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1.
Biosens Bioelectron ; 252: 116041, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38401280

RESUMO

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the ß-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Ouro/química , DNA de Cadeia Simples/genética , Espectroscopia Dielétrica , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , DNA/genética , RNA Mensageiro , Expressão Gênica , Impedância Elétrica
2.
Plant Sci ; 330: 111632, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36758729

RESUMO

Plants possess an efficient, two-tiered immune system to combat pathogens and pests. Several decades of research have characterized different features of these two well-known tiers, PTI and ETI (Pattern/ Effector-triggered Immunity). NLR (Nucleotide-binding domain Leucine-rich Repeat) receptors have been found to link PTI to ETI, and be required for full potentiation of plant immune responses in several systems. Intra-cellular helper-NLRs (h-NLRs) mediate ETI and have been focused on extensively in recent research. Previously, we investigated the roles of the h-NLR SlNRC4a in tomato immunity, finding that a specific mutation in this gene results in gain of function constitutive defense activation and broad disease resistance. Deletion of the entire NRC4 clade, which contains 3 genes, can compromise tomato immunity. Here, we decided to investigate the role of an additional clade member, SlNRC4b, in basal immunity. We generated a gain of function mutant in SlNRC4b using CRISPR-Cas9, as well as a double gain of function mutant in both genes. Similarly to the slnrc4a mutant, a slnrc4b mutant also possessed increased basal immunity and broad spectrum disease resistance. The double mutant displayed additive effects in some cases, with significant increases in resistance to fungal phytopathogens as compared with each of the single mutants. Our work confirms that the NRC4 family h-NLRs are important in the plant immune system, suggesting that this gene family has the potential to be promising in targeted agricultural adaptation in the Solanaceae family, promoting disease resistance and prevention of yield loss to pathogens.


Assuntos
Resistência à Doença , Solanum lycopersicum , Resistência à Doença/genética , Solanum lycopersicum/genética , Proteínas NLR/genética , Imunidade Vegetal/genética , Plantas , Doenças das Plantas
3.
Membranes (Basel) ; 12(8)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-36005675

RESUMO

Pattern recognition receptor (PRR) trafficking to the plasma membrane and endocytosis plays a crucial role in pattern triggered immunity (PTI). Dynamin-related proteins (DRPs) participate in endocytosis and recycling. In Arabidopsis, DRP1 and DRP2 are involved in plasma membrane scission during endocytosis. They are required for the PRR FLS2 endocytosis induction and PTI activation after elicitation with flg22, the MAMP recognized by FLS2. In tomato, SlDRP2A regulates the PRR LeEIX2 endocytosis and PTI activation in response to EIX, the MAMP recognized by LeEIX2. However, it is unknown if other DRPs participate in these processes. Taking advantage of bioinformatics tools, we selected SlDRP2B among the eight DRP2 tomato orthologues to study its functionality in trafficking and plant immunity. Through transient expression of SlDRP1B and its dominant-negative mutant on Nicotiana benthamiana and Nicotiana tabacum, we analyzed SlDRP1B function. We observed that SlDRP1B is physically associated with the LeEIX2 and modifies LeEIX2 trafficking, increasing its presence in endosomes. An enhancement of EIX-elicitated defense responses accompanies the role of SlDRP1B on LeEIX endocytosis. In addition, SlDRP1B overexpression enhanced flg22-elicited defense response. With these results, we conclude that SlDRP1B regulates PRR trafficking and, therefore, plant immunity, similarly to the SlDRP2A role.

4.
Plant J ; 104(5): 1369-1381, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33048397

RESUMO

The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe-associated molecular patterns. Plant PRRs are either receptor-like kinases (RLKs), which have an extracellular domain for ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor-like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene-inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two-hybrid screen and found a tomato protein that we termed SlRLK-like. The interaction of LeEIX2 with SlRLK-like was verified using co-immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK-like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK-like using the CRISPR/Cas9 system increased EIX-induced ethylene production and 1-aminocyclopropane-1-carboxylate synthase (SlACS2) gene expression in tomato. Co-expression of SlRLK-like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum-associated degradation process. Notably, truncation of SlRLK-like protein revealed that the malectin-like domain is sufficient and essential for its function. Moreover, SlRLK-like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)-induced burst of reactive oxygen species. In addition, SlRLK-like co-expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK-like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses.


Assuntos
Imunidade Vegetal/fisiologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Degradação Associada com o Retículo Endoplasmático , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/imunologia , Solanum lycopersicum/metabolismo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Técnicas do Sistema de Duplo-Híbrido , Xilosidases/imunologia , Xilosidases/metabolismo
5.
Front Plant Sci ; 10: 936, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379912

RESUMO

The endocytic trafficking pathway is employed by the plant to regulate immune responses, and is often targeted by pathogen effectors to promote virulence. The model system of the tomato receptor-like protein (RLP) LeEIX2 and its ligand, the elicitor EIX, employs endocytosis to transmit receptor-mediated signals, with some of the signaling events occurring directly from endosomal compartments. Here, to explore the trafficking mechanism of LeEIX2-mediated immune signaling, we used a proteomic approach to identify LeEIX2-associating proteins. We report the identification of SlDRP2A, a dynamin related protein, as an associating partner for LeEIX2. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 in VHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune responses.

6.
Biosens Bioelectron ; 117: 493-500, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29982119

RESUMO

The ability to interact with plants, both to sense and to actuate, would open new opportunities for precision agriculture. These interactions can be achieved by using the plant as part of the sensing system. The present work demonstrates real-time monitoring of ß-glucuronidase (GUS) expression in transgenic tobacco plants using its activity as a biomarker for functional sensing. As "proof of concept", we demonstrated GUS enzyme biosensing under constitutive expression in Msk8 tomato cells and transgenic tobacco plants and in heat shock inducible BY2 tobacco cells and tobacco plants. The sensing was done using a three-electrode microchip in Msk8 or BY2 cell culture or in tobacco plant leaves. The electrode microchip was used to transduce the expression of the GUS enzyme by chronoamperometry to a measurable electrical current signal. For the constitutive expression of GUS in Msk8 cells, the system sensitivity was 0.076 mA/mM-cm2 and the limit of detection was 0.1 mM. For the heat shock inducible BY2 cells the GUS enzyme activity was detected 12-26 h after the heat shock was applied (40 °C for 2 h) using two different substrates: p-nitrophenyl-ß-glucuronide (with sensitivity of 0.051 mA/mM-cm2) and phenolphthalein-ß-glucuronide (with sensitivity of 0.029 mA/mM-cm2).


Assuntos
Técnicas Biossensoriais/instrumentação , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Dispositivos Lab-On-A-Chip , Nicotiana/enzimologia , Estresse Fisiológico , Células Cultivadas , Fenômenos Eletrofisiológicos , Limite de Detecção , Solanum lycopersicum/citologia , Solanum lycopersicum/enzimologia , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas , Nicotiana/genética
7.
Plant Cell Environ ; 41(10): 2313-2327, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29790585

RESUMO

Plant recognition and defence against pathogens employs a two-tiered perception system. Surface-localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR required for hypersensitive response-associated cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.


Assuntos
Proteínas NLR/fisiologia , Imunidade Vegetal , Proteínas de Plantas/fisiologia , Western Blotting , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Etilenos/metabolismo , Edição de Genes , Imunoprecipitação , Solanum lycopersicum/imunologia , Solanum lycopersicum/fisiologia , Espectrometria de Massas , Microscopia Confocal , Proteínas NLR/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/fisiologia , Transdução de Sinais
8.
Front Plant Sci ; 9: 257, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29545816

RESUMO

Plants recognize microbial/pathogen associated molecular patterns (MAMP/PAMP) through pattern recognition receptors (PRRs) triggering an immune response against pathogen progression. MAMP/PAMP triggered immune response requires PRR endocytosis and trafficking for proper deployment. LeEIX2 is a well-known Solanum lycopersicum RLP-PRR, able to recognize and respond to the fungal MAMP/PAMP ethylene-inducing xylanase (EIX), and its function is highly dependent on intracellular trafficking. Identifying protein machinery components regulating LeEIX2 intracellular trafficking is crucial to our understanding of LeEIX2 mediated immune responses. In this work, we identified a novel trafficking protein, SlPRA1A, a predicted regulator of RAB, as an interactor of LeEIX2. Overexpression of SlPRA1A strongly decreases LeEIX2 endosomal localization, as well as LeEIX2 protein levels. Accordingly, the innate immune responses to EIX are markedly reduced by SlPRA1A overexpression, presumably due to a decreased LeEIX2 availability. Studies into the role of SlPRA1A in LeEIX2 trafficking revealed that LeEIX2 localization in multivesicular bodies/late endosomes is augmented by SlPRA1A. Furthermore, inhibiting vacuolar function prevents the LeEIX2 protein level reduction mediated by SlPRA1A, suggesting that SlPRA1A may redirect LeEIX2 trafficking to the vacuole for degradation. Interestingly, SlPRA1A overexpression reduces the amount of several RLP-PRRs, but does not affect the protein level of receptor-like kinase PRRs, suggesting a specific role of SlPRA1A in RLP-PRR trafficking and degradation.

9.
Methods Mol Biol ; 1578: 167-172, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28220423

RESUMO

Plant-pathogen interactions involve a large number of wide regulatory systems, necessary for plant defense responses against pathogen attack. The fungal protein ethylene-inducing xylanase (EIX) elicits defense responses in specific cultivars of tobacco and tomato. The response to EIX is controlled by a single locus encoding for LeEIX2, a leucine-rich-repeat receptor-like-protein (LRR-RLP). As an RLP, LeEIX2 does not possess an obvious cytoplasmic signaling moiety such as a kinase domain. To study LeEIX2 mode of action, it is essential to identify the potential interactors involved after EIX perception. Here, we describe the in vivo co-IP methodology used for protein interaction verification and ethylene and ROS (reactive oxygen species) measurements used for physiological effects assessment.


Assuntos
Proteínas Fúngicas/imunologia , Nicotiana/imunologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/imunologia , Etilenos/análise , Proteínas Fúngicas/metabolismo , Imunoprecipitação , Solanum lycopersicum/metabolismo , Imunidade Vegetal , Ligação Proteica , Domínios Proteicos , Espécies Reativas de Oxigênio/análise , Transdução de Sinais , Nicotiana/metabolismo
10.
Plant Mol Biol ; 84(4-5): 509-18, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24154852

RESUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Endocitose , Proteínas/metabolismo , Transdução de Sinais , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Etilenos/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Repetições Ricas em Leucina , Microscopia Confocal , Modelos Biológicos , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas/genética , Sumoilação , Nicotiana/genética , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-Híbrido
11.
Plant Physiol ; 164(2): 819-27, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24351686

RESUMO

Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.


Assuntos
Nicotiana/imunologia , Nicotiana/microbiologia , Receptores de Reconhecimento de Padrão/metabolismo , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Esteróis/metabolismo , Trichoderma/fisiologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Etilenos/biossíntese , Filipina/farmacologia , Inativação Gênica/efeitos dos fármacos , Liases Intramoleculares/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trichoderma/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido
12.
PLoS One ; 8(1): e54533, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23342166

RESUMO

Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.


Assuntos
Proteínas de Arabidopsis/metabolismo , Endossomos/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Endocitose/efeitos dos fármacos , Tolerância ao Sal , Plântula/efeitos dos fármacos , Plântula/metabolismo
13.
Plant J ; 68(3): 413-23, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21736652

RESUMO

Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.


Assuntos
Endossomos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Solanum lycopersicum/metabolismo , Morte Celular , Endocitose , Proteínas de Repetições Ricas em Leucina , Limoninas , Transdução de Sinais
14.
Plant Mol Biol ; 74(1-2): 33-45, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20521085

RESUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant's response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/química , Sequência de Bases , Primers do DNA/genética , DNA de Plantas/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Temperatura Alta , Lisina/química , Mutagênese Sítio-Dirigida , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Plântula/genética , Plântula/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
15.
PLoS One ; 4(11): e7973, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19936242

RESUMO

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/química , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Transdução de Sinais , Actinas/metabolismo , Motivos de Aminoácidos , Proteínas de Arabidopsis/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Citosol/metabolismo , Endocitose , Exocitose , Microscopia de Fluorescência/métodos , Modelos Biológicos , Mutação , Proteínas de Plantas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
16.
Mar Biotechnol (NY) ; 9(5): 543-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17624575

RESUMO

The molecular mechanisms involved in the establishment and maintenance of sponge photosymbiosis, and in particular the association with cyanobacteria, are unknown. In the present study we analyzed gene expression in a common Mediterranean sponge (Petrosia ficiformis) in relation to its symbiotic (with cyanobacteria) or aposymbiotic status. A screening approach was applied to identify genes expressed differentially in symbiotic specimens growing in the light and aposymbiotic specimens growing in a dark cave at a short distance from the illuminated specimens. Out of the various differentially expressed sequences, we isolated two novel genes (here named PfSym1 and PfSym2) that were up-regulated when cyanobacterial symbionts were harbored inside the sponge cells. The sequence of one of these genes (PfSym2) was found to contain a conserved domain: the scavenger receptor cysteine rich (SRCR) domain. This is the first report on the expression of sponge genes in relation to symbiosis and, according to the presence of an SRCR domain, we suggest possible functions for one of the genes found in the sponge-cyanobacteria symbiosis.


Assuntos
Regulação da Expressão Gênica/fisiologia , Petrosia/genética , Petrosia/microbiologia , Simbiose/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cianobactérias/fisiologia , DNA/química , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/genética , Dados de Sequência Molecular , Petrosia/fisiologia , Alinhamento de Sequência , Simbiose/fisiologia
17.
J Exp Bot ; 57(11): 2775-84, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16829546

RESUMO

The genus Nepenthes represents carnivorous plants with pitcher traps capable of efficient prey capture and digestion. The possible involvement of plant chitinases in this process was studied in Nepenthes khasiana. Two different types of endochitinases were identified in the liquid of closed traps exhibiting substrate specificity for either long chitin polymers or N-acetylglucosamine (GlcNAc) oligomers. Injection of chitin into such closed sterile pitchers induced the appearance of additional endochitinase isoenzymes, with substrate specificity only for long chitin polymers. No significant exochitinase (N-acetyl-beta-glucosaminidase) or chitobiosidase activity could be detected in the non-induced or induced trap liquid. Four genes representing two subgroups of basic chitinases, denoted as Nkchit1b and Nkchit2b, were isolated from the secretory region of N. khasiana pitchers. The main differences between the two subgroups are the presence of a proline-rich hinge region only in NkCHIT1b and a C-terminal putative vacuole targeting extension only in NkCHIT2b, indicating different compartmentalization of the two enzymes. Reverse transcription-polymerase chain reaction (RT-PCR) evaluation of mRNA levels showed that the Nkchit2b genes are constitutively expressed in the secretory cells while transcription of Nkchit1b genes is induced by chitin injection. These results show for the first time the involvement of genes encoding chitinases in prey-trap interaction and their differential expression and activity during prey trapping.


Assuntos
Quitinases/metabolismo , Magnoliopsida/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Quitina/metabolismo , Quitina/farmacologia , Quitinases/química , Quitinases/genética , Expressão Gênica/efeitos dos fármacos , Magnoliopsida/anatomia & histologia , Magnoliopsida/genética , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência
18.
Plant Cell ; 17(4): 1205-16, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15749766

RESUMO

The hormone ethylene influences plant growth, development, and some defense responses. The fungal elicitor Ethylene-Inducing Xylanase (EIX) elicits ethylene biosynthesis in tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum) leaves by induction of 1-aminocyclopropane-1-caboxylic acid synthase (Acs) gene expression. A minimal promoter element in the LeAcs2 gene required for EIX responsiveness was defined by deletion analysis in transgenic tomato plants. The sequence between -715 and -675 of the tomato Acs2 gene was found to be essential for induction by EIX. A Cys protease (LeCp) was isolated that specifically binds to this cis element in vitro. Ectopic expression of LeCp in tomato leaves induced the expression of Acs2. Moreover, chromatin immunoprecipitation showed that LeCp binds in vivo to the Acs promoter. We propose a mechanism for the dual function of the LeCp protein. The protease acts enzymatically in the cytoplasm. Then, upon signaling, a small ubiquitin-related modifier protein binds to it, enabling entrance into the nucleus, where it acts as a transcription factor. Thus, LeCp can be considered a dual-function protein, having enzymatic activity and, upon elicitor signaling, exhibiting transcriptional factor activity that induces LeAcs2 expression.


Assuntos
Cisteína Endopeptidases/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Liases/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/enzimologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , DNA Complementar/análise , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Liases/genética , Liases/isolamento & purificação , Solanum lycopersicum/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/fisiologia , Elementos de Resposta/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação
19.
Plant J ; 32(6): 1049-55, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12492845

RESUMO

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.


Assuntos
Nicotiana/genética , Doenças das Plantas/genética , Xilosidases/biossíntese , Sequência de Aminoácidos , Anticorpos/imunologia , Anticorpos/metabolismo , Bacteriófagos/genética , Bacteriófagos/imunologia , Sítios de Ligação/genética , Ligação Competitiva , Endo-1,4-beta-Xilanases , Indução Enzimática , Mapeamento de Epitopos , Regulação da Expressão Gênica de Plantas , Imunidade Inata/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Nicotiana/virologia , Xilosidases/genética , Xilosidases/metabolismo
20.
Arch Pharm (Weinheim) ; 335(11-12): 535-46, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12596218

RESUMO

A series of N-alkyl-N'-(phenethyl- and cyclohexenylethyl) guanidines and N(2)- and N(2), 4-substituted imidazolin-2-amine hydrochlorides with triazasterol-related structures was designed and synthesized as stable analogues to mimic high energy intermediates of ergosterol biosynthesis. The in vitro antifungal susceptibility tests with a standard panel of pathogenic fungi revealed moderate to strong antimycotic effects of the sixteen prepared compounds, in some cases comparable with the activity observed for itraconazole.


Assuntos
Antifúngicos/síntese química , Guanidinas/síntese química , Imidazóis/síntese química , Antifúngicos/farmacologia , Ergosterol/biossíntese , Fungos/efeitos dos fármacos , Guanidinas/farmacologia , Imidazóis/farmacologia , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
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