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1.
Plant Mol Biol ; 101(1-2): 149-162, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267255

RESUMO

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.


Assuntos
Resistência à Doença , Malus/genética , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ascomicetos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutas/genética , Frutas/imunologia , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes Reporter , Malus/imunologia , Malus/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Pseudomonas syringae/fisiologia , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Transdução de Sinais , Tabaco/genética , Tabaco/imunologia , Tabaco/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
2.
Plant Mol Biol ; 101(1-2): 203-220, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297725

RESUMO

KEY MESSAGE: Here, a functional characterization of a wheat MSR has been presented: this protein makes a contribution to the plant's tolerance of abiotic stress, acting through its catalytic capacity and its modulation of ROS and ABA pathways. The molecular mechanism and function of certain members of the methionine sulfoxide reductase (MSR) gene family have been defined, however, these analyses have not included the wheat equivalents. The wheat MSR gene TaMSRA4.1 is inducible by salinity and drought stress and in this study, we demonstrate that its activity is restricted to the Met-S-SO enantiomer, and its subcellular localization is in the chloroplast. Furthermore, constitutive expression of TaMSRA4.1 enhanced the salinity and drought tolerance of wheat and Arabidopsis thaliana. In these plants constitutively expressing TaMSRA4.1, the accumulation of reactive oxygen species (ROS) was found to be influenced through the modulation of genes encoding proteins involved in ROS signaling, generation and scavenging, while the level of endogenous abscisic acid (ABA), and the sensitivity of stomatal guard cells to exogenous ABA, was increased. A yeast two-hybrid screen, bimolecular fluorescence complementation and co-immunoprecipitation assays demonstrated that heme oxygenase 1 (HO1) interacted with TaMSRA4.1, and that this interaction depended on a TaHO1 C-terminal domain. In plants subjected to salinity or drought stress, TaMSRA4.1 reversed the oxidation of TaHO1, activating ROS and ABA signaling pathways, but not in the absence of HO1. The aforementioned properties advocate TaMSRA4.1 as a candidate for plant genetic enhancement.


Assuntos
Heme Oxigenase-1/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Triticum/enzimologia , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Secas , Perfilação da Expressão Gênica , Heme Oxigenase-1/genética , Metionina Sulfóxido Redutases/genética , Oxirredução , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Tolerância ao Sal , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Triticum/genética , Triticum/fisiologia , Técnicas do Sistema de Duplo-Híbrido
3.
Dis Aquat Organ ; 135(1): 59-70, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31244485

RESUMO

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate-early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.


Assuntos
Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Antivirais , Dissulfetos , Técnicas do Sistema de Duplo-Híbrido
4.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
5.
Plant Sci ; 285: 165-174, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203881

RESUMO

The TPL/TPR co-repressor is involved in many plant signaling pathways, including those regulating the switch from vegetative to reproductive growth. Here, a TPL homolog (TPL 1-2) was isolated from chrysanthemum. Its product was found to be deposited in the nucleus. The abundance of TPL1-2 transcript varied across the plant, with its highest level being recorded in the stem apex, and its lowest in the root and stem. In the leaf, the abundance of TPL1-2 transcript was highest at dusk in plants exposed to long days, and at dawn in those exposed to short days. Site-directed mutagenesis was used to induce an N176H mutation in TPL1-2. The constitutive expression in Arabidopsis thaliana of the wild type and the mutated alleles of TPL1-2 had a contrasting effect on flowering time, with the mutant transgene expressors flowering later than the wild type transgene expressors. The flowering-related genes FT, TSF, FUL and AP1 were all more strongly transcribed in the mutant transgene expressors than in the wild type transgene expressors.


Assuntos
Chrysanthemum/genética , Flores/crescimento & desenvolvimento , Genes de Plantas/genética , Proteínas de Plantas/genética , Arabidopsis , Chrysanthemum/crescimento & desenvolvimento , Chrysanthemum/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/fisiologia , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido
6.
Plant Sci ; 285: 184-192, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203883

RESUMO

Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.


Assuntos
Diterpenos/metabolismo , Farnesiltranstransferase/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Clonagem Molecular , Filogenia , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Tripterygium/enzimologia , Tripterygium/genética , Técnicas do Sistema de Duplo-Híbrido
7.
Plant Sci ; 285: 26-33, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203891

RESUMO

Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHsp90s family in soybean and that GmHsp90A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHsp90A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T3 transgenic soybean plants overexpressing GmHsp90A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T2 homozygous GmHsp90A2-knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1.


Assuntos
Proteínas de Choque Térmico/fisiologia , Proteínas de Plantas/fisiologia , Soja/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Clorofila/metabolismo , Edição de Genes , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Malondialdeído/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Soja/fisiologia , Técnicas do Sistema de Duplo-Híbrido
8.
Nat Commun ; 10(1): 2848, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253759

RESUMO

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.


Assuntos
Medicago truncatula/genética , Medicago truncatula/microbiologia , Proteínas de Plantas/metabolismo , Sinorhizobium meliloti/fisiologia , Agrobacterium , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Nodulação/genética , Nodulação/fisiologia , Raízes de Plantas , Simbiose/fisiologia , Técnicas do Sistema de Duplo-Híbrido
9.
Plant Mol Biol ; 101(1-2): 41-61, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31183604

RESUMO

KEY MESSAGE: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.


Assuntos
Defensinas/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Triticum/genética , DNA Complementar/genética , Grão Comestível/genética , Grão Comestível/metabolismo , Genes Reporter , Hordeum/genética , Hordeum/metabolismo , Especificidade de Órgãos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Triticum/metabolismo , Técnicas do Sistema de Duplo-Híbrido
10.
Plant Physiol Biochem ; 141: 231-239, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195253

RESUMO

Anther/pollen development is a highly programmed process in flowering plants. However, the molecular mechanism of regulating anther/pollen development is still largely unclear so far. Here, we report a cotton WRKY transcription factor (GhWRKY22) that functions in anther/pollen development. Quantitative RT-PCR and GUS activity analyses revealed that GhWRKY22 is predominantly expressed in the late developing anther/pollen of cotton. The transgenic Arabidopsis plants expressing GhWRKY22 displayed the male fertility defect with the fewer viable pollen grains. Expression of the genes involved in jasmonate (JA) biosynthesis was up-regulated, whereas expression of the JA-repressors (JAZ1 and JAZ8) was down-regulated in the transgenic Arabidopsis plants expressing GhWRKY22, compared with those in wild type. Yeast one-hybrid and ChIP-qPCR assays demonstrated that GhWRKY22 modulated the expression of JAZ genes by directly binding to their promoters for regulating anther/pollen development. Yeast two-hybrid assay indicated that GhMYB24 could interact with GhJAZ8-A and GhJAZ13-A. Furthermore, expression of AtMYB24, AtPAL2 and AtANS2 was enhanced in the transgenic Arabidopsis plants, owing to GhWRKY22 overexpression. Taking the data together, our results suggest that GhWRKY22 acts as a transcriptional repressor to regulate anther/pollen development possibly by modulating the expression of the JAZ genes.


Assuntos
Gossypium/metabolismo , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hipocótilo/metabolismo , Oxilipinas/metabolismo , Fenótipo , Infertilidade das Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/metabolismo , Ativação Transcricional , Transgenes , Técnicas do Sistema de Duplo-Híbrido
11.
Plant Sci ; 284: 1-8, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084862

RESUMO

Tanshinones are the main bioactive diterpenes in Salvia miltiorrhiza Bunge, are widely used for treating cardiovascular and cerebrovascular diseases. However, the biosynthetic mechanisms of these compounds have not yet been fully explained. In this study, a transcription factor named SmWRKY2 was isolated and functionally characterized. Multiple sequence analysis indicated it was classified into subgroup I of the WRKY family. Expression pattern showed that SmWRKY2 was mainly expressed in the stem and leaf and was inducible by methyl jasmonate (MeJA) treatment. Subcellular localization showed that SmWRKY2 was localized in the nucleus. Overexpression of SmWRKY2 in S. miltiorrhiza hairy roots significantly increased the expression of SmDXS2 and SmCPS, resulting in increased accumulation of tanshinones and the highest total tanshinone content was detected in OE-SmWRKY2-1 line, which was 1.83 times of the control. Meanwhile, tanshinone production was slightly reduced in the antisense-SmWRKY2 line. Dual-Luciferase assay showed that SmWRKY2 can positively regulate SmDXS2 and SmCPS expression, However, Y1H and EMSA experiments indicate that SmWRKY2 only binds to the W-box of the SmCPS promoter. Our study shows that SmWRKY2 is a positive regulator of tanshinone biosynthesis by mainly activating SmCPS. This study thus sheds new light on the regulatory role of SmWRKY2 in tanshinone biosynthesis.


Assuntos
Diterpenos de Abietano/biossíntese , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Salvia miltiorrhiza/metabolismo , Fatores de Transcrição/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Técnicas do Sistema de Duplo-Híbrido
12.
Plant Sci ; 284: 127-134, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084865

RESUMO

Avr9/Cf-9-INDUCED F-BOX1 (ACIF1) was first identified during screening of Avr9/Cf-9-elicited genes in tobacco. Further analysis revealed that ACIF1 was required for hypersensitive responses triggered by various elicitors in tobacco and tomato, indicating that it may be involved in various disease resistance. Here, we cloned its cotton (Gossypium hirsutum) homolog GhACIF1, which encodes an F-box protein. We show that GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. Disease resistance assays show that GhACIF1 enhances resistance to Verticillium dahliae in Arabidopsis plants, while silencing of GhACIF1 confers sensitivity to V. dahliae in cotton. Further analysis show that PevD1 elicitor activates hypersensitive and acquired immune response mediated by GhACIF1. Collectively, these results indicate that GhACIF1 contributes to protection against V. dahliae infection.


Assuntos
Resistência à Doença , Proteínas F-Box/fisiologia , Gossypium/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/fisiologia , Verticillium , Resistência à Doença/fisiologia , Proteínas F-Box/genética , Inativação Gênica , Gossypium/genética , Gossypium/microbiologia , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-Híbrido , Verticillium/metabolismo
13.
Plant Sci ; 284: 192-202, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084872

RESUMO

In rice, OsBBX14, a B-box (BBX) transcription factor, reportedly delays heading. Here, we revealed that OsBBX14 positively regulates rice photomorphogenesis. The OsBBX14-overexpressing (OsBBX14-OX) seedlings were hypersensitive to light, especially blue light, and exhibited dwarfism, while the OsBBX14 knock-out plants (osbbx14) were taller than wild-type plants under blue light. Histological analyses indicated that the observed dwarfism was mainly due to decreased cell length. Additionally, OsBBX14 abundance (mRNA and protein levels) was influenced by different light wavelengths in a time-dependent manner. The expression levels of HY5Ls (LONG HYPOCOTYL 5 LIKE) and ELIPs (EARLY LIGHT-INDUCIBLE PROTEIN) genes, whose Arabidopsis thaliana homologs function as positive regulators in the light signaling pathway, were significantly upregulated in OsBBX14-OX lines. In contrast, the expression of genes related to cell wall organization and dwarfism was downregulated in OsBBX14-OX lines. Chromatin immunoprecipitation (ChIP) assays confirmed that OsBBX14 binds to the T/G-box of HY5L1 (LONG HYPOCOTYL 5 LIKE 1) promoter. LUC complementation imaging (LCI) results suggested that OsBBX14 had physical interaction with OsCRY2 protein. Collectively, in response to blue light, OsBBX14 promotes photomorphogenesis, probably by directly or indirectly regulating the expression of HY5L1 or other genes related to cell wall organization and dwarfism.


Assuntos
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Tabaco , Técnicas do Sistema de Duplo-Híbrido
14.
Plant Sci ; 284: 91-98, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084884

RESUMO

Seeds germination or dormancy is strictly controlled by endogenous phytohormone signal and environment cues. High temperature (HT) suppresses seeds germination or triggers seeds dormancy but underlying mechanism by which HT mediates seeds germination thermoinhibition needs more investigating. SOM is reported as the critical factor negatively controls light-irradiation seeds germination by altering Abscisic acid (ABA) and gibberellin acid (GA) biosynthesis. Here we found that HT accelerates SOM expressing through ABA signal transduction component ABI3, both of abi3 and som mutants seeds show high germination rate under HT in contrast to wild type seeds. Using ABI3 as the bait, we identified the epigenetic factor Powerdress (PWR) as the ABI3 interaction protein. Genetic and physiological analysis showed that PWR negatively control the expressing of SOM, and overexpressing PWR enhanced, while pwr mutant reduced, seeds germination thermotolerance. Without HT stress, PWR accelerated the histone H3 deacetylation level and H2A.Z deposition at SOM locus, and thus suppressed ABI3-dependent SOM transcription for seeds germination, HT stress block PWR transcriptional level, thus attenuated the inhibition effect of PWR on SOM expressing, resulting into seeds germination thermoinhibition. Thus our finding propose a new function of PWR in controlling seeds germination under HT through histone acetylation modification and H2A.Z deposition.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Germinação , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Proteínas de Transporte/fisiologia , Germinação/fisiologia , Resposta ao Choque Térmico , Código das Histonas , Plantas Geneticamente Modificadas , Sementes/metabolismo , Fatores de Transcrição/fisiologia , Técnicas do Sistema de Duplo-Híbrido
15.
BMC Plant Biol ; 19(1): 202, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096905

RESUMO

BACKGROUND: The Fertilization-related kinases (FRK) form a class that belongs to the MEKK subfamily of plant MAPKKKs. It was recently shown that FRK class kinases expanded during angiosperm evolution, reaching their maximum numbers in the lineage leading to solanaceous species and culminating in the Solanum genus where they account for more than 40% of the total MEKKs. The first members studied, ScFRK1 and ScFRK2 were shown to play a pivotal role in gametophyte development in the wild potato species Solanum chacoense. RESULTS: ScFRK3 is also involved in gametophyte development. ScFRK3 is expressed in developing pollen and young ovules, reaching its highest level immediately after meiosis and during the mitosis steps in both gametophytes. Hence, three independent lines of ScFRK3 RNAi mutant plants showed decreased number of seeds per fruit. We also observed an important number of degenerated embryo sac in mature ovary. Analysis of ovule development showed that most embryo sac did not enter mitosis I in ScFRK3 RNAi mutant plants. Severe lethality was also observed during male gametophyte development, pollen being arrested before mitosis I, as observed in the female gametophyte. Obvious defects in vegetative organs were not observed, emphasizing the reproductive roles of the FRK class kinases. To isolate MAP kinases acting downstream of ScFRK3, a de novo S. chacoense transcriptome from male and female reproductive organs was assembled. Of the five ScMKKs and 16 ScMPKs retrieved, only the ScMKK3 interacted with ScFRK3, while only the ScMPK13 interacted with ScMKK3, leading to an apparent single three-tiered canonical MAP kinase cascade combination involving ScFRK3-ScMKK3-ScMPK13. CONCLUSIONS: The ScFRK3 MAPKKK is involved in a signaling cascade that regulates both male and female gamete development, and most probably act upstream of ScMKK3 and ScMPK13.


Assuntos
Óvulo Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Pólen/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Solanum/crescimento & desenvolvimento , Hibridização In Situ , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , RNA de Plantas/metabolismo , Solanum/enzimologia , Solanum/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052176

RESUMO

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Canais de Sódio/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Canais de Potássio/genética , Saccharomyces cerevisiae/metabolismo , Canais de Sódio/genética
17.
Plant Sci ; 283: 11-22, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128680

RESUMO

Chlorophyll plays a vital role in harvesting light and turning it into chemical energy. In this study, we isolated and characterized a chlorophyll-deficient mutant, which we named cs3 (chlorotic seedling 3). The cs3 mutant seedlings exhibit a yellowish phenotype at germination, and they do not survive at the seedling stage. In addition, brown necrotic spots appear on the surface of the leaves and leaf sheaths during development. DAB staining and H2O2 content measurement showed that there was excessive H2O2 accumulation in the cs3 mutant leaf. Accompanying the chlorophyll deficiency, the chloroplasts in cs3 leaf cells were abnormal. Using a map-based cloning strategy, we mapped the CS3 gene, which encodes a Ycf54 domain-containing protein, to a locus on chromosome 3. CS3 is mainly expressed in green tissues and the S136 F would influence CS3 interacting with YGL8 and its chloroplast localization. qRT-PCR analysis revealed the changes in the expression of genes involved in chlorophyll biosynthesis and degradation, chloroplast development, senescence, and photosynthesis in the cs3 mutant. In addition, our study also supports the notion that the mutation in the CS3/Ycf54 gene arrests chlorophyll biosynthesis by negatively affecting the activity of magnesium protoporphyrin IX monomethylester cyclase (MgPME-cyclase).


Assuntos
Clorofila/biossíntese , Oryza/metabolismo , Proteínas de Plantas/fisiologia , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Genes de Plantas/genética , Mutação , Oryza/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Técnicas do Sistema de Duplo-Híbrido
18.
Plant Sci ; 283: 116-126, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128681

RESUMO

Ethylene response factors (ERFs) are known to regulate fruit ripening. However, the ERF regulatory networks are not clear. In this study, we have shown that peach (Prunus persica) PpeERF2 regulates fruit ripening through suppressing the expression of two ABA biosynthesis genes (PpeNCED2, PpeNCED3) and a cell wall degradation gene (PpePG1). The transcript levels of PpeERF2 in fruit were opposite to that of PpeNCED2, PpeNCED3 and PpePG1 during ripening and in response to various ripening treatments. PpeERF2 was found to bind to the PpeNCED2, PpeNCED3 and PpePG1 promotors as demonstrated by yeast one-hybrid (Y1H) and EMSA assays; and further found to repress the promoter activities of the three genes in tobacco leaf tissues after Agrobacterium infiltration. Taken together, these results provide new information for a better understanding of the crosstalk network between ethylene signaling, cell wall degradation and ABA biosynthesis during fruit ripening.


Assuntos
Ácido Abscísico/biossíntese , Parede Celular/metabolismo , Frutas/metabolismo , Proteínas de Plantas/fisiologia , Prunus persica/metabolismo , Proteínas Repressoras/fisiologia , Ácido Abscísico/metabolismo , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Frutas/crescimento & desenvolvimento , Frutas/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Prunus persica/genética , Prunus persica/crescimento & desenvolvimento , Prunus persica/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido
19.
Plant Sci ; 283: 147-156, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128684

RESUMO

Harpin proteins secreted by plant-pathogenic gram-negative bacteria induce diverse plant defenses against different pathogens. Harpin-induced 1 (HIN1) gene highly induced in tobacco after application of Harpin protein is involved in a common plant defense pathway. However, the role of HIN1 against Tobacco mosaic virus (TMV) remains unknown. In this study, we functionally characterized the Nicotiana benthamiana HIN1 (NbHIN1) gene and generated the transgenic tobacco overexpressing the NbHIN1 gene. In a subcellular localization experiment, we found that NbHIN1 localized in the plasma membrane and cytosol. Overexpression of NbHIN1 did not lead to observed phenotype compared to wild type tobacco plant. However, the NbHIN1 overexpressing tobacco plant exhibited significantly enhanced resistance to TMV infection. Moreover, RNA-sequencing revealed the transcriptomic profiling of NbHIN1 overexpression and highlighted the primary effects on the genes in the processes related to biosynthesis of amino acids, plant-pathogen interaction and RNA transport. We also found that overexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signaling pathway might be involved in TMV resistance. Taken together, for the first time we demonstrated that overexpressing a pathogenesis-related gene NbHIN1 in N. benthamiana significantly enhances the TMV resistance, providing a potential mechanism that will enable us to engineer tobacco with improved TMV resistance in the future.


Assuntos
Ciclopentanos/metabolismo , Resistência à Doença/genética , Oxilipinas/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Transdução de Sinais , Vírus do Mosaico do Tabaco , Tabaco/genética , Western Blotting , Clonagem Molecular , Genes de Plantas/genética , Genes de Plantas/fisiologia , Microscopia Confocal , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Tabaco/metabolismo , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido
20.
Plant Sci ; 283: 32-40, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128702

RESUMO

Light is an important environmental factor affecting plant growth and development. Additionally, HY5 is a central factor that coordinates light signal transduction and regulates the expression of flower color-related genes. However, there are few reports describing the co-regulation of apple fruit coloration by MdHY5 and MYB transcription factors. In this study, we detected a light-inducible gene, MdMYBDL1, which encodes a MYB-like domain and is homologous to AtMYBD in Arabidopsis thaliana. Moreover, we observed that MdHY5 binds to the G-box element of the MdMYBDL1 promoter to upregulate expression. The overexpression of MdMYBDL1 enhanced anthocyanin accumulation in apple calli and inhibited the expression of MdMYB16 and its homolog, MdMYB308. Furthermore, MdMYB16 can form a dimer with MdMYB308 and functions as a negative regulator of anthocyanin biosynthesis. Interestingly, MdMYB16 and MdMYB308 promoter activities were inhibited by MdMYBDL1 and MdHY5. These findings imply that MdHY5 responds to light signals and functions upstream of different types of MYB transcription factors, ultimately regulating anthocyanin accumulation in apples.


Assuntos
Antocianinas/metabolismo , Malus/metabolismo , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Glucuronidase/metabolismo , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
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