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1.
PLoS Pathog ; 17(11): e1010017, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34724007

RESUMO

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues.


Assuntos
Acetiltransferases/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Proteínas de Bactérias/antagonistas & inibidores , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Pseudomonas syringae/patogenicidade , Solanum lycopersicum/imunologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
2.
Plant Physiol ; 190(1): 860-881, 2022 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-35642916

RESUMO

Plant plastids generate signals, including some derived from lipids, that need to be mobilized to effect signaling. We used informatics to discover potential plastid membrane proteins involved in microbial responses in Arabidopsis (Arabidopsis thaliana). Among these are proteins co-regulated with the systemic immunity component AZELAIC ACID INDUCED 1, a hybrid proline-rich protein (HyPRP), and HyPRP superfamily members. HyPRPs have a transmembrane domain, a proline-rich region (PRR), and a lipid transfer protein domain. The precise subcellular location(s) and function(s) are unknown for most HyPRP family members. As predicted by informatics, a subset of HyPRPs has a pool of proteins that target plastid outer envelope membranes via a mechanism that requires the PRR. Additionally, two HyPRPs may be associated with thylakoid membranes. Most of the plastid- and nonplastid-localized family members also have pools that localize to the endoplasmic reticulum, plasma membrane, or plasmodesmata. HyPRPs with plastid pools regulate, positively or negatively, systemic immunity against the pathogen Pseudomonas syringae. HyPRPs also regulate the interaction with the plant growth-promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture, and/or biomass. Thus, HyPRPs have broad and distinct roles in immunity, development, and growth responses to microbes and reside at sites that may facilitate signal molecule transport.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Plantas/metabolismo , Plastídeos/metabolismo , Prolina/metabolismo , Pseudomonas syringae/metabolismo
3.
Plant J ; 105(6): 1615-1629, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33342031

RESUMO

The proper subcellular localization of defense factors is an important part of the plant immune system. A key component for systemic resistance, lipid transfer protein (LTP)-like AZI1, is needed for the systemic movement of the priming signal azelaic acid (AZA) and a pool of AZI1 exists at the site of AZA production, the plastid envelope. Moreover, after systemic defense-triggering infections, the proportion of AZI1 localized to plastids increases. However, AZI1 does not possess a classical plastid transit peptide that can explain its localization. Instead, AZI1 uses a bipartite N-terminal signature that allows for its plastid targeting. Furthermore, the kinases MPK3 and MPK6, associated with systemic immunity, promote the accumulation of AZI1 at plastids during priming induction. Our results indicate the existence of a mode of plastid targeting possibly related to defense responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia
4.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36232932

RESUMO

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone's accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas , Hormônios/metabolismo , Mutação , Doenças das Plantas/genética , Pseudomonas syringae , Ácido Salicílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
J Exp Bot ; 72(7): 2710-2726, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33463678

RESUMO

The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Resistência à Doença , Epiderme , Doenças das Plantas , Plastídeos , Pseudomonas syringae
6.
Mol Plant Microbe Interact ; 32(1): 86-94, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30156481

RESUMO

Local interactions between individual plant organs and diverse microorganisms can lead to whole plant immunity via the mobilization of defense signals. One such signal is the plastid lipid-derived oxylipin azelaic acid (AZA). Arabidopsis lacking AZI1 or EARLI1, related lipid transfer family proteins, exhibit reduced AZA transport among leaves and cannot mount systemic immunity. AZA has been detected in roots as well as leaves. Therefore, the present study addresses the effects on plants of AZA application to roots. AZA but not the structurally related suberic acid inhibits root growth when directly in contact with roots. Treatment of roots with AZA also induces resistance to Pseudomonas syringae in aerial tissues. These effects of AZA on root growth and disease resistance depend, at least partially, on AZI1 and EARLI1. AZI1 in roots localizes to plastids, similar to its known location in leaves. Interestingly, kinases previously shown to modify AZI1 in vitro, MPK3 and MPK6, are also needed for AZA-induced root-growth inhibition and aboveground immunity. Finally, deuterium-labeled AZA applied to the roots does not move to aerial tissues. Thus, AZA application to roots triggers systemic immunity through an AZI1/EARLI1/MPK3/MPK6-dependent pathway and AZA effects may involve one or more additional mobile signals.


Assuntos
Arabidopsis , Ácidos Dicarboxílicos , Imunidade Vegetal , Pseudomonas syringae , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Ácidos Dicarboxílicos/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Pseudomonas syringae/fisiologia
7.
Plant Physiol ; 176(3): 2515-2531, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29438088

RESUMO

Salicylic acid (SA) is a major defense signal in plants. In Arabidopsis (Arabidopsis thaliana), the chloroplast-localized isochorismate pathway is the main source of SA biosynthesis during abiotic stress or pathogen infections. In the first step of the pathway, the enzyme ISOCHORISMATE SYNTHASE1 (ICS1) converts chorismate to isochorismate. An unknown enzyme subsequently converts isochorismate to SA. Here, we show that ICS1 protein levels increase during UV-C stress. To identify proteins that may play roles in SA production by regulating ICS1, we analyzed proteins that coimmunoprecipitated with ICS1 via mass spectrometry. The ICS1 complexes contained a large number of peptides from the PROHIBITIN (PHB) protein family, with PHB3 the most abundant. PHB proteins have diverse biological functions that include acting as scaffolds for protein complex formation and stabilization. PHB3 was reported previously to localize to mitochondria. Using fractionation, protease protection, and live imaging, we show that PHB3 also localizes to chloroplasts, where ICS1 resides. Notably, loss of PHB3 function led to decreased ICS1 protein levels in response to UV-C stress. However, ICS1 transcript levels remain unchanged, indicating that ICS1 is regulated posttranscriptionally. The phb3 mutant displayed reduced levels of SA, the SA-regulated protein PR1, and hypersensitive cell death in response to UV-C and avirulent strains of Pseudomonas syringae and, correspondingly, supported increased growth of P. syringae The expression of a PHB3 transgene in the phb3 mutant complemented all of these phenotypes. We suggest a model in which the formation of PHB3-ICS1 complexes stabilizes ICS1 to promote SA production in response to stress.


Assuntos
Arabidopsis/metabolismo , Transferases Intramoleculares/metabolismo , Proteínas Repressoras/metabolismo , Ácido Salicílico/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Transferases Intramoleculares/genética , Mitocôndrias/metabolismo , Mutação , Plantas Geneticamente Modificadas , Proibitinas , Pseudomonas syringae/patogenicidade , Proteínas Repressoras/genética , Estresse Fisiológico , Raios Ultravioleta
8.
Biochim Biophys Acta Proteins Proteom ; 1866(2): 224-229, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29050961

RESUMO

Enzyme-dependent post-translational modifications (PTMs) mediate the cellular regulation of proteins and can be discovered using proteomics. However, even where the peptides of interest can be enriched for analysis with state-of-the-art LC-MS/MS tools and informatics, only a fraction of peptide ions can be identified confidently. Thus, many PTM sites remain undiscovered and unconfirmed. In this minireview, we use a case study to discuss how the use of inclusion lists, turning off isotopic exclusion, and manual validation significantly increased depth of coverage, facilitating discovery of acetylation sites in targets of an acetyltransferase virulence factor. These underutilized strategies have the potential to help answer many mechanistic biological questions that large-scale proteomic studies cannot.


Assuntos
Peptídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Acetilação , Animais , Cromatografia Líquida/métodos , Humanos , Peptídeos/química , Peptídeos/metabolismo
9.
Mol Plant Microbe Interact ; 30(2): 150-160, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28051349

RESUMO

In plants, cell surface pattern recognition receptors (PRRs) provide a first line of defense against pathogens. Although each PRR recognizes a specific ligand, they share common signaling outputs, such as callose and other cell wall-based defenses. Several PRRs are also important for callose induction in response to the defense signal salicylic acid (SA). The extent to which common components are needed for PRR signaling outputs is not known. The gain-of-function Arabidopsis mutant of ACCELERATED CELL DEATH6 (ACD6) acd6-1 shows constitutive callose production that partially depends on PRRs. ACD6-1 (and ACD6) forms complexes with the PRR FLAGELLIN SENSING2, and ACD6 is needed for responses to several PRR ligands. Thus, ACD6-1 could serve as a probe to identify additional proteins important for PRR-mediated signaling. Candidate signaling proteins (CSPs), identified in our proteomic screen after immunoprecipitation of hemagglutinin (HA)-tagged ACD6-1, include several subfamilies of receptor-like kinase (RLK) proteins and a MECHANO-SENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 4 (MSL4). In acd6-1, CSPs contribute to autoimmunity. In wild type, CSPs are needed for defense against bacteria and callose responses to two or more microbial-derived patterns and an SA agonist. CSPs may function to either i) promote the assembly of signaling complexes, ii) regulate the output of known PRRs, or both.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Autoimunidade , Membrana Celular/metabolismo , Mecanotransdução Celular , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fenótipo , Pseudomonas syringae/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Salicílico/metabolismo , Regulação para Cima/genética
10.
Mol Plant Microbe Interact ; 30(7): 515-516, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28398839

RESUMO

Reader Comments | Submit a Comment The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.


Assuntos
Agricultura/métodos , Produtos Agrícolas/crescimento & desenvolvimento , Abastecimento de Alimentos , Pesquisa Translacional Biomédica/métodos , Biotecnologia/métodos , Mudança Climática , Produtos Agrícolas/microbiologia , Produtos Agrícolas/parasitologia , Humanos , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia
11.
J Exp Bot ; 68(7): 1769-1783, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28521013

RESUMO

Diverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections. Remarkably, entry into the plant cell via endocytosis together with the FLS2 receptor is needed for delivery to vascular tissue and long-distance transport of flg22. This contrasts with known routes of long distance transport of other non-cell-permeant molecules in plants, which require membrane-localized transporters for entry to vascular tissue. Thus, a plasma membrane receptor acts as a transporter to enable access of its ligand to distal trafficking routes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flagelina/metabolismo , Proteínas Quinases/metabolismo , Transporte Proteico , Endocitose , Ligantes
12.
Plant Cell ; 26(10): 4171-87, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25315322

RESUMO

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.


Assuntos
Anquirinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ácido Salicílico/metabolismo , Anquirinas/genética , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Immunoblotting , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiadiazóis/farmacologia
13.
Plant Cell ; 26(8): 3449-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25149397

RESUMO

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Ceramidas/biossíntese , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Apoptose/genética , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagia , Botrytis/imunologia , Botrytis/fisiologia , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Espécies Reativas de Oxigênio/metabolismo
14.
PLoS Pathog ; 10(6): e1004232, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24968323

RESUMO

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.


Assuntos
Citoesqueleto de Actina/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Pseudomonas syringae/patogenicidade , Fatores de Virulência/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/antagonistas & inibidores , Actinas/química , Actinas/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endocitose/efeitos dos fármacos , Herbicidas/química , Herbicidas/metabolismo , Herbicidas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/efeitos dos fármacos , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Plântula/microbiologia , Solubilidade , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Virulência/efeitos dos fármacos , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacologia
15.
Mol Plant Microbe Interact ; 28(4): 455-66, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25372120

RESUMO

Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Arabidopsis/genética , Arabidopsis/imunologia , Resistência à Doença/genética , Transaminases/genética , Transaminases/imunologia , Arabidopsis/química , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ácidos Pipecólicos/análise , Nicotiana/genética , Nicotiana/imunologia
16.
STAR Protoc ; 5(2): 102944, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38470913

RESUMO

Understanding the generation, movement, uptake, and perception of mobile defense signals is key for unraveling the systemic resistance programs in flowering plants against pathogens. Here, we present a protocol for analyzing the movement and uptake of isotopically labeled signaling molecule azelaic acid (AZA) in Arabidopsis thaliana. We describe steps to assess 14C-AZA uptake into leaf discs and its movement from local to systemic tissues. We also detail the assay for uptake and movement of 2H-AZA from roots to the shoot. For complete details on the use and execution of this protocol, please refer to Cecchini et al.1,2.


Assuntos
Arabidopsis , Ácidos Dicarboxílicos , Arabidopsis/metabolismo , Ácidos Dicarboxílicos/metabolismo , Marcação por Isótopo/métodos , Folhas de Planta/metabolismo , Transdução de Sinais/fisiologia , Raízes de Plantas/metabolismo , Transporte Biológico
17.
Bio Protoc ; 14(7): e4964, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38618179

RESUMO

Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1-32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system. Key features • This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species. • A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55. • Requires only 16-18 days to complete with high efficacy. Graphical overview.

18.
Plant J ; 69(4): 589-600, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21988537

RESUMO

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin-related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 (+) sectors, ACD2 functions cell autonomously, implicating a pro-death ACD2 substrate as being cell non-autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light-dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro-death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Mitocôndrias/enzimologia , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Reguladoras de Apoptose/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/enzimologia , Luz , Modelos Biológicos , Mutação , Oxirredutases/genética , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Explosão Respiratória
19.
Plant Physiol ; 158(4): 1803-18, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22319072

RESUMO

The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.


Assuntos
Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Nicotiana/microbiologia , Folhas de Planta/microbiologia , Pseudomonas syringae/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Aminoácidos/metabolismo , Aderência Bacteriana/fisiologia , Biocatálise , Biofilmes/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Proteínas de Fluorescência Verde/metabolismo , Solanum lycopersicum/imunologia , Fenótipo , Células Vegetais/microbiologia , Folhas de Planta/imunologia , Mutação Puntual/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Ácido Salicílico/metabolismo , Propriedades de Superfície , Nicotiana/imunologia
20.
Proc Natl Acad Sci U S A ; 107(29): 13177-82, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20615948

RESUMO

Plant heat shock protein Hsp70 is the major target of HopI1, a virulence effector of pathogenic Pseudomonas syringae. Hsp70 is essential for the virulence function of HopI1. HopI1 directly binds Hsp70 through its C-terminal J domain and stimulates Hsp70 ATP hydrolysis activity in vitro. In plants, HopI1 forms large complexes in association with Hsp70 and induces and recruits cytosolic Hsp70 to chloroplasts, the site of HopI1 localization. Deletion of a central P/Q-rich repeat region disrupts HopI1 virulence but not Hsp70 interactions or association with chloroplasts. Thus, HopI1 must not only bind Hsp70 through its J domain, but likely actively affects Hsp70 activity and/or specificity. At high temperature, HopI1 is dispensable for P. syringae pathogenicity, unless excess Hsp70 is provided. A working hypothesis is that Hsp70 has a defense-promoting activity(s) that HopI1 or high temperature can subvert. Enhanced susceptibility of Hsp70-depleted plants to nonpathogenic strains of P. syringae supports a defense-promoting role for Hsp70.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Plantas/metabolismo , Pseudomonas syringae/patogenicidade , Estresse Fisiológico , Trifosfato de Adenosina/metabolismo , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Citosol/metabolismo , Hidrólise , Imunidade Inata/imunologia , Complexos Multiproteicos/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Temperatura , Nicotiana/metabolismo , Nicotiana/microbiologia , Virulência , Fatores de Virulência/metabolismo
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