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1.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478725

RESUMO

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.


Assuntos
Aspergillus oryzae/genética , Bioquímica/métodos , Proteínas Fúngicas/biossíntese , Mutação/genética , Aspergillus oryzae/metabolismo , DNA/metabolismo , Polietilenoglicóis/química , Protoplastos/metabolismo , Esporos Fúngicos/metabolismo
2.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218171

RESUMO

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.


Assuntos
Orchidaceae/metabolismo , Protoplastos/metabolismo , Separação Celular , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Orchidaceae/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica
3.
BMC Plant Biol ; 20(1): 11, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910821

RESUMO

BACKGROUND: NAD kinases (NADKs) are the only known enzymes that directly phosphorylate NAD(H) to generate NADP(H) in different subcellular compartments. They participate in multiple life activities, such as modulating the NADP/NAD ratio, maintaining the intracellular redox balance and responding to environmental stresses. However, the functions of individual NADK in plants are still under investigation. Here, a rice NADK, namely, OsNADK1, was identified, and its functions in plant growth regulation and stress tolerance were analysed by employing a series of transgenic plant lines. RESULTS: OsNADK1 is a cytosol-localized NADK in rice. It was expressed in all rice tissues examined, and its transcriptional expression could be stimulated by a number of environmental stress treatments. Compared with wild-type (WT) rice, the mutant plant osnadk1 in which OsNADK1 was knocked out was a dwarf at the heading stage and had decreased NADP(H)/NAD(H), ascorbic acid (ASA)/dehydroascorbate (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, which led to increased oxidation states in the rice cells and sensitivity to drought. Moreover, certain stress-related genes showed differential expression patterns in osnadk1 under both normal growth and drought-stress conditions compared with WT. Among these genes, OsDREB1B and several WRKY family transcription factors, e.g., OsWRKY21 and OsWRKY42, showed correlated co-expression patterns with OsNADK1 in osnadk1 and the plants overexpressing or underexpressing OsNADK1, implying roles for these transcription factors in OsNADK1-mediated processes. In addition, overexpression of OsNADK1 enhanced the drought tolerance of rice plants, whereas loss of function of the gene reduced the tolerance. Furthermore, the proline content was dramatically increased in the leaves of the OsNADK1-overexpressing lines under drought conditions. CONCLUSIONS: Altogether, the results suggest that an OsNADK1-mediated intracellular redox balance is involved in the tolerance of rice plants to drought.


Assuntos
Secas , NAD , Oryza/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Estresse Fisiológico/genética , Clonagem Molecular/métodos , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , NAD/genética , NAD/metabolismo , Oryza/metabolismo , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Protoplastos/citologia , Protoplastos/metabolismo , Transcriptoma
4.
Planta ; 251(1): 26, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31797121

RESUMO

MAIN CONCLUSION: Silencing of CI-sHsps by RNAi negatively affected the seed germination process and heat stress response of rice seedlings. Seed size of RNAiCI-sHsp was reduced as compared to wild-type plants. Small heat shock proteins (sHsps) are the ATP-independent chaperones ubiquitously expressed in response to diverse environmental and developmental cues. Cytosolic sHsps constitute the major repertoire of sHsp family. Rice cytosolic class I (CI)-sHsps consists of seven members (Hsp16.9A, Hsp16.9B, Hsp16.9C, Hsp17.4, Hsp17.7, Hsp17.9A and Hsp18). Purified OsHsp17.4 and OsHsp17.9A proteins exhibited chaperone activity by preventing formation of large aggregates with model substrate citrate synthase. OsHsp16.9A and OsHsp17.4 showed nucleo-cytoplasmic localization, while the localization of OsHsp17.9A was preferentially in the nucleus. Transgenic tobacco plants expressing OsHsp17.4 and OsHsp17.9A proteins and Arabidopsis plants ectopically expressing OsHsp17.4 protein showed improved thermotolerance to the respective trans-hosts during the post-stress recovery process. Single hairpin construct was designed to generate all CI-sHsp silenced (RNAiCI-sHsp) rice lines. The major vegetative and reproductive attributes of the RNAiCI-sHsp plants were comparable to the wild-type rice plants. Basal and acquired thermotolerance response of RNAiCI-sHsp seedlings of rice was mildly affected. The seed length of RNAiCI-sHsp rice plants was significantly reduced. The seed germination process was delayed and seed thermotolerance of RNAiCI-sHsp was negatively affected than the non-transgenic seeds. We, thus, implicate that sHsp genes are critical in seedling thermotolerance and seed physiology.


Assuntos
Inativação Gênica , Proteínas de Choque Térmico Pequenas/metabolismo , Oryza/genética , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Plântula/fisiologia , Sementes/fisiologia , Termotolerância/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Citrato (si)-Sintase/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Proteínas de Choque Térmico Pequenas/genética , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Multimerização Proteica , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Termotolerância/genética , Tabaco/genética , Transcriptoma/genética
5.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801204

RESUMO

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Pogostemon/genética , Proteínas Repressoras/genética , Sesquiterpenos/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Inativação Gênica , Isomerases/genética , Isomerases/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Pogostemon/classificação , Pogostemon/efeitos dos fármacos , Pogostemon/metabolismo , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Técnicas do Sistema de Duplo-Híbrido
6.
Environ Sci Pollut Res Int ; 26(36): 36680-36687, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31741272

RESUMO

Transporters play an important role in the uptake and redistribution of agrochemicals to the site of insect feeding. The product of the Arabidopsis thaliana gene AtAAP1 substantially contributes to inorganic nitrogen acquisition under ecologically relevant amino acid concentrations. Here, the transporter ability of AtAAP1 to a chlorantraniliprole-alanine conjugate (CAP-Ala-1) was tested both in planta and in vitro. Thirty-day-old and 15-day-old plants overexpressing AtAAP1 increased the uptake of CAP-Ala-1 into the roots, whereas AtAAP1 deficiency did not completely block the uptake of CAP-Ala-1. An uptake experiment carried out in Xenopus laevis oocytes expressing AtAAP1 showed that CAP-Ala-1 interacted with AtAAP1. Although little native AtAAP1 transcription was present in the leaves, constitutive expression of AtAAP1 in plants significantly increased the ability of the leaf mesophyll protoplasts to take up CAP-Ala-1. The observations supported the possibility of exploiting AtAAP1 as a component of a novel delivery and redistribution system for amino acid-based pesticide conjugates.


Assuntos
Alanina/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ortoaminobenzoatos/metabolismo , Alanina/química , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Animais , Transporte Biológico/genética , Expressão Gênica , Inseticidas/química , Inseticidas/metabolismo , Oócitos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Xenopus laevis , ortoaminobenzoatos/química
7.
Int J Mol Sci ; 20(21)2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31690047

RESUMO

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.


Assuntos
Parede Celular/efeitos dos fármacos , Daucus carota/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Parede Celular/metabolismo , Daucus carota/citologia , Pectinas/metabolismo , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo
8.
BMC Biotechnol ; 19(1): 71, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31684940

RESUMO

BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma de Planta/genética , RNA Guia/genética , Triticum/genética , Protoplastos/metabolismo
9.
Molecules ; 24(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470600

RESUMO

Small signaling peptides (SSPs) are a class of short peptides playing critical roles in plant growth and development. SSPs are also involved in the phytohormone signaling pathway. However, identification of mature SSPs is still a technical challenge because of their extremely low concentrations in plant tissue and complicated interference by many other metabolites. Here, we report an optimized protocol to extract SSPs based on protoplast extraction and to analyze SSPs based on tandem mass spectrometry peptidomics. Using plant protoplasts as the material, soluble peptides were directly extracted into phosphate buffer. The interference of non-signaling peptides was significantly decreased. Moreover, we applied the protocol to identify potential SSPs in auxin treated wild type and auxin biosynthesis defective mutant yuc2yuc6. Over 100 potential SSPs showed a response to auxin in Arabidopsis thaliana.


Assuntos
Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Oligopeptídeos/isolamento & purificação , Reguladores de Crescimento de Planta/farmacologia , Transdução de Sinais/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/classificação , Expressão Gênica , Perfilação da Expressão Gênica , Ácidos Indolacéticos/metabolismo , Oligopeptídeos/biossíntese , Oligopeptídeos/classificação , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteômica/métodos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Transdução de Sinais/genética
10.
Biochemistry (Mosc) ; 84(7): 817-828, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31509731

RESUMO

Natural competence of mitochondria for DNA uptake has been known for the last 20 years. Until the present time, all studies of this process have been conducted exclusively in isolated mitochondria, as no system for investigation of the DNA transport into the mitochondria in intact cells has been available. The objective of this work was to improve and standardize the existing approaches for investigating DNA import into plant mitochondria in an in organello system. A method for detecting the import of fluorescently labeled DNA substrates has been developed. Based on the features of DNA import into the mitochondria, we suggested an efficient method for the evaluation of the DNA import efficiency by quantitative PCR. We also developed and characterized the in vivo system that allows to detect DNA transport from the cytoplasm to the mitochondrial matrix in Arabidopsis thaliana protoplasts. A combination of the proposed techniques for studying the DNA uptake by plant mitochondria might be useful for elucidating whether the properties of the mitochondrial DNA import established in the in organello system are preserved in vivo.


Assuntos
Arabidopsis/metabolismo , Transporte Biológico/genética , Brassica napus/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Zea mays/metabolismo , Genoma Mitocondrial , Genoma de Planta , Técnicas In Vitro/métodos , Proteínas Mitocondriais/genética , Células Vegetais/metabolismo , Protoplastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Coloração e Rotulagem
11.
Plant Sci ; 286: 1-6, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300135

RESUMO

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.


Assuntos
Adaptação Fisiológica/efeitos da radiação , Arabidopsis/metabolismo , Oryza/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Oryza/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Protoplastos/metabolismo , Protoplastos/efeitos da radiação
12.
New Phytol ; 224(2): 833-847, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31318449

RESUMO

The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Padrões Moleculares Associados a Patógenos/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relação Dose-Resposta a Droga , Flagelina/química , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Padrões Moleculares Associados a Patógenos/administração & dosagem , Padrões Moleculares Associados a Patógenos/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Protoplastos/metabolismo
13.
Transgenic Res ; 28(Suppl 2): 61-64, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31321685

RESUMO

Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.


Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/tendências , Ribonucleoproteínas/genética , Agrobacterium/genética , Genoma de Planta/genética , Mutação , Protoplastos/metabolismo
14.
Curr Protoc Mol Biol ; 127(1): e89, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237422

RESUMO

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.


Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Marcação de Genes/métodos , Genes Sintéticos/genética , Oryza/genética , Ativação Transcricional , Sistemas CRISPR-Cas/genética , Genes de Plantas/genética , Regiões Promotoras Genéticas , Protoplastos/metabolismo , RNA Guia/genética
15.
Mol Biol Cell ; 30(16): 2053-2064, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31216223

RESUMO

The cytokinetic ring generates tensile force that drives cell division, but how tension emerges from the relatively disordered ring organization remains unclear. Long ago, a musclelike sliding filament mechanism was proposed, but evidence for sarcomeric order is lacking. Here we present quantitative evidence that in fission yeast, ring tension originates from barbed-end anchoring of actin filaments to the plasma membrane, providing resistance to myosin forces that enables filaments to develop tension. The role of anchoring was highlighted by experiments on isolated fission yeast rings, where sections of ring became unanchored from the membrane and shortened ∼30-fold faster than normal. The dramatically elevated constriction rates are unexplained. Here we present a molecularly explicit simulation of constricting partially anchored rings as studied in these experiments. Simulations accurately reproduced the experimental constriction rates and showed that following anchor release, a segment becomes tensionless and shortens via a novel noncontractile reeling-in mechanism at about the velocity of load-free myosin II. The ends are reeled in by barbed end-anchored actin filaments in adjacent segments. Other actin anchoring schemes failed to constrict rings. Our results quantitatively support a specific organization and anchoring scheme that generate tension in the cytokinetic ring.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Citocinese , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Trifosfato de Adenosina/metabolismo , Fenômenos Biomecânicos , Modelos Biológicos , Miosina Tipo II/metabolismo , Protoplastos/metabolismo , Sarcômeros/metabolismo
16.
BMC Biotechnol ; 19(1): 36, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208390

RESUMO

BACKGROUND: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. RESULTS: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. CONCLUSIONS: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Mutação , Folhas de Planta/citologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Protoplastos/citologia , Tabaco/citologia , Tabaco/genética
17.
New Phytol ; 224(1): 306-320, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31225911

RESUMO

Rice (Oryza sativa) is a facultative short-day (SD) plant, flowering early under SD and late under long-day (LD) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD. Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post-transcriptional level. We found that constitutive expression of Ghd7 delays flowering in the wild-type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co-expressing OsGIGANTEA (OsGI) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and OsGI play antagonist roles in regulating Ghd7 protein stability and flowering time. We show that OsPHYA, OsPHYB and OsGI could directly interact with Ghd7. Interestingly, OsPHYA and OsPHYB could inhibit the interaction between OsGI and Ghd7, thus helping to stabilize Ghd7 protein. Our results revealed a new level of Ghd7 regulation by phytochromes and OsGI in photoperiodic control of flowering in rice.


Assuntos
Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/fisiologia , Fotoperíodo , Fitocromo/metabolismo , Proteínas de Plantas/genética , Transcrição Genética , Transporte Ativo do Núcleo Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Modelos Biológicos , Oryza/anatomia & histologia , Oryza/efeitos da radiação , Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Protoplastos/metabolismo , Protoplastos/efeitos da radiação , Transcrição Genética/efeitos da radiação
18.
Methods Mol Biol ; 1998: 163-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250301

RESUMO

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Protoplastos/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Western Blotting/métodos , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Vetores Genéticos/genética , Mutação , Raízes de Plantas/citologia , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
19.
Methods Mol Biol ; 2014: 253-266, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31197802

RESUMO

Sucrose transport across membranes requires the activity of transport proteins. Sucrose-specific SWEET proteins mediate sugar efflux out of the cytosol and SUC proteins catalyze the uptake of sucrose from the apoplast. Both transport processes are involved in phloem loading in source leaves as well as in the post-phloem pathway in sink tissues. An important step during the characterization of new sucrose transporters is to analyze their transport activity. This is usually achieved by heterologous expression of the respective gene in yeast cells or Xenopus oocytes and subsequent uptake measurements. However, in some cases, mistargeting to internal membranes or the lack of protein modifications and/or interaction partners in the heterologous system can interfere with uptake analyses. Therefore, a new in planta method was developed that is based on mesophyll protoplasts as expression system and the fluorescent sucrose analog esculin to monitor uptake activities by confocal microscopy. In this chapter we describe the design of constructs required to analyze sucrose transporters in protoplasts, the experimental setup of the protoplast-esculin assay, and the quantitative evaluation of the obtained data. The quantification of esculin uptake allows the application of the new assay to a variety of questions, e.g., by comparison of point mutants, splice variants, or transporters with and without interaction partners.


Assuntos
Bioensaio , Esculina/metabolismo , Protoplastos/metabolismo , Sacarose/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Microscopia Confocal , Floema/metabolismo , Folhas de Planta/metabolismo
20.
Genome Res ; 29(8): 1343-1351, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31186303

RESUMO

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.


Assuntos
DNA/genética , Análise em Microsséries/métodos , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Animais , DNA/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Marcadores Genéticos , Humanos , Meiose , Análise em Microsséries/instrumentação , Plasmídeos/química , Plasmídeos/metabolismo , Ploidias , Populus/citologia , Ligação Proteica , Protoplastos/citologia , Protoplastos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo
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