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1.
Nat Commun ; 11(1): 4393, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879321

RESUMO

Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.


Assuntos
Cladosporium , Resistência à Doença/genética , Peptídeo Hidrolases/genética , Imunidade Vegetal/genética , Solanum , Tabaco , Cladosporium/genética , Cladosporium/metabolismo , Cladosporium/patogenicidade , Evolução Molecular , Proteínas Fúngicas/metabolismo , Genes de Plantas , Interações Hospedeiro-Parasita , Peptídeo Hidrolases/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inibidores de Proteases/metabolismo , Solanum/genética , Solanum/metabolismo , Solanum/microbiologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/microbiologia
2.
PLoS Pathog ; 16(8): e1008780, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866188

RESUMO

Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N. benthamiana and rice. Further analysis showed that NbUBL5.1 impaired the function of p3 as a suppressor of silencing by degrading it through the 26S proteasome. NbUBL5.1 and OsUBL5 interacted with RPN10 and RPN13, the receptors of ubiquitin in the 26S proteasome. Furthermore, silencing of NbRPN10 or NbRPN13 compromised the degradation of p3 mediated by NbUBL5.1. Together, the results suggest that UBL5 mediates the degradation of RSV p3 protein through the 26S proteasome, a previously unreported plant defense strategy against RSV infection.


Assuntos
Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Tenuivirus/metabolismo , Tabaco/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Repressoras/genética , Tenuivirus/genética , Tabaco/genética , Ubiquitinas/genética , Proteínas Virais/genética
3.
PLoS One ; 15(7): e0236943, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735612

RESUMO

Halophyte Lobularia maritima LmSAP encodes an A20AN1 zinc-finger stress-associated protein which expression is up-regulated by abiotic stresses and heavy metals in transgenic tobacco. To deepen our understanding of LmSAP function, we isolated a 1,147 bp genomic fragment upstream of LmSAP coding sequence designated as PrLmSAP. In silico analyses of PrLmSAP revealed the presence of consensus CAAT and TATA boxes and cis-regulatory elements required for abiotic stress, phytohormones, pathogen, and wound responses, and also for tissue-specific expression. The PrLmSAP sequence was fused to the ß-glucuronidase (gusA) reporter gene and transferred to rice. Histochemical GUS staining showed a pattern of tissue-specific expression in transgenic rice, with staining observed in roots, coleoptiles, leaves, stems and floral organs but not in seeds or in the root elongation zone. Wounding strongly stimulated GUS accumulation in leaves and stems. Interestingly, we observed a high stimulation of the promoter activity when rice seedlings were exposed to NaCl, PEG, ABA, MeJA, GA, cold, and heavy metals (Al3+, Cd2+, Cu2+ and Zn2+). These results suggest that the LmSAP promoter can be a convenient tool for stress-inducible gene expression and is a potential candidate for crop genetic engineering.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Regiões Promotoras Genéticas , Plantas Tolerantes a Sal/genética , Estresse Fisiológico/genética , Dedos de Zinco/genética , Produtos Agrícolas/genética , Engenharia Genética , Glucuronidase/metabolismo , Metais Pesados/metabolismo , Especificidade de Órgãos , Oryza/genética , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
4.
PLoS One ; 15(8): e0236246, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804956

RESUMO

K+ is an essential nutrient for plant growth and is responsible for many important physiological processes. K+ deficiency leads to crop yield losses, and overexpression of K+ transporter genes has been proven to be an effective way to resolve this problem. However, current research on the overexpression of K+ transporter genes is limited to plant sources. TrkH is a bacterial K+ transporter whose function generally depends on the regulation of TrkA. To date, whether TrkH can improve K+ uptake in eukaryotic organisms is still unknown. In this study, a novel MbtrkH gene was cloned from marine microbial metagenomic DNA. Functional complementation and K+-depletion analyses revealed that MbTrkH functions in K+ uptake in the K+-deficient yeast strain CY162. Moreover, K+-depletion assays revealed that MbtrkH overexpression improves plant K+ uptake. K+ hydroponic culture experiments showed that, compared with WT tobacco lines, MbtrkH transgenic tobacco lines had significantly greater fresh weights, dry weights and K+ contents. These results indicate that MbTrkH promotes K+ uptake independently of TrkA in eukaryotes and provide a new strategy for improving K+-use efficiency in plants.


Assuntos
Organismos Aquáticos/genética , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Água do Mar/microbiologia , Tabaco/metabolismo , Clonagem Molecular , Metagenoma , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Tabaco/genética
5.
Nat Commun ; 11(1): 3847, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737299

RESUMO

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.


Assuntos
Aptâmeros de Nucleotídeos/genética , Arabidopsis/genética , Brassica/genética , Engenharia Genética/métodos , RNA Mensageiro/genética , Tabaco/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arabidopsis/metabolismo , Compostos de Benzil/química , Brassica/metabolismo , Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Genes Reporter , Imidazolinas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/metabolismo , Tabaco/metabolismo , Transformação Genética
6.
Nat Protoc ; 15(9): 3064-3087, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807907

RESUMO

Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a 'green' alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d.


Assuntos
DNA/química , Portadores de Fármacos/química , Engenharia , Nanoestruturas/química , RNA Interferente Pequeno/metabolismo , Tabaco/metabolismo , DNA/metabolismo , Portadores de Fármacos/metabolismo , RNA Interferente Pequeno/genética , Tabaco/genética
7.
Science ; 369(6504): 698-702, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32764072

RESUMO

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.


Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Celulase/metabolismo , Horticultura/métodos , Proteínas de Plantas/metabolismo , Tabaco/fisiologia , Adesão Celular/genética , Comunicação Celular/genética , Celulase/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tabaco/enzimologia , Tabaco/genética , Transcrição Genética
8.
Plant Mol Biol ; 104(3): 309-325, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32833148

RESUMO

KEY MESSAGE: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.


Assuntos
Antocianinas/biossíntese , Fagopyrum/metabolismo , Proteínas de Plantas/metabolismo , Proantocianidinas/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis , Fagopyrum/genética , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico , Tabaco/genética , Fatores de Transcrição/química
9.
Gene ; 760: 144990, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721476

RESUMO

The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants.


Assuntos
Tabaco/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antocianinas/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , Tabaco/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética
10.
Nature ; 584(7819): 148-153, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32699417

RESUMO

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.


Assuntos
Vias Biossintéticas , Colchicina/biossíntese , Engenharia Metabólica , Vias Biossintéticas/genética , Colchicaceae/enzimologia , Colchicaceae/genética , Colchicaceae/metabolismo , Colchicina/química , Colchicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Fenilalanina/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcriptoma , Tirosina/metabolismo
11.
PLoS One ; 15(6): e0235341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603354

RESUMO

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.


Assuntos
Aldeído Liases/química , Aldeído Liases/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Esterases/química , Esterases/genética , Hevea/genética , Hevea/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida/métodos , Proteínas de Plantas/genética , Dobramento de Proteína , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
12.
Phytochemistry ; 177: 112424, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32526514

RESUMO

In this study, we determined the pyridine alkaloid content (nicotine, nornicotine, anabasine, anatabine, cotinine, and myosmine) of 58 species and 2 subspecies of the Nicotiana genus by ultra-high-performance liquid chromatography coupled with mass spectrometry. We observed clear correlation between Noctiflorae and Suaveolentes sections and their above average accumulation of anabasine in the genus. In addition, the results demonstrated the presence of not only trace amounts but quantifiable levels of myosmine, an alkaloid previously detected in only minute quantities, in the leaves and roots of 16 species. In this study, analysis of gene expression of 58 species and 2 subspecies from the Nicotiana genus by mRNA sequencing was performed for the first time. Sequencing reads were mapped against annotated genes of a Nicotiana tabacum reference genome and expression values were subsequently calculated. Hierarchical clustering of alkaloid biosynthesis pathway genes and alkaloid content composition revealed patterns clearly segregating Nicotiana sections. Correlation of gene expression with alkaloid accumulation phenotypes was evident, including low putrescine methyltransferase expression for all species in the Suaveolentes section or clear correlation of nicotine demethylase with conversion rates of nicotine to nornicotine in the majority of species. Multiple additional correlations between alkaloid accumulation and gene expression values were identified, which makes this study an important fundament toward future scientific exploration of the Nicotiana genus.


Assuntos
Alcaloides , Tabaco/genética , Anabasina , Folhas de Planta , Transcriptoma
13.
PLoS One ; 15(6): e0233911, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479550

RESUMO

Promoters are very important for transcriptional regulation and gene expression, and have become invaluable tools for genetic engineering. Owing to the characteristics of obligate biotrophs, molecular research into obligate biotrophic fungi is seriously lagging behind, and very few of their endogenous promoters have been developed. In this study, a WY7 fragment was predicted in the genome of Oidium heveae Steinmann using PromoterScan. Its promoter function was verified with transient transformations (Agrobacterium tumefaciens-mediated transformation, ATMT) in Nicotiana tabacum cv. Xanthi nc. The analysis of the transcription range showed that WY7 could regulate GUS expression in both monocots (Zea mays Linn and Oryza sativa L. spp. Japonica cv. Nipponbare) and dicots (N. tabacum and Hylocereus undulates Britt). The results of the quantitative detection showed that the GUS transient expression levels when regulated by WY7 was more than 11.7 times that of the CaMV 35S promoter in dicots (N. tabacum) and 5.13 times that of the ACT1 promoter in monocots (O. sativa). GUS staining was not detected in the T1 generation of the WY7-GUS transgenic N. tabacum. This showed that WY7 is an inducible promoter. The cis elements of WY7 were predicted using PlantCARE, and further experiments indicated that WY7 was a low temperature- and salt-inducible promoter. Soluble proteins produced by WY7-hpa1Xoo transgenic tobacco elicited hypersensitive responses (HR) in N. tabacum leaves. N. tabacum transformed with pBI121-WY7-hpa1Xoo exhibited enhanced resistance to the tobacco mosaic virus (TMV). The WY7 promoter has a lot of potential as a tool for plant genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms of O. heveae.


Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Doenças das Plantas/prevenção & controle , Regiões Promotoras Genéticas , Fungos/patogenicidade , Genoma Fúngico , Hevea/genética , Hevea/microbiologia , Interações Hospedeiro-Patógeno/genética , Magnoliopsida/genética , Magnoliopsida/microbiologia , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/microbiologia , Transformação Genética , Zea mays/genética , Zea mays/microbiologia
14.
Nucleic Acids Res ; 48(11): 6234-6250, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396204

RESUMO

Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3' end of the syn-tasiRNA and the 5' end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Interferência de RNA , RNA Interferente Pequeno/genética , Pareamento de Bases , Sequência de Bases , Vetores Genéticos , RNA Interferente Pequeno/metabolismo , Tabaco/genética , Tabaco/virologia
16.
PLoS One ; 15(5): e0232986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407419

RESUMO

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.


Assuntos
Antocianinas/biossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Solanum melongena/genética , Solanum melongena/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reguladores , Família Multigênica , Filogenia , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(23): 13127-13137, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434921

RESUMO

Stomatal guard cells control gas exchange that allows plant photosynthesis but limits water loss from plants to the environment. In Arabidopsis, stomatal development is mainly controlled by a signaling pathway comprising peptide ligands, membrane receptors, a mitogen-activated protein kinase (MAPK) cascade, and a set of transcription factors. The initiation of the stomatal lineage requires the activity of the bHLH transcription factor SPEECHLESS (SPCH) with its partners. Multiple kinases were found to regulate SPCH protein stability and function through phosphorylation, yet no antagonistic protein phosphatase activities have been identified. Here, we identify the conserved PP2A phosphatases as positive regulators of Arabidopsis stomatal development. We show that mutations in genes encoding PP2A subunits result in lowered stomatal production in Arabidopsis Genetic analyses place the PP2A function upstream of SPCH. Pharmacological treatments support a role for PP2A in promoting SPCH protein stability. We further find that SPCH directly binds to the PP2A-A subunits in vitro. In plants, nonphosphorylatable SPCH proteins are less affected by PP2A activity levels. Thus, our research suggests that PP2A may function to regulate the phosphorylation status of the master transcription factor SPCH in stomatal development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Estômatos de Plantas/crescimento & desenvolvimento , Proteína Fosfatase 2/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação , Fosforilação/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/isolamento & purificação , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tabaco/genética
18.
Gene ; 753: 144809, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470503

RESUMO

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Plantas/genética , Solanaceae/enzimologia , Solanaceae/genética , Proteínas rho de Ligação ao GTP/genética , Capsicum/enzimologia , Capsicum/genética , Genoma de Planta , Estudo de Associação Genômica Ampla/métodos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Filogenia , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Tabaco/enzimologia , Tabaco/genética , Proteínas rho de Ligação ao GTP/metabolismo
19.
Nat Commun ; 11(1): 2589, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444691

RESUMO

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiões Promotoras Genéticas , Terminação da Transcrição Genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Fator Estimulador de Clivagem/genética , Regulação da Expressão Gênica de Plantas , Mutação , Plantas Geneticamente Modificadas , Poliadenilação , Isoformas de Proteínas/genética , RNA de Plantas , Tabaco/genética , Sítio de Iniciação de Transcrição
20.
Plant Mol Biol ; 103(4-5): 561-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32405802

RESUMO

KEY MESSAGE: CmHKT1;1 selectively exports Na+ from plant cells. Upon NaCl stress, its expression increased in a salt-tolerant melon cultivar. Overexpression of CmHKT1;1 increased transgenic Arabidopsis salt tolerance through improved K+/Na+ balance. High-affinity K+ transporters (HKTs) are thought to be involved in reducing Na+ in plant shoots under salt stress and modulating salt tolerance, but their function in a moderately salt-tolerant species of melon (Cucumis melo L.) remains unclear. In this study, a Na+ transporter gene, CmHKT1;1 (GenBank accession number: MK986658), was isolated from melons based on genome data. The transcript of CmHKT1;1 was relatively more abundant in roots than in stems or leaves from melon seedlings. The tobacco transient expression system showed that CmHKT1;1 was plasma-membrane localized. Upon salt stress, CmHKT1;1 expression was more strongly upregulated in a salt-tolerant melon cultivar, 'Bingxuecui' (BXC) compared with a salt-sensitive cultivar, 'Yulu' (YL). Electrophysiological evidence demonstrated that CmHKT1;1 only transported Na+, rather than K+, when expressed in Xenopus laevis oocytes. Overexpression of CmHKT1;1 increased salt sensitivity in Saccharomyces cerevisiae and salt tolerance in Arabidopsis thaliana. Under NaCl treatments, transgenic Arabidopsis plants accumulated significantly lower concentrations of Na+ in shoots than wild type plants and showed a better K+/Na+ balance, leading to better Fv/Fm, root length, biomass, and enhanced plant growth. The CmHKT1;1 gene may serve as a useful candidate for improving crop salt tolerance.


Assuntos
Arabidopsis/metabolismo , Cucumis melo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Clorofila/análise , Clonagem Molecular , Cucumis melo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal , Plântula/genética , Plântula/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Simportadores/genética , Simportadores/metabolismo , Tabaco/genética , Tabaco/metabolismo
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