RESUMO
Spray-induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double-stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole-fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii-Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis-antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a, lipase 1, and acetyl-CoA oxidase in energy production; and genes involved in manipulation of the plant host via abscisic acid metabolism (9-cis-epoxycarotenoid dioxygenase, xanthoxin dehydrogenase, and a putative abscisic acid G-protein coupled receptor) and secretion of the effector protein, effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator-Vitis vinifera system and tested six successful targets identified using the G. orontii-A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii-A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.
Assuntos
Arabidopsis , Arabidopsis/microbiologia , Ácido Abscísico/metabolismo , Sequência de Bases , Inativação Gênica , Doenças das Plantas/microbiologiaRESUMO
Extreme weather conditions associated with climate change affect many aspects of plant and animal life, including the response to infectious diseases. Production of salicylic acid (SA), a central plant defence hormone1-3, is particularly vulnerable to suppression by short periods of hot weather above the normal plant growth temperature range via an unknown mechanism4-7. Here we show that suppression of SA production in Arabidopsis thaliana at 28 °C is independent of PHYTOCHROME B8,9 (phyB) and EARLY FLOWERING 310 (ELF3), which regulate thermo-responsive plant growth and development. Instead, we found that formation of GUANYLATE BINDING PROTEIN-LIKE 3 (GBPL3) defence-activated biomolecular condensates11 (GDACs) was reduced at the higher growth temperature. The altered GDAC formation in vivo is linked to impaired recruitment of GBPL3 and SA-associated Mediator subunits to the promoters of CBP60g and SARD1, which encode master immune transcription factors. Unlike many other SA signalling components, including the SA receptor and biosynthetic genes, optimized CBP60g expression was sufficient to broadly restore SA production, basal immunity and effector-triggered immunity at the elevated growth temperature without significant growth trade-offs. CBP60g family transcription factors are widely conserved in plants12. These results have implications for safeguarding the plant immune system as well as understanding the concept of the plant-pathogen-environment disease triangle and the emergence of new disease epidemics in a warming climate.
Assuntos
Aclimatação , Proteínas de Arabidopsis , Arabidopsis , Meio Ambiente , Aquecimento Global , Imunidade Vegetal , Temperatura , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/genética , Regulação da Expressão Gênica de Plantas , Aquecimento Global/estatística & dados numéricos , Interações Hospedeiro-Patógeno , Fitocromo B , Doenças das Plantas/genética , Imunidade Vegetal/genética , Ácido Salicílico/metabolismo , Fatores de TranscriçãoRESUMO
Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re-envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.
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 UltravioletaRESUMO
The GH3 family of adenylating enzymes conjugate acyl substrates such as the growth hormone indole-3-acetic acid (IAA) to amino acids via a two-step reaction of acyl substrate adenylation followed by amino acid conjugation. Arabidopsis thaliana GH3.5 was previously shown to be unusual in that it could adenylate both IAA and the defense hormone salicylic acid (SA, 2-hydroxybenzoate). Our detailed studies of the kinetics of GH3.5 on a variety of auxin and benzoate substrates provides insight into the acyl preference and reaction mechanism of GH3.5. For example, we found GH3.5 activity on substituted benzoates is not defined by the substitution position as it is for GH3.12/PBS3. Most importantly, we show that GH3.5 strongly prefers Asp as the amino acid conjugate and that the concentration of Asp dictates the functional activity of GH3.5 on IAA vs. SA. Not only is Asp used in amino acid biosynthesis, but it also plays an important role in nitrogen mobilization and in the production of downstream metabolites, including pipecolic acid which propagates defense systemically. During active growth, [IAA] and [Asp] are high and the catalytic efficiency (kcat/Km) of GH3.5 for IAA is 360-fold higher than with SA. GH3.5 is expressed under these conditions and conversion of IAA to inactive IAA-Asp would provide fine spatial and temporal control over local auxin developmental responses. By contrast, [SA] is dramatically elevated in response to (hemi)-biotrophic pathogens which also induce GH3.5 expression. Under these conditions, [Asp] is low and GH3.5 has equal affinity (Km) for SA and IAA with similar catalytic efficiencies. However, the concentration of IAA tends to be very low, well below the Km for IAA. Therefore, GH3.5 catalyzed formation of SA-Asp would occur, fine-tuning localized defensive responses through conversion of active free SA to SA-Asp. Taken together, we show how GH3.5, with dual activity on IAA and SA, can integrate cellular metabolic status via Asp to provide fine control of growth vs. defense outcomes and hormone homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Aspártico/análise , Ligases/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/análise , Ácidos Indolacéticos/metabolismo , Cinética , Ácido Salicílico/metabolismoRESUMO
Diverse plant biotrophs that establish a sustained site of nutrient acquisition induce localized host endoreduplication. Endoreduplication is a process by which cells successively replicate their genomes without mitosis, resulting in an increase in nuclear DNA ploidy. Elevated ploidy is associated with enhanced cell size, metabolic capacity, and the capacity to differentiate. Localized host endoreduplication induced by adapted plant biotrophs promotes biotroph colonization, development, and/or proliferation. When induced host endoreduplication is limited, biotroph growth and/or development are compromised. Herein, we examine a diverse set of plant-biotroph interactions to identify (a) common host components manipulated to promote induced host endoreduplication and (b) biotroph effectors that facilitate this induced host process. Shared mechanisms to promote host endoreduplication and development of nutrient exchange/feeding sites include manipulation centered on endocycle entry at the G2-M transition as well as yet undefined roles for differentiation regulators (e.g., CLE peptides) and pectin/cell wall modification.
Assuntos
Endorreduplicação , Plantas/genética , Plantas/microbiologia , Ploidias , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Mitose , SimbioseRESUMO
Laser microdissection of cells allows for isolation of specific cells of interest for downstream analyses including transcriptional profiling. Plant cells present unique challenges for laser microdissection due to their cellulosic cell walls and large vacuoles. Here we present protocols for plant tissue preparation, laser microdissection of select plant cells, and linear amplification of RNA from dissected cells. Linear amplification of RNA from dissected cells allows sufficient RNA for subsequent quantitative analysis by RT-PCR, microarray, or RNA sequencing.
Assuntos
Microdissecção e Captura a Laser/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Células Vegetais , RNA de Plantas/genética , RNA de Plantas/isolamento & purificaçãoRESUMO
In plants, the activation of immunity is often inversely correlated with growth. Mechanisms that control plant growth in the context of pathogen challenge and immunity are unclear. Investigating Arabidopsis infection with the powdery mildew fungus, we find that the Arabidopsis atypical E2F DEL1, a transcriptional repressor known to promote cell proliferation, represses accumulation of the hormone salicylic acid (SA), an established regulator of plant immunity. DEL1-deficient plants are more resistant to pathogens and slightly smaller than wild-type. The resistance and size phenotypes of DEL1-deficient plants are due to the induction of SA and activation of immunity in the absence of pathogen challenge. Moreover, Enhanced Disease Susceptibility 5 (EDS5), a SA transporter required for elevated SA and immunity, is a direct repressed target of DEL1. Together, these findings indicate that DEL1 control of SA levels contributes to regulating the balance between growth and immunity in developing leaves.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Ácido Salicílico/metabolismo , Fatores de Transcrição/imunologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Arabidopsis/imunologia , Proliferação de Células , Regulação da Expressão Gênica de Plantas , Imunidade Inata , Proteínas de Membrana Transportadoras/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/imunologia , Saccharomycetales/imunologia , Saccharomycetales/patogenicidade , Transdução de Sinais/imunologia , Fatores de Transcrição/genéticaRESUMO
Golovinomyces orontii is an obligate biotrophic powdery mildew (PM) that colonizes Arabidopsis thaliana and agronomic species. It establishes a specialized feeding structure in epidermal cells to fuel its extensive surface hyphal growth and reproduction. Previously, endoreduplication was identified in Arabidopsis mesophyll cells underlying the fungal feeding site, presumably to meet the metabolic demands imposed by the fungus. Furthermore, the cell cycle transcription factor MYB3R4 was shown to regulate this process. Herein, PM-induced endoreduplication is further characterized and three additional factors influencing host ploidy in cells underlying the fungal feeding site are identified. While mutations in PUX2 and PMR6 reduce basal ploidy, mutations in PMR5 (and MYB3R4) abrogate the PM-induced ploidy increase. Moreover, analysis of pmr5 microarray data suggests that PMR5 acts upstream of a MYB3R transcription factor such as MYB3R4 to control PM-induced ploidy. Induced endoreduplication occurs exclusively in mesophyll cells underlying the fungal feeding site at 5 days postinoculation, concomitant with PM reproduction. Gene copy number increases and chromatin remains decondensed, suggesting active, elevated gene expression. Cell ploidy underlying the fungal feeding site is highly correlated with the extent of PM growth and reproduction for these mutants, indicating that (induced) mesophyll cell ploidy is a PM susceptibility determinant.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Doenças das Plantas/microbiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Interações Hospedeiro-Patógeno , Ploidias , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , TransativadoresRESUMO
GH3 amino acid conjugases have been identified in many plant and bacterial species. The evolution of GH3 genes in plant species is explored using the sequenced rosids Arabidopsis, papaya, poplar, and grape. Analysis of the sequenced non-rosid eudicots monkey flower and columbine, the monocots maize and rice, as well as spikemoss and moss is included to provide further insight into the origin of GH3 clades. Comparison of co-linear genes in regions surrounding GH3 genes between species helps reconstruct the evolutionary history of the family. Combining analysis of synteny with phylogenetics, gene expression and functional data redefines the Group III GH3 genes, of which AtGH3.12/PBS3, a regulator of stress-induced salicylic acid metabolism and plant defense, is a member. Contrary to previous reports that restrict PBS3 to Arabidopsis and its close relatives, PBS3 syntelogs are identified in poplar, grape, columbine, maize and rice suggesting descent from a common ancestral chromosome dating to before the eudicot/monocot split. In addition, the clade containing PBS3 has undergone a unique expansion in Arabidopsis, with expression patterns for these genes consistent with specialized and evolving stress-responsive functions.
Assuntos
Arabidopsis/genética , Evolução Molecular , Nucleotidiltransferases/genética , Proteínas de Plantas/genética , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Carica/genética , Funções Verossimilhança , Família Multigênica , Nucleotidiltransferases/classificação , Nucleotidiltransferases/metabolismo , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Populus/genética , Alinhamento de Sequência , Sintenia , Vitis/genéticaRESUMO
High-affinity iron scavenging through the use of siderophores is a well-established virulence determinant in mammalian pathogenesis. However, few examples have been reported for plant pathogens. Here, we use a genetic approach to investigate the role of siderophores in Pseudomonas syringae pv. tomato DC3000 (DC3000) virulence in tomato. DC3000, an agronomically important pathogen, has two known siderophores for high-affinity iron scavenging, yersiniabactin and pyoverdin, and we uncover a third siderophore, citrate, required for growth when iron is limiting. Though growth of a DC3000 triple mutant unable to either synthesize or import these siderophores is severely restricted in iron-limited culture, it is fully pathogenic. One explanation for this phenotype is that the DC3000 triple mutant is able to directly pirate plant iron compounds such as heme/hemin or iron-nicotianamine, and our data indicate that DC3000 can import iron-nicotianamine with high affinity. However, an alternative explanation, supported by data from others, is that the pathogenic environment of DC3000 (i.e., leaf apoplast) is not iron limited but is iron replete, with available iron of >1 µM. Growth of the triple mutant in culture is restored to wild-type levels by supplementation with a variety of iron chelates at >1 µM, including iron(III) dicitrate, a dominant chelate of the leaf apoplast. This suggests that lower-affinity iron import would be sufficient for DC3000 iron nutrition in planta and is in sharp contrast to the high-affinity iron-scavenging mechanisms required in mammalian pathogenesis.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade , Sideróforos/metabolismo , Ácido Cítrico/metabolismo , Técnicas de Inativação de Genes , Solanum lycopersicum/microbiologia , Oligopeptídeos/metabolismo , Fenóis/metabolismo , Pseudomonas syringae/genética , Sideróforos/genética , Tiazóis/metabolismo , VirulênciaRESUMO
The molecular mechanisms that mediate the intimate interaction of an adapted obligate biotroph, such as the powdery mildew Golovinomyces orontii, on its host plant are spatially and temporally distinct. As G. orontii exclusively infects epidermal cells with a dominant host response occurring in the underlying mesophyll cells, we sought to develop a method to accurately and reproducibly perform global expression profiling on Arabidopsis thaliana leaf epidermal and mesophyll cells at the site of infection. Specific stages of G. orontii disease progression on Arabidopsis are visible by microscopy thus allowing distinct phases of the interaction to be studied. Tissue preparation, laser microdissection, and RNA isolation protocols that allow for temporally and spatially defined global expression profiling are described. By using these procedures to examine the growth and reproduction phase (5 days postinfection) of G. orontii on Arabidopsis, we identified known and novel processes, process components, and putative regulators of these processes that mediate the sustained growth and reproduction of this adapted obligate biotroph.
Assuntos
Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno , Lasers , Microdissecção/métodos , RNA/isolamento & purificação , Arabidopsis/citologia , Arabidopsis/genética , Ascomicetos/genética , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Microdissecção/instrumentação , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/microbiologiaRESUMO
Global expression profiling of RNA isolated from laser microdissected cells allows one to profile a specific set of cells allowing for enhanced sensitivity and for cell- or site-specific patterns of expression to emerge. In Chapter 19, we detail our optimized methods of tissue preparation, laser microdissection (LMD), and RNA isolation of cells at the site of Golovinomyces orontii infection of mature Arabidopsis leaves. Here, we describe (1) amplification of the RNA to obtain sufficient starting material for microarray analysis, (2) microarray hybridization and associated quality control assessments. As tissue preparation, LMD, and/or RNA amplification could impact mRNA quality, distribution, and/or microarray processing and output, it is important to include quality control assessments at every step of the protocol to ensure that the final data is a reproducible and accurate readout of the biological source material. The collection of parallel samples to evaluate these components of the experimental protocol allows one to determine their impact on mRNA quality and distribution (described in Chapter 19) and on microarray output (discussed here). In addition, one likely wants to compare similarly processed whole leaf samples to LMD-isolated samples in order to identify genes and processes specifically impacted or more highly impacted at the infection site compared with the whole leaf. Using the procedures described herein to profile cells specifically at the site of powdery mildew infection of Arabidopsis (Chandran et al., Proc Natl Acad Sci U S A 107(1):460-465, 2010), we determined that our site-specific global expression data was a highly reproducible, sensitive, and accurate readout of the infection site. Furthermore, this site-specific analysis allowed us to identify novel processes (e.g., endore-duplication), regulators (e.g., MYB3R4), and process components associated with the sustained growth and reproduction of the powdery mildew G. orontii on Arabidopsis thaliana at 5 days postinfection that were hidden in whole leaf analyses (Chandran et al., Proc Natl Acad Sci U S A 107(1):460-465, 2010).
Assuntos
Ascomicetos/genética , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno/genética , Lasers , Microdissecção/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/genética , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Perfilação da Expressão Gênica/instrumentação , Microdissecção/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Folhas de Planta/genética , Folhas de Planta/microbiologia , RNA/isolamento & purificaçãoRESUMO
Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented.
RESUMO
Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance.
Assuntos
Arabidopsis/imunologia , Ascomicetos/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanidinas/farmacologia , Imidazóis/farmacologia , Inseticidas/farmacologia , Nitrocompostos/farmacologia , Ácido Salicílico/imunologia , Tiazóis/farmacologia , Arabidopsis/microbiologia , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica de Plantas/imunologia , Guanidinas/química , Guanidinas/imunologia , Imidazóis/química , Imidazóis/imunologia , Inseticidas/química , Inseticidas/imunologia , Espectrometria de Massas , Estrutura Molecular , Neonicotinoides , Nitrocompostos/química , Nitrocompostos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/metabolismo , Tiazóis/química , Tiazóis/imunologiaRESUMO
Plant biotrophs often establish highly specialized and localized interaction sites where sustained nutrient exchange occurs. Increased plant nuclear DNA ploidy at or adjacent to these sites has now been reported for a diverse set of interactions, including those with fungal and bacterial symbionts and parasitic fungi and nematodes. Also, novel regulators of induced endoreduplication have recently been identified. When localized host endoreduplication is reduced, so too is the growth and/or development of the biotroph, suggesting endoreduplication supports the enhanced metabolic demands imposed by these interactions. Transcriptome analyses support this function and further identify specific ploidy-impacted processes. Remarkably, notwithstanding differences in time scales, the ploidy-impacted processes are consistent with the Gene Balance Hypothesis, which can also be used to predict effector targets. As effector influence may diminish with enhanced ploidy, these interaction sites may be uniquely suited to identify effector-impacted processes as well as elucidate endocycle regulation and function.
Assuntos
Núcleo Celular/genética , Plantas/genética , Ploidias , Animais , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Plantas/microbiologia , Plantas/parasitologia , SimbioseRESUMO
To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana, laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host.
Assuntos
Arabidopsis , Ascomicetos/fisiologia , Interações Hospedeiro-Patógeno , Microdissecção/métodos , Micoses/metabolismo , Doenças das Plantas , Arabidopsis/citologia , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Sinalização do Cálcio/fisiologia , Carbono/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lasers , Pinças Ópticas , Oxirredução , Fotossíntese/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Fatores de Transcrição/genéticaRESUMO
Salicylate (SA, 2-hydroxybenzoate) is a phytohormone best known for its role as a critical mediator of local and systemic plant defense responses. In response to pathogens such as Pseudomonas syringae, SA is synthesized and activates widespread gene expression. In gh3.12/pbs3 mutants of Arabidopsis thaliana, induced total SA accumulation is significantly compromised as is SA-dependent gene expression and plant defense. AtGH3 subfamily I and II members have been shown to conjugate phytohormone acyl substrates to amino acids in vitro, with this role supported by in planta analyses. Here we sought to determine the in vitro biochemical activity and kinetic properties of GH3.12/avrPphB susceptible 3 (PBS3), a member of the uncharacterized AtGH3 subfamily III. Using a novel high throughput adenylation assay, we characterized the acyl substrate preference of PBS3. We found PBS3 favors 4-substituted benzoates such as 4-aminobenzoate and 4-hydroxybenzoate, with moderate activity on benzoate and no observed activity with 2-substituted benzoates. Similar to known GH3 enzymes, PBS3 catalyzes the conjugation of specific amino acids (e.g. Glu) to its preferred acyl substrates. Kinetic analyses indicate 4-aminobenzoate and 4-hydroxybenzoate are preferred acyl substrates as PBS3 exhibits both higher affinities (apparent K(m) = 153 and 459 microm, respectively) and higher catalytic efficiencies (k(cat)/K(m) = 0.0179 and 0.0444 microm(-1) min(-1), respectively) with these acyl substrates compared with benzoate (apparent K(m) = 867 microm, k(cat)/K(m) = 0.0046 microm(-1) min(-1)). Notably, SA specifically and reversibly inhibits PBS3 activity with an IC(50) of 15 microm. This suggests a general mechanism for the rapid, reversible regulation of GH3 activity and small molecule cross-talk. For PBS3, this may allow for coordination of flux through diverse chorismate-derived pathways.
Assuntos
Aminoácidos/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Benzoatos/química , Ácido Salicílico/química , Carbono-Oxigênio Liases/química , Catálise , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Concentração Inibidora 50 , Cinética , Espectrometria de Massas/métodos , Modelos Químicos , Mutação , Pseudomonas syringae/metabolismoRESUMO
Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wild-type and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T(2) statistic). Functional analyses of T(2)-selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.