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1.
Plant Mol Biol ; 101(4-5): 499-506, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31621004

RESUMO

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different auxin response factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos Orgânicos , Proteínas de Plantas/metabolismo , Soja/metabolismo , Fatores de Transcrição/metabolismo , Fluorescência , Ligação Proteica , Sensibilidade e Especificidade
2.
Plant Sci ; 287: 110172, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481220

RESUMO

Plants must protect themselves from abiotic stresses such as drought, cold, and high salinity. The common thread of all three stresses is that they cause dehydration, which in turn promotes the formation of reactive oxygen species (ROS). Dehydrin proteins (dehydrins) are a large family of proteins that have been identified in nearly all land plants, and whose presence is correlated with plant protection from abiotic stresses. Several dehydrin studies have shown that some dehydrins localize to the nucleus, as well as the cytoplasm, but a functional role for nuclear dehydrins has not yet been determined. We show here that the Vitis riparia dehydrin VrDHN1 localizes to the nucleus and is able to bind to DNA to protect it from damage caused by hydrogen peroxide, an ROS source. We also show that the binding to DNA is not DNA-sequence specific, suggesting that the protein is able to protect any exposed DNA without interfering with its normal function. NMR studies show that the binding is largely driven by the lysine-rich nature of dehydrins located in the conserved K-segments. Unlike other, previously studied dehydrins, VrDHN1 binding to DNA is not enhanced through the presence of metals. Lastly, we demonstrate that the Y-segment does not bind ATP, as has long been proposed.


Assuntos
DNA de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Vitis/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Espectroscopia de Ressonância Magnética , Espécies Reativas de Oxigênio/metabolismo
3.
Nat Commun ; 10(1): 3916, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477705

RESUMO

Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA de Plantas/química , RNA de Plantas/genética
4.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370193

RESUMO

Prior experiments illustrated reactive oxygen species (ROS) overproduction in maize plants infested with bird-cherry-oat (Rhopalosiphum padi L.) aphids. However, there is no available data unveiling the impact of aphids feeding on oxidative damages of crucial macromolecules in maize tissues. Therefore, the purpose of the current study was to evaluate the scale of oxidative damages of genomic DNA, total RNA and mRNA, proteins, and lipids in seedling leaves of two maize genotypes (Zlota Karlowa and Waza cvs-susceptible and relatively resistant to the aphids, respectively). The content of oxidized guanosine residues (8-hydroxy-2'-deoxyguanosine; 8-OHdG) in genomic DNA, 8-hydroxyguanosine (8-OHG) in RNA molecules, protein carbonyl groups, total thiols (T-SH), protein-bound thiols (PB-SH), non-protein thiols (NP-SH), malondialdehyde (MDA) and electrolyte leakage (EL) levels in maze plants were determined. In addition, the electrical penetration graphs (EPG) technique was used to monitor and the aphid stylet positioning and feeding modes in the hosts. Maize seedlings were infested with 0 (control), 30 or 60 R. padi adult apterae per plant. Substantial increases in the levels of RNA, protein and lipid oxidation markers in response to aphid herbivory, but no significant oxidative damages of genomic DNA, were found. Alterations in the studied parameters were dependent on maize genotype, insect abundance and infestation time.


Assuntos
Afídeos/fisiologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Parasita/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Zea mays/genética , /metabolismo , Animais , Afídeos/patogenicidade , DNA de Plantas/genética , DNA de Plantas/metabolismo , Genótipo , Guanosina/análogos & derivados , Guanosina/metabolismo , Lipídeos/química , Malondialdeído/metabolismo , Oxirredução , Estresse Oxidativo , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/genética , Plântula/parasitologia , Compostos de Sulfidrila/metabolismo , Zea mays/parasitologia
5.
Gene ; 718: 144018, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31454543

RESUMO

Cytosine DNA methylation (5mC) is an epigenetic mark that regulates gene expression in plant responses to environmental stresses. Zinc-finger protein (ZFP) is the largest family of DNA-binding transcription factors that also plays an essential role in eukaryote. In plant we have already identified and characterized different useful ZFP-genes. While, the main objective of this research was to observe and identify more targeted stress responsive genes of ZFPs epigenetically throughout genome in rice for the first time. A comprehensive correlation analysis was performed through methylated DNA immunoprecipitation (MeDIP)-chip hybridization in rice under salt and osmotic stresses. High salinity and drought are two major abiotic hazards that are destroying the crop world-wide. As a result, Through-out genome 14 unique stress responsive transcription factors of ZFP-genes with varying level of methylation and expression under two conditions (control vs. stress) were isolated. All the identified genes were confirmed from different databases for their specific structure, cis-regulatory elements, phylogenetic analysis, and synteny analysis. Moreover, the tissue-specific expression patterns, and expression under abiotic and phytohormones stresses were also investigated. Phylogenetically all the genes were divided into 6 distinct subgroups with Arabidopsis and orthologous proteins were find-out through synteny analysis. Available RNA-seq data in response to various phytohormones provided hormone inducible gene expression profile. Through Reverse Transcriptase qPCR (RT-qPCR) analysis tissue-specific expression in shoot and root over various time points against salt and osmotic stresses exhibited the diverse expression patterns of identified genes. Overall, the present study providing a foundation for in-depth characterization of identified genes and to further understand the epigenetic role of DNA methylation for genes expression and environmental stresses regulation in higher plant.


Assuntos
Metilação de DNA/fisiologia , DNA de Plantas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza , Proteínas de Plantas , Estresse Fisiológico/fisiologia , Fatores de Transcrição , DNA de Plantas/genética , DNA de Plantas/metabolismo , Estudo de Associação Genômica Ampla , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340536

RESUMO

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the 'sun loving' C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.


Assuntos
Adaptação Biológica/efeitos dos fármacos , Cistus/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/efeitos da radiação , RNA de Plantas/genética , Ácido Abscísico/metabolismo , Adaptação Biológica/genética , Antioxidantes/metabolismo , Clorofila/biossíntese , Cistus/genética , Cistus/metabolismo , Dano ao DNA , Reparo do DNA , DNA de Plantas/genética , DNA de Plantas/metabolismo , Flavonoides/biossíntese , Transdução de Sinal Luminoso/genética , Região do Mediterrâneo , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/metabolismo , Análise de Sequência de RNA , Energia Solar , Luz Solar , Água/metabolismo , Zeaxantinas/biossíntese
7.
Food Chem ; 292: 350-358, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054687

RESUMO

The objective of this study was to quantitatively assess potato omics profiles of new varieties for meaningful differences from analogous profiles of commercial varieties through the SIMCA one-class classification model. Analytical profiles of nine commercial potato varieties, eleven experimental potato varieties, one GM potato variety that had acquired Phytophtora resistance based on a single insert with potato-derived DNA sequences, and its non-GM commercial counterpart were generated. The ten conventional varieties were used to construct the one-class model. Omics profiles from experimental non-GM and GM varieties were assessed using the one-class SIMCA models. No potential unintended effects were identified in the case of the GM variety. The model showed that varieties that were genetically more distant from the commercial varieties were recognized as aberrant, highlighting its potential in determining whether additional evaluation is required for the risk assessment of materials produced from any breeding technique, including genetic modification.


Assuntos
Metaboloma , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/metabolismo , Transcriptoma , DNA de Plantas/química , DNA de Plantas/metabolismo , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Metabolômica , Plantas Geneticamente Modificadas/genética , Análise de Componente Principal , Medição de Risco , Análise de Sequência de RNA , Solanum tuberosum/genética
8.
Molecules ; 24(10)2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126138

RESUMO

Guanine-rich DNA strands can adopt tertiary structures known as G-quadruplexes (G4s) that form when Hoogsteen base-paired guanines assemble as planar stacks, stabilized by a central cation like K+. In this study, we investigated the conformational heterogeneity of a G-rich sequence from the 5' untranslated region of the Zea mays hexokinase4 gene. This sequence adopted an extensively polymorphic G-quadruplex, including non-canonical bulged G-quadruplex folds that co-existed in solution. The nature of this polymorphism depended, in part, on the incorporation of different sets of adjacent guanines into a quadruplex core, which permitted the formation of the different conformations. Additionally, we showed that the maize homolog of the human nucleoside diphosphate kinase (NDPK) NM23-H2 protein-ZmNDPK1-specifically recognizes and promotes formation of a subset of these conformations. Heteromorphic G-quadruplexes play a role in microorganisms' ability to evade the host immune system, so we also discuss how the underlying properties that determine heterogeneity of this sequence could apply to microorganism G4s.


Assuntos
DNA de Plantas/química , Hexoquinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Zea mays/enzimologia , Regiões 5' não Traduzidas , Sítios de Ligação , Dicroísmo Circular , DNA de Plantas/metabolismo , Quadruplex G , Hexoquinase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espectrofotometria Ultravioleta , Zea mays/genética
9.
Food Chem ; 294: 73-78, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126507

RESUMO

The increased use of genetically modified organisms (GMOs) is accompanied by increased complexity of the matrices that contain GMOs. The most common DNA-based approach for GMO detection and quantification is real-time quantitative polymerase chain reaction (qPCR). However, as qPCR is sensitive to inhibitors and relies on standard curves for quantification, it has limited application in GMO quantification for complex matrices. To overcome this hurdle in DNA quantification, we present droplet digital PCR (ddPCR) assays that were designed to target 'Roundup Ready' soybean and the soybean reference gene. Three ddPCR assays were transferred from qPCR to QX100/QX200 ddPCR platforms and characterised. Together, the fitness-for-purpose study on four real-life samples and the use of a chamber-based PCR system, showed that dPCR has great potential to improve such measurements in GMO testing and monitoring of food authenticity.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real , DNA de Plantas/metabolismo , Limite de Detecção , Soja/genética
10.
Nucleic Acids Res ; 47(13): 6714-6725, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31127286

RESUMO

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.


Assuntos
Proteínas de Arabidopsis/fisiologia , RNA Polimerase II/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Sintéticos , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Mensageiro/biossíntese , RNA de Plantas/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Elongação da Transcrição Genética , Sítio de Iniciação de Transcrição
11.
BMC Genomics ; 20(1): 262, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940088

RESUMO

BACKGROUND: The cytogenomic study of repetitive regions is fundamental for the understanding of morphofunctional mechanisms and genome evolution. Passiflora edulis a species of relevant agronomic value, this work had its genome sequenced by next generation sequencing and bioinformatics analysis performed by RepeatExplorer pipeline. The clusters allowed the identification and characterization of repetitive elements (predominant contributors to most plant genomes). The aim of this study was to identify, characterize and map the repetitive DNA of P. edulis, providing important cytogenomic markers, especially sequences associated with the centromere. RESULTS: Three clusters of satellite DNAs (69, 118 and 207) and seven clusters of Long Terminal Repeat (LTR) retrotransposons of the superfamilies Ty1/Copy and Ty3/Gypsy and families Angela, Athila, Chromovirus and Maximus-Sire (6, 11, 36, 43, 86, 94 and 135) were characterized and analyzed. The chromosome mapping of satellite DNAs showed two hybridization sites co-located in the 5S rDNA region (PeSat_1), subterminal hybridizations (PeSat_3) and hybridization in four sites, co-located in the 45S rDNA region (PeSat_2). Most of the retroelements hybridizations showed signals scattered in the chromosomes, diverging in abundance, and only the cluster 6 presented pericentromeric regions marking. No satellite DNAs and retroelement associated with centromere was observed. CONCLUSION: P. edulis has a highly repetitive genome, with the predominance of Ty3/Gypsy LTR retrotransposon. The satellite DNAs and LTR retrotransposon characterized are promising markers for investigation of the evolutionary patterns and genetic distinction of species and hybrids of Passiflora.


Assuntos
DNA Satélite/genética , Passiflora/genética , Retroelementos/genética , Mapeamento Cromossômico , Cromossomos de Plantas , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA Satélite/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Filogenia , RNA Ribossômico/genética , RNA Ribossômico 5S/genética , Análise de Sequência de DNA
12.
PLoS One ; 14(4): e0214964, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31026257

RESUMO

The transcription factor selectively binds with the cis-regulatory elements of the promoter and regulates the differential expression of genes. In this study, we aimed to identify and validate the presence of GCC-box and TCC-box motifs in the promoters of upregulated differentially expressed genes (UR-DEGs) and downregulated differentially expressed genes (DR-DEGs) under anoxia using molecular beacon probe (MBP) based real-time PCR. The GCC-box motif was detected in UR-DEGs (DnaJ and 60S ribosomal protein L7 genes), whereas, the TCC-box was detected in DR-DEGs (DnaK and CPuORF11 genes). In addition, the mechanism of interaction of AP2/EREBP family transcription factor (LOC_Os03g22170) with GCC-box promoter motif present in DnaJ gene (LOC_Os06g09560) and 60S ribosomal protein L7 gene (LOC_Os08g42920); and TCC-box promoter motif of DnaK gene (LOC_Os02g48110) and CPuORF11 gene (LOC_Os02g01240) were explored using molecular dynamics (MD) simulations analysis including binding free energy calculations, principal component analyses, and free energy landscapes. The binding free energy analysis revealed that AP2/EREBP model residues such as Arg68, Arg72, Arg83, Lys87, and Arg90 were commonly involved in the formation of hydrogen bonds with GCC and TCC-box promoter motifs, suggesting that these residues are critical for strong interaction. The movement of the entire protein bound to DNA was restricted, confirming the stability of the complex. This study provides comprehensive binding information and a more detailed view of the dynamic interaction between proteins and DNA.


Assuntos
Regulação da Expressão Gênica de Plantas , Motivos de Nucleotídeos , Oryza , Proteínas de Plantas , Regiões Promotoras Genéticas , Fatores de Transcrição , Simulação por Computador , DNA de Plantas/genética , DNA de Plantas/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
PLoS One ; 14(4): e0214953, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30951558

RESUMO

Autotetraploid rice is a useful germplasm for polyploid rice breeding, however, low seed setting is the major barrier in commercial utilization of autotetraploid rice. Our research group has developed neo-tetraploid rice lines, which have the characteristics of high fertility and heterosis when crossed with autotetraploid rice. In the present study, re-sequencing and RNA-seq were employed to detect global DNA variations and differentially expressed genes (DEGs) during meiosis stage in three neo-tetraploid rice lines compared to their parents, respectively. Here, a total of 4109881 SNPs and 640592 InDels were detected in neo-tetraploid lines compared to the reference genome, and 1805 specific presence/absence variations (PAVs) were detected in three lines. Approximately 12% and 0.5% of the total SNPs and InDels identified in three lines were located in genic regions, respectively. A total of 28 genes, harboring at least one of the large-effect SNP and/or InDel which affect the integrity of the encoded protein, were identified in the three lines. Together, 324 specific mutation genes, including 52 meiosis-related genes and 8 epigenetics-related genes were detected in neo-tetraploid rice compared to their parents. Of these 324 genes, five meiosis-related and three epigenetics-related genes displayed differential expressions during meiosis stage. Notably, 498 specific transcripts, 48 differentially expressed transposons and 245 differentially expressed ncRNAs were also detected in neo-tetraploid rice. Our results suggested that genomic structural reprogramming, DNA variations and differential expressions of some important meiosis and epigenetics related genes might be associated with high fertility in neo-tetraploid rice.


Assuntos
DNA de Plantas , Regulação da Expressão Gênica de Plantas , Oryza , Tetraploidia , DNA de Plantas/genética , DNA de Plantas/metabolismo , Fertilidade/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Oryza/genética , Oryza/metabolismo
14.
mBio ; 10(2)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837342

RESUMO

Histone-linked extracellular DNA (exDNA) is a component of neutrophil extracellular traps (NETs). NETs have been shown to play a role in immune response to bacteria, fungi, viruses, and protozoan parasites. Mutation of genes encoding group A Streptococcus extracellular DNases (exDNases) results in reduced virulence in animals, a finding that implies that exDNases are deployed as counter defense against host DNA-containing NETs. Is the exDNA/exDNase mechanism also relevant to plants and their pathogens? It has been demonstrated previously that exDNA is a component of a matrix secreted from plant root caps and that plants also carry out an extracellular trapping process. Treatment with DNase I destroys root tip resistance to infection by fungi, the most abundant plant pathogens. We show that the absence of a single gene encoding a candidate exDNase results in significantly reduced virulence of a fungal plant pathogen to its host on leaves, the known infection site, and on roots. Mg2+-dependent exDNase activity was demonstrated in fungal culture filtrates and induced when host leaf material was present. It is speculated that the enzyme functions to degrade plant-secreted DNA, a component of a complex matrix akin to neutrophil extracellular traps of animals.IMPORTANCE We document that the absence of a single gene encoding a DNase in a fungal plant pathogen results in significantly reduced virulence to a plant host. We compared a wild-type strain of the maize pathogen Cochliobolus heterostrophus and an isogenic mutant lacking a candidate secreted DNase-encoding gene and demonstrated that the mutant is reduced in virulence on leaves and on roots. There are no previous reports of deletion of such a gene from either an animal or plant fungal pathogen accompanied by comparative assays of mutants and wild type for alterations in virulence. We observed DNase activity, in fungal culture filtrates, that is Mg2+ dependent and induced when plant host leaf material is present. Our findings demonstrate not only that fungi use extracellular DNases (exDNases) for virulence, but also that the relevant molecules are deployed in above-ground leaves as well as below-ground plant tissues. Overall, these data provide support for a common defense/counter defense virulence mechanism used by animals, plants, and their fungal and bacterial pathogens and suggest that components of the mechanism might be novel targets for the control of plant disease.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/crescimento & desenvolvimento , DNA de Plantas/metabolismo , Desoxirribonucleases/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Fatores de Virulência/metabolismo , Animais , Hidrólise , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Zea mays
15.
PLoS One ; 14(2): e0211471, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707722

RESUMO

Miscanthus lutarioriparius is a native perennial Miscanthus species of China, which is currently used as raw material of papermaking and bioenergy crop. It also has been considered as a promising eco-bioindustrial plant, which can offer raw material and gene for the biomass industry. However, lack of germplasm resources and genetic diversity information of M. lutarioriparius have become the bottleneck that prevents the stable and further development of the biomass industry. In the present study, genetic diversity of 153 M. lutarioriparius individuals nine populations was studied using 27 Start Codon Targeted (SCoT) markers. High polymorphic bands (97.67%), polymorphic information content (0.26) and allele number (1.88) showed SCoT as a reliable marker system for genetic analysis in M. lutarioriparius. At the species, the percentage of polymorphic loci [PPL] was 97.2%, Nei's gene diversity [H] was 0.36, Shannon index [I] was 0.54 and Expected Heterozygosity [He] was 0.56. Genetic variation within populations (84.91%) was higher than among populations (15.09%) based on analysis of molecular variance (AMOVA). Moderate level of genetic differentiation was found in M. lutarioriparius populations (Fst = 0.15), which is further confirmed by STRUCTURE, principal coordinates analysis (PCoA) and an unweighted pair group method with arithmetic mean (UPGMA) analysis that could reveal a clear separation between groups of the north and south of Yangtze River. The gene flow of the populations within the respective south and north of Yangtze River area was higher, but lower between the areas. There was no obvious correlation between genetic distance and geographic distance. The breeding systems, geographical isolation and fragmented habitat of M. lutarioriparius may be due to the high level of genetic diversity, moderate genetic differentiation, and the population, structure. The study further suggests some measure for conservation of genetic resources and provides the genetic basis for improving the efficiency of breeding based on the results of diversity analysis.


Assuntos
Variação Genética , Poaceae/genética , Teorema de Bayes , China , Análise por Conglomerados , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Fluxo Gênico , Repetições de Microssatélites/genética , Polimorfismo Genético , Análise de Componente Principal
16.
Biochim Biophys Acta Gene Regul Mech ; 1862(5): 582-597, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30753903

RESUMO

Co-ordinated interplay between Polycomb group (PcG) and Trithorax group (TrxG) of proteins regulate chromatin state and maintain the transcription "off" and "on" state of a gene in higher eukaryotes. Targeting PcG complex to a specific locus is mediated by DNA sequences known as Polycomb response elements. Interestingly, these PREs are also recognized by TrxG proteins to antagonise PcG mediated gene repression. In this study, we have characterised DNA binding property of rice trithorax group factor ULTRAPETALA1 (OsULT1) which has a SAND domain and B-box motif. Chromatin immunoprecipitation assay indicates cold induced enrichment of OsULT1 occupancy and a decrease in H3K27me3 mark in the promoter region of OsDREB1b gene, during transcription activation. OsULT1 binds to the cis motif "GAGAG", and the sequence specificity is contributed mainly by the SAND domain. GAGAG is one of the cis motifs present in PREs that are recognized by Drosophila GAGA factor and Pipsqueak. Thus, binding of OsULT1 to GAGAG motif, along with a decrease in H3K27me3 suggests that OsULT1 antagonises the repressive effect of PcG complex for transcriptional activation of OsDREB1b. Moreover, OsULT1 interacts with rice SET domain-containing methyltransferase TRX1, suggesting OsULT1 is an integral part of plant Trithorax group complex. Furthermore, the increase in ULT1 levels during environmental cues suggests its involvement in the transcriptional regulation of stress responsive genes. Collectively, these results suggest that the antagonistic functions of PcG and TrxG proteins and the mechanism of recruitment of these complexes to target loci are evolutionarily conserved for gene expression regulation across kingdoms.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Elementos de Resposta , Sítios de Ligação , Temperatura Baixa , DNA de Plantas/química , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona Metiltransferases/metabolismo , Motivos de Nucleotídeos , Oryza/enzimologia , Oryza/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Multimerização Proteica , Estresse Fisiológico/genética , Fatores de Transcrição/genética
17.
Nucleic Acids Res ; 47(8): 4308-4318, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30783673

RESUMO

In rice, the critical regulator of the salicylic acid signalling pathway is OsWRKY45, a transcription factor (TF) of the WRKY TF family that functions by binding to the W-box of gene promoters, but the structural basis of OsWRKY45/W-box DNA recognition is unknown. Here, we show the crystal structure of the DNA binding domain of OsWRKY45 (OsWRKY45-DBD, i.e. the WRKY and zinc finger domain) in complex with a W-box DNA. Surprisingly, two OsWRKY45-DBD molecules exchange ß4-ß5 strands to form a dimer. The domain swapping occurs at the hinge region between the ß3 and ß4 strands, and is bridged and stabilized by zinc ion via coordinating residues from different chains. The dimer contains two identical DNA binding domains that interact with the major groove of W-box DNA. In addition to hydrophobic and direct hydrogen bonds, water mediated hydrogen bonds are also involved in base-specific interaction between protein and DNA. Finally, we discussed the cause and consequence of domain swapping of OsWRKY45-DBD, and based on our work and that of previous studies present a detailed mechanism of W-box recognition by WRKY TFs. This work reveals a novel dimerization and DNA-binding mode of WRKY TFs, and an intricate picture of the WRKY/W-box DNA recognition.


Assuntos
DNA de Plantas/química , Proteínas de Ligação a DNA/química , Oryza/genética , Proteínas de Plantas/química , Subunidades Proteicas/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Food Chem ; 283: 596-603, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30722917

RESUMO

The spice made from the fruits of Piper nigrum L. (Piperaceae) has high economic value since the beginnings of international trade. Because its price has been increasing, adulterations with papaya seeds, cayenne pepper and maize flour were reported. These have been screened by methodologies dedicated to the detection of single adulterants lacking sensitivity and specificity. Herein we propose a specific, highly-sensitive, high-throughput and affordable qPCR-based methodology for the detection of P. nigrum contaminants (Carica papaya, Zea mays and Capsicum annuum) using plant DNA barcodes trnL and psbA-trnH. The method enables the specific detection of contaminants in a short time with low limits of detection (LOD6 values of 1, 2 and 10 Haploid Genome Equivalents). A market survey (29 samples) revealed 41% of samples contaminated, though about ¾ at very low levels indicating accidental contamination. The proposed tool will contribute to the improvement of quality of this much traded spice.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/análise , Piper nigrum/genética , Capsicum/genética , Carica/genética , Primers do DNA/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Frutas/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Zea mays/genética
19.
Plant Mol Biol ; 99(1-2): 17-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30511330

RESUMO

KEY MESSAGE: Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain. Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5' end maturation of mRNAs ascribed to the combined action of 5'→3' exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5'→3' ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-ß-lactamase and ß-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5'→3' exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5' end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Estabilidade de RNA , Ribonucleases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , DNA de Plantas/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Mutação , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/genética
20.
Food Chem ; 272: 635-642, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30309592

RESUMO

Among spices, Saffron is among the most extensively interrogated for purity and authenticity. Numerous methods have been recommended for authentication of Saffron samples and for detection of adulterants for codex compliance. However, none of these methods can fulfill both of these important quality criteria. This study describes a three step approach to achieving this goal by including the established ISO3632 method and two additional methods based on microscopic examination and DNA barcoding. We provide results showing the utility of these methods both independently and in combination for quality evaluation of 36 commercial saffron samples. Our results show that use of the ISO3632 approach alone can reveal the color and aroma but not the genetic origin of the material or distinguish between synthetic components versus natural ingredients. Also, the microscopic observation method can give a preliminary indication of saffron authenticity, but used alone it is unable to quantify purity. Finally, a relatively new method based on the use of DNA barcodes can authenticate the biological origin of the saffron, but here results may be misleading if auto-adulterating materials are present. Overall, our study reveals that through the combined use of all three methods, saffron authentication can substantially improved.


Assuntos
Crocus/química , Qualidade dos Alimentos , Crocus/classificação , Crocus/genética , Código de Barras de DNA Taxonômico , DNA de Plantas/química , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Microscopia , Filogenia , Espectrofotometria
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