peptide systems in a typical real-world drug discovery project, we report an application of the thermodynamic integration (TI) method to the RBFE calculation of ghrelin receptor and its peptide agonists. Along with the calculation, the synthesis and in vitro EC50 activity of relamorelin and 17 new peptide derivatives were also reported. A cost-effective criterion to determine the data collection time was proposed for peptides in the TI simulation. The average of three TI repeats yielded a mean absolute error of 0.98 kcal/mol and Pearson's correlation coefficient (R) of 0.77 against the experimental free energy derived from the in vitro EC50 activity, showing good repeatability of the proposed method and a slightly better agreement than the results obtained from the arbitrary time frames up to 20 ns. Although it is limited by having one target and a deduced binding pose, we hope that this study can add some insights into alchemical free energy calculation of protein-peptide systems, providing theoretical assistance to the development of peptide drugs.
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Diseño de Fármacos , Péptidos , Receptores de Ghrelina , Termodinámica , Receptores de Ghrelina/agonistas , Receptores de Ghrelina/metabolismo , Péptidos/química , Péptidos/farmacología , Humanos , Unión Proteica , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Extensins are hydroxyproline-rich glycoproteins and generally play a structural role in cell wall integrity. In this study, we determined a novel role of tomato (Solanum lycopersicum) SENESCENCE-ASSOCIATED EXTENSIN1 (SAE1) in leaf senescence. Both gain- and loss-of-function analyses suggest that SAE1 plays a positive role in leaf senescence in tomato. Transgenic plants overexpressing SAE1 (SAE1-OX) exhibited premature leaf senescence and enhanced dark-induced senescence, whereas SAE1 knockout (SAE1-KO) plants displayed delayed development-dependent and dark-induced leaf senescence. Heterologous overexpression of SlSAE1 in Arabidopsis also led to premature leaf senescence and enhanced dark-induced senescence. In addition, the SAE1 protein was found to interact with the tomato ubiquitin ligase SlSINA4, and SlSINA4 promoted SAE1 degradation in a ligase-dependent manner when co-expressed in Nicotiana benthamiana leaves, suggesting that SlSINA4 controls SAE1 protein levels via the ubiquitin-proteasome pathway. Introduction of an SlSINA4-overexpression construct into the SAE1-OX tomato plants consistently completely eliminated accumulation of the SAE1 protein and suppressed the phenotypes conferred by overexpression of SAE1. Taken together, our results suggest that the tomato extensin SAE1 plays a positive role in leaf senescence and is regulated by the ubiquitin ligase SINA4.
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Arabidopsis , Solanum lycopersicum , Ubiquitina/genética , Solanum lycopersicum/genética , Ligasas/genética , Senescencia de la Planta , Arabidopsis/genética , Hojas de la Planta , Regulación de la Expresión Génica de las PlantasRESUMEN
Chinese cherry industry has developed rapidly over the past few years, with the planting acreage continuously expanding, from Shandong province to Liaoning, Shaanxi, Hebei, Sichuan etc. Monilia spp. are the most important causal agents of brown rot of cherry, to date, M. fructicola, M. mumecola, and M. fructigena were reported to cause brown rot of cherry in China (Chen et al. 2013; Yin et al. 2014; Liu et al. 2012). In May 2023, fruit of sweet cherry cultivar 'Hongdeng' (Prunus avium L.) with symptoms resembling brown rot were collected from Tongchuan City, Shaanxi Province. Conidia on diseased tissues were spread on a water agar (WA, 1.5% agar and distilled water) medium and isolated with a glass needle under a professional single spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China). If no conidia were present, fruit pieces (5 × 5 mm) at the intersection of healthy and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for 30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then washed three times in sterilized water. After the tissue pieces were dried, they were placed on potato dextrose agar (PDA; 200 g of potato, 20 g of dextrose, and agar at 20 g/L) and incubated at 22 °C for about twenty days to produce spores and then single spore isolation was carried out. Thirty single-spore isolates were obtained and all were morphologically similar. The isolates produced white-gray colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the PDA medium after 15 days of incubation at 22°C. Conidia were one-celled, hyaline, ellipsoid to lemon shape (14.12 × 10.37 µm), with 1-2 germs which is similar to M. yunnanensis on peach. The genomic DNA of the isolates was extracted as described previously (Chi et al. 2009). The pathogen identity was confirmed by multiplex PCR which resulted in a 237bp amplicon, which is diagnostic of M. yunnanensis (Hu et al. 2011). Further sequencing of the internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene (accession number: OR192774) indicated 100% identity with that of M. yunnanensis isolates (accession numbers: MW355895, ON024742). The average daily growth of mycelium on PDA at 22°C was 11.44 mm. Koch's postulates were fulfilled by inoculating 20 mature sweet cherry fruits of cv. 'Van' with mycelial plugs in a drilled hole. After 3 days of incubation at 22â in an airtight plastic tray with wet paper, the inoculated fruit developed typical brown rot symptoms. The developing spores on inoculated fruit were confirmed to be M. yunnanensis based on ITS sequence. All control fruit inoculated with a PDA plug remained healthy. M. yunnanensis was first reported as the causal agent of brown rot of peach in China (Hu et al. 2011). Later studies demonstrated that it is also pathogen on other fruits, e.g. hawthorn (Zhao et al. 2013), plum (Yin et al. 2015), apricot (Yin et al. 2017), apple, and pear in China (Zhu et al. 2016). To our knowledge, this is the first report of cherry brown fruit rot caused by M. yunnanensis, indicating the high risk of this species to cherry production, and effective strategies must be taken to prevent the possible control failure in practice.
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An essential step in the analysis of single-cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single-cell predictive marker (SPmarker) to identify novel cell-type marker genes in the Arabidopsis root. Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers. Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single-cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non-Arabidopsis species, which expands the number of marker genes for this cell type by 35-154%. Our results represent a new approach to identifying cell-type marker genes from scRNA-seq data and pave the way for cross-species mapping of scRNA-seq data in plants.
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Arabidopsis , Análisis de la Célula Individual , Arabidopsis/genética , Biomarcadores , Perfilación de la Expresión Génica/métodos , Aprendizaje Automático , RNA-Seq , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Secuenciación del ExomaRESUMEN
BACKGROUND: Intercropping is often used in the tea producing areas where land resources are not so abundant, and the produced green tea is tasted more delicious through a tea-Chinese chestnut intercropping system according to the experience of indigenous farmers. The length and weight of tea leaf increase under this intercropping system and their root systems are stratified vertically and coordinate symbiosis. However, the delicacy mechanism under the intercropping is not fully understood. RESULTS: Green tea from the Chinese chestnut-tea intercropping system established in the 1980s ranked highest compared with a pure tea plantation from the same region. Based on the non-targeted metabolomics, 100 differential metabolites were upregulated in the tea leaves from intercropping system relative to monoculture system. Twenty-one amino acids were upregulated and three downregulated in response to the intercropping based on the targeted metabolomics; half of the upregulated amino acids had positive effects on the tea taste. Levels of allantoic acid, sugars, sugar alcohols, and oleic acid were higher and less bitter flavonoids in the intercropping system than those in monoculture system. The upregulated metabolites could promote the quality of tea and its health-beneficial health effects. Flavone and flavonol biosynthesis and phenylalanine metabolism showed the greatest difference. Numerous pathways associated with amino acid metabolism altered, suggesting that the intercropping of Chinese chestnut-tea could greatly influence amino acid metabolism in tea plants. CONCLUSIONS: These results enhance our understanding of the metabolic mechanisms by which tea quality is improved in the Chinese chestnut-tea intercropping system and demonstrate that there is great potential to improve tea quality at the metabolomic level by adopting such an intercropping system.
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Aminoácidos/metabolismo , Camellia sinensis/metabolismo , Producción de Cultivos/métodos , Metaboloma , Té/normas , Camellia sinensis/química , Cromatografía Liquida , Flavonoides/metabolismo , Espectrometría de Masas , Redes y Vías Metabólicas , Metabolómica , Hojas de la Planta/química , Hojas de la Planta/metabolismoRESUMEN
Use of paclitaxel as monotherapy or in combination with other therapeutic agents is a widely employed front-line chemotherapeutic strategy for cervical cancer. However, previous reports have shown that approximately 70% of the patients with cervical cancer develop resistance to paclitaxel. Epithelial-mesenchymal transition (EMT) contributes to the occurrence of chemoresistance in several types of cancer, including cervical cancer. Identification of the critical signaling pathway that regulates the EMT process may provide a novel strategy for avoiding or delaying the emergence of paclitaxel resistance during the treatment of cervical cancer. Herein, we established a paclitaxel-resistant cervical cancer cell line (HeLa-229PTR cells) by culturing parental HeLa-229 cells with increasing concentrations of paclitaxel. We observed elevated expression of Notch1 in HeLa-229PTR cells compared with their parental HeLa-229 cells, indicating its potential involvement in the EMT phenotype of the paclitaxel-resistant cells. Furthermore, silencing of the NOTCH1 gene, as well as treatment with a γ-secretase inhibitor (DAPT) partially reversed the EMT phenotype and significantly enhanced the sensitivity of HeLa-229PTR cells to paclitaxel. Moreover, we found that DAPT could significantly inhibit invasiveness, reduce colony formation activity, and promote apoptosis of HeLa-229PTR cells. Taken together, these results indicated that HeLa-229PTR cells develop the EMT phenotype partly through activation of Notch1 signaling. Thus, inhibition of Notch1 signaling can be a strategy for the reversal of the EMT phenotype and may increase the sensitivity of cervical cancer cells to treatment with paclitaxel.
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Transición Epitelial-Mesenquimal , Neoplasias del Cuello Uterino , Línea Celular Tumoral , Movimiento Celular , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Paclitaxel/farmacología , Transducción de Señal , Neoplasias del Cuello Uterino/genéticaRESUMEN
Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely while maintaining pluripotency. Krüppel-like factor (Klf) 4 is an important member of the Klf family that is known to play a key role in pluripotency and somatic cell reprogramming. However, the identification and functional comparison of Klf4 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we identified three novel alternative splicing variants of Klf4 in mESCs-mKlf4-108, mKlf4-375 and mKlf4-1482-that are distinct from the previously known mKlf4-1449. mKlf4-1449 and mKlf4-1482 may stimulate the growth of ESCs, while mKlf4-108 can only promote the growth of ESCs in LIFlow/serum conditions. In addition, both mKlf4-1449 and mKlf4-1482 can inhibit the differentiation of mESCs. However, the ability of mKlf4-1482 to promote self-renewal and inhibit differentiation is not as strong as that of mKlf4-1449. In contrast, both mKlf4-108 and mKlf4-375 may have the ability to induce endodermal differentiation. Taken together, we have identified for the first time the existence of alternative splicing variants of mKlf4 and have revealed their different roles, which provide new insights into the contribution of Klf4 to the self-renewal and pluripotency of mouse ESCs.
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Empalme Alternativo , Autorrenovación de las Células/genética , Autorrenovación de las Células/fisiología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/fisiología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Codón sin Sentido , Factor 4 Similar a Kruppel , Ratones , Modelos Biológicos , Poli A/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologíaRESUMEN
Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.
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Proteínas de Arabidopsis , Arabidopsis , Proteasas de Cisteína , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteasas de Cisteína/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Pseudomonas syringae/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.
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Proteínas Bacterianas/metabolismo , NAD/metabolismo , Fosfotransferasas/metabolismo , Enfermedades de las Plantas/microbiología , Xanthomonas/enzimología , Xanthomonas/patogenicidad , Proteínas Bacterianas/genética , Oryza/microbiología , Fosforilación , Fosfotransferasas/genética , Nicotiana/microbiología , Virulencia , Xanthomonas/genéticaRESUMEN
Polyploidization is a significant source of genomic and organism diversification during plant evolution, and leads to substantial alterations in plant phenotypes and natural fitness. To help understand the phenotypic and molecular impacts of autopolyploidization, we conducted epigenetic and full-transcriptomic analyses of a synthesized autopolyploid accession of switchgrass (Panicum virgatum) in order to interpret the molecular and phenotypic changes. We found that mCHH levels were decreased in both genic and transposable element (TE) regions, and that TE methylation near genes was decreased as well. Among 142 differentially expressed genes involved in cell division, cellulose biosynthesis, auxin response, growth, and reproduction processes, 75 of them were modified by 122 differentially methylated regions, 10 miRNAs, and 15 siRNAs. In addition, up-regulated PvTOE1 and suppressed PvFT probably contribute to later flowering time of the autopolyploid. The expression changes were probably associated with modification of nearby methylation sites and siRNAs. We also experimentally demonstrated that expression levels of PvFT and PvTOE1 were regulated by DNA methylation, supporting the link between alterations in methylation induced by polyploidization and the phenotypic changes that were observed. Collectively, our results show epigenetic modifications in synthetic autopolyploid switchgrass for the first time, and support the hypothesis that polyploidization-induced methylation is an important cause of phenotypic alterations and is potentially important for plant evolution and improved fitness.
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Epigenoma/genética , Panicum/genética , Elementos Transponibles de ADN/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Transcriptoma/genéticaRESUMEN
The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary elements. Yet overexpression of XND1 blocks differentiation of tracheary elements. The molecular mechanism of XND1 action was investigated. Phylogenetic and motif analyses indicated that XND1 and its homologs are present only in angiosperms and possess a highly conserved C-terminal region containing linear motifs (CKII-acidic, LXCXE, E2FTD -like and LXCXE-mimic) predicted to interact with the cell cycle and differentiation regulator RETINOBLASTOMA-RELATED (RBR). Protein-protein interaction and functional analyses of XND1 deletion mutants were used to test the importance of RBR-interaction motifs. Deletion of either the LXCXE or the LXCXE-mimic motif reduced both the XND1-RBR interaction and XND1 efficacy as a repressor of differentiation, with loss of the LXCXE motif having the strongest negative impacts. The function of the XND1 C-terminal domain could be partially replaced by RBR fused to the N-terminal domain of XND1. XND1 also transactivated gene expression in yeast and plants. The properties of XND1, a transactivator that depends on multiple linear RBR-interaction motifs to inhibit differentiation, have not previously been described for a plant protein. XND1 harbors an apparently angiosperm-specific combination of interaction motifs potentially linking the general differentiation regulator RBR with a xylem-specific pathway for inhibition of differentiation.
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Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Xilema/citología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis , Fenotipo , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , Transactivadores/metabolismoRESUMEN
BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. RESULTS: In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain, jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from 'Alamo', a rust-resistant switchgrass cultivar, and 'Dacotah', a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar 'Summer' plants indicated that the expression of some of these RGHs was developmentally regulated. CONCLUSIONS: Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.
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Resistencia a la Enfermedad/genética , Perfilación de la Expresión Génica , Genes de Plantas , Estudios de Asociación Genética , Panicum/genética , Alelos , Secuencia de Aminoácidos , Biología Computacional/métodos , Bases de Datos de Ácidos Nucleicos , Predisposición Genética a la Enfermedad , Genoma de Planta , Genómica/métodos , Panicum/clasificación , Filogenia , Polimorfismo de Nucleótido Simple , Posición Específica de Matrices de Puntuación , Dominios y Motivos de Interacción de Proteínas/genética , Reproducibilidad de los ResultadosRESUMEN
The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein-protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses.
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Regulación de la Expresión Génica de las Plantas , Oryza/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Transcripción Genética , Triticum/genética , Ácido Abscísico/metabolismo , Redes Reguladoras de Genes , Oryza/metabolismo , Oryza/efectos de la radiación , Estrés Oxidativo , Inmunidad de la Planta/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Factores de Transcripción/metabolismo , Triticum/metabolismo , Triticum/efectos de la radiación , Rayos UltravioletaRESUMEN
Acidovorax citrulli causes bacterial fruit blotch of cucurbits, a serious economic threat to watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide. Based on genetic and biochemical traits, A. citrulli strains have been divided into two distinct groups: group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from and are highly virulent on watermelon. The pathogen depends on a functional type III secretion system for pathogenicity. Annotation of the genome of the group II strain AAC00-1 revealed 11 genes encoding putative type III secreted (T3S) effectors. Due to the crucial role of type III secretion for A. citrulli pathogenicity, we hypothesized that group I and II strains differ in their T3S effector repertoire. Comparative analysis of the 11 effector genes from a collection of 22 A. citrulli strains confirmed this hypothesis. Moreover, this analysis led to the identification of a third A. citrulli group, which was supported by DNA:DNA hybridization, DNA fingerprinting, multilocus sequence analysis of conserved genes, and virulence assays. The effector genes assessed in this study are homologous to effectors from other plant-pathogenic bacteria, mainly belonging to Xanthomonas spp. and Ralstonia solanacearum. Analyses of the effective number of codons and gas chromatography content of effector genes relative to a representative set of housekeeping genes support the idea that these effector genes were acquired by lateral gene transfer. Further investigation is required to identify new T3S effectors of A. citrulli and to determine their contribution to virulence and host preferential association.
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Citrullus/microbiología , Comamonadaceae/genética , Cucumis melo/microbiología , Enfermedades de las Plantas/microbiología , Polimorfismo de Nucleótido Simple , Sistemas de Secreción Tipo III/genética , Secuencia de Bases , Comamonadaceae/metabolismo , Comamonadaceae/patogenicidad , Frutas/microbiología , Datos de Secuencia Molecular , Fenotipo , Filogenia , Plantones/microbiología , Análisis de Secuencia de ADN , Sistemas de Secreción Tipo III/metabolismo , VirulenciaRESUMEN
A new style of tofu coagulated through the fermentation of Lactobacillus plantarum SJ-L-1 was produced. L. plantarum SJ-L-1 with a high growth rate and excellent acid production ability was isolated and identified from naturally fermented soy yellow whey. The gene annotation indicated the potential outstanding isoflavone conversion capacity of L. plantarum SJ-L-1. Furthermore, fermentation tofu was prepared using L. plantarum SJ-L-1 and Lactobacillus rhamnosus 1-16 as the starter microbiota. Compared to traditional MgCl2 tofu and fermented soy whey tofu, SJ-L-1 tofu exhibited a slight increase in hardness and better structure uniformity. SJ-L-1 tofu also possessed the highest levels of total isoflavone content (76.33 µg/g) and volatile compounds (561.54 µg/kg) among the four styles of tofu. This research indicated that this new type of tofu coagulated through a combination of heat and fermentation of L. plantarum SJ-L-1 represents a promising candidate for future functional foods.
RESUMEN
The type VI secretion system (T6SS) of avian pathogenic Escherichia coli (APEC) can affect the functions of eukaryotic cells by secreting or injecting effectors. Hemolysin co-regulatory protein (Hcp), one of the markers of the T6SS, is both a structural protein and an effector protein of the T6SS. According to previous studies, mitochondria in eukaryotic cells are targeted by pathogenic bacteria. However, little is known about the regulation of mitochondria in eukaryotic host cells by the T6SS effector protein Hcp of APEC. In our study, DF-1 cells co-incubated with Hcp2a protein for 6 h showed decreased mitochondrial membrane potential, increased Ca2+ concentration, and increased cellular reactive oxygen species (ROS) levels. We therefore conclude that Hcp2a protein causes dysfunction to mitochondria in DF-1 cells. To explain the mechanism that causes mitochondrial dysfunction, we reanalyzed the Hcp2a interaction protein dataset in DF-1 cells, and the Leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), which is associated with mitochondria, was screened. The protein and molecular docking results showed that Hcp2a protein and LETM1 protein have better binding. Finally, subcellular localization results showed that Hcp2a was localized to mitochondria. In summary, Hcp2a effector proteins caused dysfunction to DF-1 cellular mitochondria, and we hypothesize that the interaction of Hcp2a protein with LETM1 protein induces mitochondrial dysfunction and promotes mitochondrial localization of Hcp2a in DF-1 cells.
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Proteínas Aviares , Enfermedades Mitocondriales , Animales , Escherichia coli , Simulación del Acoplamiento Molecular , Pollos/microbiología , Enfermedades Mitocondriales/veterinariaRESUMEN
Effectors of the bacterial type III secretion system provide invaluable molecular probes to elucidate the molecular mechanisms of plant immunity and pathogen virulence. In this report, we focus on the AvrBs2 effector protein from the bacterial pathogen Xanthomonas euvesicatoria (Xe), the causal agent of bacterial spot disease of tomato and pepper. Employing homology-based structural analysis, we generate a three-dimensional structural model for the AvrBs2 protein and identify catalytic sites in its putative glycerolphosphodiesterase domain (GDE). We demonstrate that the identified catalytic region of AvrBs2 was able to functionally replace the GDE catalytic site of the bacterial glycerophosphodiesterase BhGlpQ cloned from Borrelia hermsii and is required for AvrBs2 virulence. Mutations in the GDE catalytic domain did not disrupt the recognition of AvrBs2 by the cognate plant resistance gene Bs2. In addition, AvrBs2 activation of Bs2 suppressed subsequent delivery of other Xanthomonas type III effectors into the host plant cells. Investigation of the mechanism underlying this modulation of the type III secretion system may offer new strategies to generate broad-spectrum resistance to bacterial pathogens.
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Proteínas Bacterianas/química , Modelos Moleculares , Factores de Virulencia/química , Xanthomonas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Capsicum/microbiología , Solanum lycopersicum/microbiología , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Estructura Terciaria de Proteína , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo , Xanthomonas/patogenicidadRESUMEN
We analyze a fully stochastic model of heterogeneous nucleation and self-assembly in a closed system with a fixed total particle number M, and a fixed number of seeds Ns. Each seed can bind a maximum of N particles. A discrete master equation for the probability distribution of the cluster sizes is derived and the corresponding cluster concentrations are found using kinetic Monte-Carlo simulations in terms of the density of seeds, the total mass, and the maximum cluster size. In the limit of slow detachment, we also find new analytic expressions and recursion relations for the cluster densities at intermediate times and at equilibrium. Our analytic and numerical findings are compared with those obtained from classical mass-action equations and the discrepancies between the two approaches analyzed.
Asunto(s)
Procesos Estocásticos , Algoritmos , Cinética , Método de MontecarloRESUMEN
The bath industry has multiple attributes, such as economic, health, and cultural communication. Therefore, exploring this industry's spatial pattern evolution is crucial to forming a healthy and balanced development model. Based on POI (Points of Interest) and population migration data, this paper uses spatial statistics and radial basis function neural network to explore the spatial pattern evolution and influencing factors of the bath industry in mainland China. The results show that: (1) The bath industry presents a strong development pattern in the north, south-northeast, and east-northwest regions and weak development in the rest of the country. As a result, the spatial development of new bath space is more malleable. (2) The input of bathing culture has a guiding role in developing the bath industry. The growth of market demand and related industries has a specific influence on the development of the bath industry. (3) Improving the bath industry's adaptability, integration, and service level are feasible to ensure healthy and balanced development. (4) Bathhouses should improve their service system and risk management control during the pandemic.