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1.
Cell ; 179(1): 205-218.e21, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31522888

RESUMO

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mimetismo Molecular/imunologia , Imunidade Vegetal/fisiologia , Adenosina Trifosfatases/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/química , Células HEK293 , Proteínas de Choque Térmico HSP90/química , Células HeLa , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Fosforilação , Plasmídeos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Quinases/metabolismo , Pseudomonas syringae/metabolismo , Saccharomyces cerevisiae/metabolismo
2.
New Phytol ; 235(3): 1179-1195, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35491734

RESUMO

Knowledge of the immune mechanisms responsible for viral recognition is critical for understanding durable disease resistance and successful crop protection. We determined how potato virus Y (PVY) coat protein (CP) is recognised by Rysto , a TNL immune receptor. We applied structural modelling, site-directed mutagenesis, transient overexpression, co-immunoprecipitation, infection assays and physiological cell death marker measurements to investigate the mechanism of Rysto -CP interaction. Rysto associates directly with PVY CP in planta that is conditioned by the presence of a CP central 149 amino acids domain. Each deletion that affects the CP core region impairs the ability of Rysto to trigger defence. Point mutations in the amino acid residues Ser125 , Arg157 , and Asp201 of the conserved RNA-binding pocket of potyviral CP reduce or abolish Rysto binding and Rysto -dependent responses, demonstrating that appropriate folding of the CP core is crucial for Rysto -mediated recognition. Rysto recognises the CPs of at least 10 crop-damaging viruses that share a similar core region. It confers immunity to plum pox virus and turnip mosaic virus in both Solanaceae and Brassicaceae systems, demonstrating potential utility in engineering virus resistance in various crops. Our findings shed new light on how R proteins detect different viruses by sensing conserved structural patterns.


Assuntos
Potyvirus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Resistência à Doença , Potyvirus/fisiologia
3.
J Exp Bot ; 73(1): 94-109, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34522949

RESUMO

In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx and swelling, eventually leading to pollen membrane rupture. In metazoans, calcium- and phospholipid-binding proteins, referred to as annexins, participate in the repair of plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of pollen membrane integrity. We analyzed the cellular dynamics of ANN5 in pollen grains undergoing hydration in favorable or stress conditions. We observed a transient association of ANN5 with the pollen membrane during in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at the pollen membrane. Interestingly, calcium or magnesium application affected pollen membrane properties differently, causing rupture or shrinkage of pollen membrane, respectively. Both treatments, however, induced ANN5 recruitment to the pollen membrane. Our data suggest a model in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Anexina A5 , Anexinas , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Permeabilidade , Pólen/metabolismo , Tubo Polínico/metabolismo
4.
Plant J ; 104(3): 645-661, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32772469

RESUMO

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.


Assuntos
Potyvirus/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/virologia , Regulação da Expressão Gênica de Plantas/genética , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potyvirus/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo
5.
Plant Biotechnol J ; 18(3): 655-667, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31397954

RESUMO

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.


Assuntos
Resistência à Doença , Genes de Plantas , Doenças das Plantas/virologia , Potyvirus/patogenicidade , Solanum tuberosum/imunologia , Animais , Afídeos/virologia , Cruzamento , Proteínas NLR/imunologia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/virologia , Solanum tuberosum/virologia
6.
BMC Plant Biol ; 18(1): 183, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189843

RESUMO

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.


Assuntos
Anexina A5/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/farmacologia , Plastídeos/fisiologia , Pólen/crescimento & desenvolvimento , Proteínas rab1 de Ligação ao GTP/farmacologia , Anexina A5/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacologia , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Técnicas de Silenciamento de Genes , Genes de Plantas , Homeostase , Pólen/anatomia & histologia , Pólen/genética , Tubo Polínico/crescimento & desenvolvimento , Plântula/metabolismo , Nicotiana/genética , Nicotiana/fisiologia , Transcriptoma , Proteínas rab1 de Ligação ao GTP/genética
7.
Plant Physiol ; 174(3): 1913-1930, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28522456

RESUMO

Acclimation to water deficit (WD) enables plants to maintain growth under unfavorable environmental conditions, although the mechanisms are not completely understood. In this study, the natural variation of long-term acclimation to moderate and severe soil WD was investigated in 18 Arabidopsis (Arabidopsis thaliana) accessions using PHENOPSIS, an automated phenotyping platform. Soil water content was adjusted at an early stage of plant development and maintained at a constant level until reproductive age was achieved. The accessions were selected based on the expression levels of ANNEXIN1, a drought-related marker. Severe WD conditions had a greater effect on most of the measured morphophysiological traits than moderate WD conditions. Multivariate analyses indicated that trait responses associated with plant size and water management drove most of the variation. Accessions with similar responses at these two levels were grouped in clusters that displayed different response strategies to WD The expression levels of selected stress-response genes revealed large natural variation under WD conditions. Responses of morphophysiological traits, such as projected rosette area, transpiration rate, and rosette water content, were correlated with changes in the expression of stress-related genes, such as NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 and N-MYC DOWNREGULATED-LIKE1 (NDL1), in response to WD Interestingly, the morphophysiological acclimation response to WD also was reflected in the gene expression levels (most notably those of NDL1, CHALCONE SYNTHASE, and MYB DOMAIN PROTEIN44) in plants cultivated under well-watered conditions. Our results may lead to the development of biomarkers and predictors of plant morphophysiological responses based on gene expression patterns.


Assuntos
Arabidopsis/anatomia & histologia , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico/genética , Água/fisiologia , Arabidopsis/genética , Ecótipo , Fenótipo , Transpiração Vegetal/genética , Análise de Componente Principal , Solo
8.
Plant Physiol ; 161(4): 2049-61, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23396834

RESUMO

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Interações Hospedeiro-Patógeno , Proteínas de Plantas/metabolismo , Pseudomonas syringae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Cromatografia Líquida , Sequência Conservada/genética , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Massas , Dados de Sequência Molecular , Phaseolus/metabolismo , Phaseolus/microbiologia , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Pseudomonas syringae/patogenicidade , Frações Subcelulares/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologia , Virulência
9.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 1): 52-62, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23275163

RESUMO

Plant endo-1,3-ß-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (1→3)-ß-glucans, with very limited activity towards mixed (1→3,1→4)-ß-glucans and branched (1→3,1→6)-ß-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-ß-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (1→3)-ß-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein-saccharide interactions and were determined at resolutions of 1.68 and 1.55 Å, respectively. The geometry of the active-site cleft clearly precludes any (1→4)-ß-glucan topology at the subsites from -3 to +4 and could possibly accommodate ß-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-ß-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there.


Assuntos
Substituição de Aminoácidos/genética , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/genética , Oligossacarídeos/química , Solanum tuberosum/enzimologia , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Hidrólise , Ligantes , Oligossacarídeos/genética , Solanum tuberosum/genética , Trissacarídeos/química
10.
Plant Biotechnol J ; 11(4): 459-69, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23231480

RESUMO

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.


Assuntos
Secas , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Solanum tuberosum/genética
11.
New Phytol ; 200(1): 158-171, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23731343

RESUMO

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK.


Assuntos
Núcleo Celular , Resistência à Doença , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotiana/fisiologia , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Vírus do Mosaico do Tabaco , Fosforilação , Transdução de Sinais , Nicotiana/metabolismo , Nicotiana/virologia
12.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 6): 713-23, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22683794

RESUMO

Endo-1,3-ß-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted ß-1,3-linked glucosyl residues. The plant enzymes belong to glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. X-ray diffraction data were collected to 1.40 and 1.26 Å resolution from two crystals of endo-1,3-ß-glucanase from Solanum tuberosum (potato, cultivar Désirée) which, despite having a similar packing framework, represented two separate crystal forms. In particular, they differed in the Matthews coefficient and are consequently referred to as higher density (HD; 1.40 Å resolution) and lower density (LD; 1.26 Å resolution) forms. The general fold of the protein resembles that of other known plant endo-1,3-ß-glucanases and is defined by a (ß/α)(8)-barrel with an additional subdomain built around the C-terminal half of the barrel. The structures revealed high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with structures of other GH17 endo-1,3-ß-glucanases revealed differences in the arrangement of the secondary-structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate-binding patterns. The crystal structures revealed an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His(6) tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighbouring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.


Assuntos
Domínio Catalítico , Glucana Endo-1,3-beta-D-Glucosidase/química , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
13.
Physiol Plant ; 135(4): 351-64, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19292825

RESUMO

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria (P. syringae) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.


Assuntos
Álcoois/metabolismo , Nicotiana/metabolismo , Terpenos/metabolismo , Peróxido de Hidrogênio/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/virologia , Plastoquinona/metabolismo , Pseudomonas syringae/fisiologia , Ácido Salicílico/farmacologia , Estresse Fisiológico , Nicotiana/efeitos dos fármacos , Nicotiana/microbiologia , Nicotiana/virologia , Vírus do Mosaico do Tabaco/fisiologia
14.
J Exp Bot ; 59(8): 2205-19, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18467325

RESUMO

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 muM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of gamma-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in gamma-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.


Assuntos
Aminoaciltransferases/metabolismo , Cádmio/metabolismo , Expressão Gênica , Nicotiana/genética , Nicotiana/fisiologia , Aminoaciltransferases/genética , Animais , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cádmio/farmacologia , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Glutationa , Estresse Oxidativo , Fitoquelatinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/crescimento & desenvolvimento
15.
Acta Biochim Pol ; 55(4): 791-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19081847

RESUMO

The synthesis and degradation of (1-->3)-beta-glycosidic bonds between glucose moieties are essential metabolic processes in plant cell architecture and function. We have found that a unique, conserved cysteine residue, positioned outside the catalytic centre of potato endo-(1-->3)-beta-glucanase - product of the gluB20-2 gene, participates in determining the substrate specificity of the enzyme. The same residue is largely responsible for endo-(1-->3)-beta-glucanase inhibition by mercury ions. Our results confirm that the spatial adjustment between an enzyme and its substrate is one of the essential factors contributing to the specificity and accuracy of enzymatic reactions.


Assuntos
Cisteína/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Catálise , Cisteína/química , Glucana 1,3-beta-Glucosidase/antagonistas & inibidores , Glucana 1,3-beta-Glucosidase/química , Mercúrio/farmacologia , Dados de Sequência Molecular , Especificidade por Substrato
16.
Bio Protoc ; 8(4): e2739, 2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-34179267

RESUMO

High-throughput phenotyping of plant traits is a powerful tool to further our understanding of plant growth and its underlying physiological, molecular, and genetic determinisms. This protocol describes the methodology of a standard phenotyping experiment in PHENOPSIS automated platform, which was engineered in INRA-LEPSE (https://www6.montpellier.inra.fr/lepse) and custom-made by Optimalog company. The seminal method was published by Granier et al. (2006). The platform is used to explore and test various ecophysiological hypotheses (Tisné et al., 2010; Baerenfaller et al., 2012; Vile et al., 2012; Bac-Molenaar et al., 2015; Rymaszewski et al., 2017). Here, the focus concerns the preparation and management of experiments, as well as measurements of growth-related traits (e.g., projected rosette area, total leaf area and growth rate), water status-related traits (e.g., leaf dry matter content and relative water content), and plant architecture-related traits (e.g., stomatal density and index and lamina/petiole ratio). Briefly, a completely randomized (block) design is set up in the growth chamber. Next, the substrate is prepared, its initial water content is measured and pots are filled. Seeds are sown onto the soil surface and germinated prior to the experiment. After germination, soil watering and image (visible, infra-red, fluorescence) acquisition are planned by the user and performed by the automaton. Destructive measurements may be performed during the experiment. Data extraction from images and estimation of growth-related trait values involves semi-automated procedures and statistical processing.

17.
Front Plant Sci ; 9: 978, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30042777

RESUMO

Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant - microbe interactions.

18.
PLoS One ; 10(7): e0132683, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26172952

RESUMO

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress.


Assuntos
Anexinas/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Anexinas/genética , Clorofila/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Luz , Estresse Oxidativo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Estresse Fisiológico , Xantofilas/metabolismo
19.
FEBS Lett ; 571(1-3): 61-6, 2004 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-15280018

RESUMO

Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development. Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants. Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones. The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined. Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl Superose columns. After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a dimer. The protein participated in NO-degradation activity with NAD(P)H as a cofactor. After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses.


Assuntos
Hemoglobinas/genética , Hemoglobinas/metabolismo , Medicago sativa/metabolismo , Nicotiana/genética , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Bases , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Primers do DNA , DNA de Plantas/genética , Eletroforese em Gel de Poliacrilamida , Hemoglobinas/isolamento & purificação , Folhas de Planta/metabolismo , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/metabolismo , Pseudomonas syringae/patogenicidade , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/metabolismo , Transcrição Gênica
20.
J Plant Physiol ; 161(5): 621-9, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15202719

RESUMO

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.


Assuntos
Cucumis/genética , Nicotiana/genética , Temperatura Baixa , Cucumis/efeitos dos fármacos , Cucumis/microbiologia , Flores/genética , Flores/metabolismo , Fungos/genética , Genes Reporter , Glucosídeos/farmacologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Salicilatos/farmacologia , Ácido Salicílico/farmacologia , Transformação Genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
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