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1.
Plant Mol Biol ; 100(3): 215-230, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053988

RESUMO

KEY MESSAGE: Two homologs PsnSuSy1 and PsnSuSy2 from poplar played largely similar but little distinct roles in modulating sink strength, accelerating vegetative growth and modifying secondary growth of plant. Co-overexpression of them together resulted in small but perceptible additive effects. Sucrose synthase (SuSy) acts as a crucial determinant of sink strength by controlling the conversion of sucrose into UDP-glucose, which is not only the sole precursor for cellulose biosynthesis but also an extracellular signaling molecule for plants growth. Therefore, modification of SuSy activity in plants is of utmost importance. We have isolated two SuSy genes from poplar, PsnSuSy1 and PsnSuSy2, which were preferentially expressed in secondary xylem/phloem. To investigate their functions, T2 tobacco transgenic lines of PsnSuSy1 and PsnSuSy2 were generated and then crossed to generate PsnSuSy1/PsnSuSy2 dual overexpression transgenic lines. SuSy activities in all lines were significantly increased though PsnSuSy1/PsnSuSy2 lines only exhibited slightly higher SuSy activities than either PsnSuSy1 or PsnSuSy2 lines. The significantly increased fructose and glucose, engendered by augmented SuSy activities, caused the alternations of many physiological, biochemical measures and phenotypic traits that include accelerated vegetative growth, thickened secondary cell wall, and increased stem breaking force, accompanied with altered expression levels of related pathway genes. The correlation relationships between SuSy activities and many of these traits were statistically significant. However, differences of almost all traits among three types of transgenic lines were insignificant. These findings clearly demonstrated that PsnSuSy1 and PsnSuSy2 had similar but little distinct functions and insubstantial additive effects on modulating sink strength and affecting allocation of carbon elements among secondary cell wall components.


Assuntos
Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucosiltransferases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Parede Celular/ultraestrutura , Celulose/biossíntese , Clorofila/análise , Clonagem Molecular , Perfilação da Expressão Gênica , Glucosiltransferases/metabolismo , Lignina/metabolismo , Floema/metabolismo , Fotossíntese , Polissacarídeos/metabolismo , Populus/genética , Análise de Sequência , Sacarose/metabolismo , Tabaco/citologia , Tabaco/crescimento & desenvolvimento , Xilema/metabolismo
2.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
3.
J Biotechnol ; 295: 41-48, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30826446

RESUMO

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.


Assuntos
Vacinas contra Ebola , Ebolavirus/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Proteínas do Envelope Viral , Células Cultivadas , Vacinas contra Ebola/química , Vacinas contra Ebola/genética , Vacinas contra Ebola/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vegetais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
4.
Planta ; 250(1): 79-94, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919065

RESUMO

MAIN CONCLUSION: Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Sesquiterpenos/metabolismo , Tabaco/metabolismo , Triglicerídeos/metabolismo , Animais , Vias Biossintéticas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/química , Proteínas de Membrana/genética , Camundongos , Especificidade de Órgãos , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteômica , Sesquiterpenos/análise , Terpenos/metabolismo , Tabaco/citologia , Tabaco/genética , Triglicerídeos/análise
5.
Viruses ; 10(12)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30558257

RESUMO

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation.


Assuntos
Caulimovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Caulimovirus/genética , Núcleo Celular/química , Núcleo Celular/virologia , Citoplasma/química , Citoplasma/virologia , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Fases de Leitura Aberta , Doenças das Plantas/virologia , Fator de Iniciação 3 em Procariotos/genética , Interferência de RNA , Tabaco/citologia , Tabaco/virologia , Transativadores/genética , Proteínas Virais/genética
6.
Virology ; 523: 6-14, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30056212

RESUMO

Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.


Assuntos
Núcleo Celular/virologia , Citosol/virologia , Drosophila melanogaster/virologia , Macrófagos/virologia , Células Vegetais/virologia , Rhabdoviridae/genética , Tabaco/virologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Clonagem Molecular/métodos , Citosol/metabolismo , Citosol/ultraestrutura , Drosophila melanogaster/citologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Imagem Óptica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhabdoviridae/crescimento & desenvolvimento , Rhabdoviridae/metabolismo , Tabaco/citologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
7.
Int J Mol Sci ; 19(6)2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29882931

RESUMO

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.


Assuntos
Agrobacterium/metabolismo , Inativação Gênica , Células Vegetais/metabolismo , Proteínas Recombinantes/biossíntese , Supressão Genética , Tabaco/citologia , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Biomassa , Técnicas de Cocultura , Humanos , Cinética , Plantas Geneticamente Modificadas , Suspensões , Fatores de Tempo
8.
Virology ; 520: 103-110, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29843054

RESUMO

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation.


Assuntos
Caulimovirus/genética , Proteínas de Ligação a DNA/metabolismo , Doenças das Plantas/virologia , Interferência de RNA , Proteínas Virais/metabolismo , Agrobacterium/genética , Caulimovirus/metabolismo , Caulimovirus/patogenicidade , Núcleo Celular/virologia , Citoplasma/virologia , Proteínas de Ligação a DNA/genética , Inativação Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Folhas de Planta/virologia , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/virologia , Potexvirus/genética , RNA/química , RNA/genética , Tabaco/citologia , Tabaco/virologia , Proteínas Virais/genética
9.
Int J Mol Sci ; 19(4)2018 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-29659495

RESUMO

Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier for plant-based production of human therapeutics is that different glycosylation patterns are found on plant and mammalian proteins. Since glycosylation can affect the efficacy, safety and stability of a therapeutic protein, methods to control glycan structures and distributions in plant-based systems would be beneficial. In these studies, we performed Agrobacterium-mediated transient expression in glycoengineered plant cell suspension cultures. To reduce the presence of plant-specific glycans on the product, we generated and characterized cell suspension cultures from β-1,2-xylosyltransferase and α-1,3-fucosyltransferase knockdown Nicotiana benthamiana. An anthrax decoy fusion protein was transiently produced in these glycoengineered plant cell suspension cultures through co-culture with genetically engineered Agrobacterium. The mass ratio of Agrobacterium to plant cells used was shown to impact recombinant protein expression levels. N-glycosylation analysis on the anthrax decoy fusion protein produced in glycoengineered N. benthamiana showed a dramatic reduction in plant-specific N-glycans. Overall, the results presented here demonstrate the feasibility of a simple, rapid and scalable process for transient production of recombinant proteins without plant-specific glycans in a glycoengineered plant cell culture host.


Assuntos
Técnicas de Cultura de Células/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Tabaco/citologia , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Técnicas de Cocultura , Glicosilação , Cinética , Mutação/genética , Polissacarídeos/metabolismo , Suspensões , Tabaco/crescimento & desenvolvimento
10.
Plant Physiol Biochem ; 127: 287-298, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29649745

RESUMO

Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hydroxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the undifferentiated cell culture system.


Assuntos
Aciltransferases , Cynara/enzimologia , Metabolômica , Células Vegetais/metabolismo , Proteínas de Plantas , Tabaco , Aciltransferases/genética , Aciltransferases/metabolismo , Cynara/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo
11.
Biotechnol Lett ; 40(6): 981-987, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29619743

RESUMO

OBJECTIVE: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. RESULTS: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g FW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g FW-1 activity towards CBCA production). CONCLUSION: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.


Assuntos
Canabinoides/metabolismo , Espaço Intracelular/enzimologia , Oxirredutases Intramoleculares , Engenharia Metabólica/métodos , Proteínas de Plantas , Tabaco/enzimologia , Cannabis/enzimologia , Cannabis/genética , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo
12.
Plant Physiol Biochem ; 126: 206-216, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29525444

RESUMO

Cladosporium herbarum is a plant pathogen associated with passion fruit scab and mild diseases in pea and soybean. In this study, a peptidogalactomannan (pGM) of C. herbarum mycelium was isolated and structurally characterized, and its role in plant-fungus interactions was evaluated. C. herbarum pGM is composed of carbohydrates (76%) and contains mannose, galactose and glucose as its main monosaccharides (molar ratio, 52:36:12). Methylation and 13C-nuclear magnetic resonance (13C-NMR) spectroscopy analysis have shown the presence of a main chain containing (1 → 6)-linked α-D-Manp residues, and ß-D-Galf residues are present as (1 → 5)-interlinked side chains. ß-Galactofuranose containing similar structures were characterized by our group in A. fumigatus, A. versicolor, A. flavus and C. resinae. Tobacco BY-2 cells were used as a model system to address the question of the role of C. herbarum pGM in cell viability and induction of the expression of plant defense-related genes. Native and partially acid hydrolyzed pGMs (lacking galactofuranosyl side-chain residues) were incubated with BY-2 cell suspensions at different concentrations. Cell viability drastically decreased after exposure to more than 400 µg ml-1 pGM; however no cell viability effect was observed after exposure to a partially acid hydrolyzed pGM. BY-2 cell contact with pGM strongly induce the expression of plant defense-related genes, such as phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX), as well as the pathogen-related PR-1a, PR-2 and PR-3 genes, suggesting that pGM activates defense responses in tobacco cells. Interestingly, contact with partially hydrolyzed pGM also induced defense-related gene expression at earlier times than native pGM. These results show that the side chains of the (1 → 5)-linked ß-D-galactofuranosyl units from pGM play an important role in the first line fungus-plant interactions mediating plant responses against C. herbarum. In addition, it was observed that pGM and/or C. herbarum conidia are able to induced HR when in contact with tobacco leaves and in vitro plantlets roots, producing necrotic lesions and peroxidase and NO burst, respectively.


Assuntos
Cladosporium , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/microbiologia , Folhas de Planta , Raízes de Plantas , Tabaco , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Raízes de Plantas/microbiologia , Tabaco/citologia , Tabaco/enzimologia , Tabaco/microbiologia
13.
Plant Cell Rep ; 37(5): 809-818, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29502206

RESUMO

KEY MESSAGE: Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca2+) as shown by comparison of transport assays in Ca2+-rich and Ca2+-free buffers and upon treatment with inhibitors of plasma membrane Ca2+-permeable channels Al3+ and ruthenium red, both abolishing the effect of AgNO3. Confocal microscopy of Ca2+-sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca2+-permeable channels at the plasma membrane.


Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espaço Intracelular/metabolismo , Células Vegetais/metabolismo , Prata/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácidos Indolacéticos/metabolismo , Íons , Células Vegetais/efeitos dos fármacos
14.
Plant Sci ; 270: 47-57, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29576086

RESUMO

Ubiquitination is a post-translational modification that plays a crucial role during the regulation of plant immune signalling. The plant ATL family consists of a large number of putative RING type ubiquitin ligases. We show that potato ATL family gene StRFP1 and its orthologue NbATL60 from N. benthamiana both respond to Phytophthora infestans culture filtrate (CF) and flg22 induction. StRFP1 positively regulates immunity against P. infestans in potato. Ectopic transient expression of StRFP1 or expression of NbATL60 in N. benthamiana also enhances late blight resistance. By contrast, silencing NbATL60 in N. benthamiana reduces late blight resistance and leads to plant growth inhibition. Both StRFP1 and NbATL60 localize to the plasma membrane and intracellular puncta and possess E3 Ligase activity in vitro. Furthermore we demonstrate that the RING finger domain mutants of StRFP1 and NbATL60 lost E3 ligase activity and fail to suppress P. infestans colonization in N. benthamiana, indicating that E3 ligase activity is critical for StRFP1 and NbATL60 to regulate immunity. Overexpression or RNA interference of StRFP1 in transgenic potato led to increased or decreased expression of PTI maker genes (WRKY7, WRKY8, ACRE31 and Pti5) respectively. Similarly silencing of NbATL60 in N. benthamiana decreases expression of these PTI marker genes. Moreover, VIGS of NbATL60 in N. benthamiana did not compromise P. infestans PAMP INF1 or R2/Avr2, R3a/AVR3a, Rx/Cp and Pto/AvrPto triggered cell death. These results indicate that ATL genes StRFP1 and NbATL60 contribute to basal immunity (PTI) in Solanaceous plants.


Assuntos
Resistência à Doença , Phytophthora infestans/fisiologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Tabaco/genética , Morte Celular , Expressão Gênica , Genes Reporter , Padrões Moleculares Associados a Patógenos , Fenótipo , Doenças das Plantas/microbiologia , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Interferência de RNA , Plântula/citologia , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Solanum tuberosum/citologia , Solanum tuberosum/imunologia , Solanum tuberosum/microbiologia , Tabaco/citologia , Tabaco/imunologia , Tabaco/microbiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
15.
Planta ; 247(6): 1323-1338, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29511814

RESUMO

MAIN CONCLUSION: Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression. Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK-WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.


Assuntos
Brassica napus/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Morte Celular , Flores/genética , Flores/fisiologia , Genes Reporter , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Plântula/genética , Plântula/fisiologia , Fatores de Tempo , Tabaco/citologia , Tabaco/genética , Tabaco/fisiologia , Fatores de Transcrição/genética , Regulação para Cima
16.
Biochem Biophys Res Commun ; 499(2): 196-201, 2018 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-29555475

RESUMO

Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.


Assuntos
Bacteriófago T7/genética , Expressão Gênica , Células Vegetais/metabolismo , Tabaco/citologia , Agrobacterium/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Biossíntese de Proteínas , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Transcrição Genética
17.
ACS Synth Biol ; 7(3): 774-781, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29439563

RESUMO

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.


Assuntos
Bioengenharia/métodos , Vias Biossintéticas , Compartimento Celular , Terpenos/metabolismo , Geraniltranstransferase/metabolismo , Células do Mesofilo/metabolismo , Plantas Geneticamente Modificadas , Esqualeno/metabolismo , Frações Subcelulares/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/crescimento & desenvolvimento
18.
PLoS Comput Biol ; 14(2): e1005959, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29394250

RESUMO

Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Simulação por Computador , Microtúbulos/metabolismo , Células Vegetais , Software , Algoritmos , Anisotropia , Membrana Celular/metabolismo , Biologia Computacional , Hedera/citologia , Microscopia Confocal , Modelos Biológicos , Distribuição Normal , Folhas de Planta/citologia , Probabilidade , Processos Estocásticos , Tabaco/citologia , Tubulina (Proteína)/metabolismo
19.
Mol Plant Pathol ; 19(1): 129-142, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27768829

RESUMO

The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.


Assuntos
Arabidopsis/imunologia , Proteínas de Bactérias/metabolismo , Imunidade Vegetal , Ralstonia solanacearum/metabolismo , Tabaco/imunologia , Sistemas de Secreção Tipo III/metabolismo , Arabidopsis/microbiologia , Núcleo Celular/metabolismo , Cisteína/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Oxirredução , Células Vegetais/metabolismo , Ralstonia solanacearum/patogenicidade , Tiorredoxinas/metabolismo , Tabaco/citologia , Tabaco/microbiologia , Virulência , gama-Glutamilciclotransferase/metabolismo
20.
Mol Plant Microbe Interact ; 31(3): 374-385, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29106332

RESUMO

Effector proteins are exported to the interior of host cells by diverse plant pathogens. Many oomycete pathogens maintain large families of candidate effector genes, encoding proteins with a secretory leader followed by an RxLR motif. Although most of these genes are very divergent between oomycete species, several genes are conserved between Phytophthora species and Hyaloperonospora arabidopsidis, suggesting that they play important roles in pathogenicity. We describe a pair of conserved effector candidates, HaRxL23 and PsAvh73, from H. arabidopsidis and P. sojae respectively. We show that HaRxL23 is expressed early during infection of Arabidopsis. HaRxL23 triggers an ecotype-specific defense response in Arabidopsis, suggesting that it is recognized by a host surveillance protein. HaRxL23 and PsAvh73 can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Nicotiana benthamiana and effector-triggered immunity (ETI) in soybean. Transgenic Arabidopsis constitutively expressing HaRxL23 or PsAvh73 exhibit suppression of PTI and enhancement of bacterial and oomycete virulence. Together, our experiments demonstrate that these conserved oomycete RxLR effectors suppress PTI and ETI across diverse plant species.


Assuntos
Sequência Conservada , Oomicetos/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Phytophthora/metabolismo , Imunidade Vegetal , Plantas/imunologia , Plantas/microbiologia , Proteínas/metabolismo , Sequência de Aminoácidos , Apoptose , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ecótipo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oomicetos/patogenicidade , Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , Domínios Proteicos , Proteínas/química , Pseudomonas syringae/fisiologia , Soja/imunologia , Soja/microbiologia , Sintenia/genética , Tabaco/citologia , Tabaco/microbiologia , Transformação Genética
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