RESUMO
With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.
Assuntos
Solanum lycopersicum , Ativação Transcricional , Clavibacter/genética , Sistemas CRISPR-Cas , Edição de Genes , Produtos Agrícolas/genética , Doenças das Plantas/genéticaRESUMO
Priming is a mechanism of defense that prepares the plant's immune system for a faster and/or stronger activation of cellular defenses against future exposure to different types of stress. This enhanced resistance can be achieved by using inorganic and organic compounds which imitate the biological induction of systemic acquired resistance. INA (2,6 dichloro-isonicotinic acid) was the first synthetic compound created as a resistance inducer for plant-pathogen interactions. However, the use of INA to activate primed resistance in common bean, at the seed stage and during germination, remains experimentally unexplored. Here, we test the hypothesis that INA-seed treatment would induce resistance in common bean plants to Pseudomonas syringae pv. phaseolicola, and that the increased resistance is not accompanied by a tradeoff between plant defense and growth. Additionally, it was hypothesized that treating seeds with INA has a transgenerational priming effect. We provide evidence that seed treatment activates a primed state for disease resistance, in which low nucleosome enrichment and reduced histone activation marks during the priming phase, are associated with a defense-resistant phenotype, characterized by symptom appearance, pathogen accumulation, yield, and changes in gene expression. In addition, the priming status for induced resistance can be inherited to its offspring.
Assuntos
Resistência à Doença/imunologia , Germinação/imunologia , Ácidos Isonicotínicos/metabolismo , Phaseolus/imunologia , Phaseolus/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/imunologia , Produtos Agrícolas/imunologia , Produtos Agrícolas/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidadeRESUMO
Symbiotic Rhizobium-legume associations are mediated by exchange of chemical signals that eventually result in the development of a nitrogen-fixing nodule. Such signal interactions are thought to be at the center of the plants' capacity either to activate a defense response or to suppress the defense response to allow colonization by symbiotic bacteria. In addition, the colonization of plant roots by rhizobacteria activates an induced condition of improved defensive capacity in plants known as induced systemic resistance, based on "defense priming," which protects unexposed plant tissues from biotic stress.Here, we demonstrate that inoculation of common bean plants with Rhizobium etli resulted in a robust resistance against Pseudomonas syringae pv. phaseolicola. Indeed, inoculation with R. etli was associated with a reduction in the lesion size caused by the pathogen and lower colony forming units compared to mock-inoculated plants. Activation of the induced resistance was associated with an accumulation of the reactive oxygen species superoxide anion (O2 -) and a faster and stronger callose deposition. Transcription of defense related genes in plants treated with R. etli exhibit a pattern that is typical of the priming response. In addition, R. etli-primed plants developed a transgenerational defense memory and could produce offspring that were more resistant to halo blight disease. R. etli is a rhizobacteria that could reduce the proliferation of the virulent strain P. syringae pv. phaseolicola in common bean plants and should be considered as a potentially beneficial and eco-friendly tool in plant disease management.
RESUMO
The legume-rhizobium symbiotic relationship has been widely studied and characterized. However, little information is available about the role of histone lysine methyltransferases in the legume-rhizobium interaction and in the formation of nitrogen-fixing nodules in the common bean. Thus, this study aimed to gain a better understanding of the epigenetic control of nodulation in the common bean. Specifically, we studied the role of PvTRX1h, a histone lysine methyltransferase coding gene, in nodule development and auxin biosynthesis. Through a reverse genetics approach, we generated common bean composite plants to knock-down PvTRX1h expression. Here we found that the down-regulation of PvTRX1h increased the number of nodules per plant, but reduced the number of colony-forming units recovered from nodules. Genes coding for enzymes involved in the synthesis of the indole-3-acetic acid were up-regulated, as was the concentration of this hormone. In addition, PvTRX1h down-regulation altered starch accumulation as determined by the number of amyloplasts per nodule. Metabolic fingerprinting by direct liquid introduction-electrospray ionization-mass spectrometry (DLI-ESI-MS) revealed that the root nodules were globally affected by PvTRX1h down-regulation. Therefore, PvTRX1h likely acts through chromatin histone modifications that alter the auxin signaling network to determine bacterial colonization, nodule number, starch accumulation, hormone levels, and cell proliferation.
Assuntos
Ácidos Indolacéticos/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Amido/metabolismo , Western Blotting , Regulação para Baixo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Throughout evolution, plants have developed diverse mechanisms of defense that "prime" their innate immune system for more robust and active induction of defense responses against different types of stress. Nowadays there are numerous reports concerning the molecular bases of priming, as well as the generational priming mechanisms. Information concerning transgenerational priming, however, remains deficient. Some reports have indicated, nonetheless, that the priming status of a plant can be inherited to its offspring. Here, we show that the priming agent ß-aminobutyric acid induced resistance to Pseudomonas syringae pv. phaseolicola infection in the common bean (Phaseolus vulgaris L.) We have analyzed the transgenerational patterns of gene expression of the PvPR1 gene (Phaseolus vulgaris PR1), a highly responsive gene to priming, and show that a transgenerational priming response against pathogen attack can last for at least two generations. We hypothesize that a defense-resistant phenotype and easily identifiable, generational and transgenerational, "primed patterns" of gene expression are excellent indicators of the priming response in crop plants. Furthermore, we propose here that modern plant breeding methods and crop improvement efforts must include the use of elicitors to prime induced resistance in the field and, above all, to select for induced heritable states in progeny that is primed for defense.
RESUMO
To survive in adverse conditions, plants have evolved complex mechanisms that "prime" their defense system to respond and adapt to stresses. Their competence to respond to such stresses fundamentally depends on its capacity to modulate the transcriptome rapidly and specifically. Thus, chromatin dynamics is a mechanism linked to transcriptional regulation and enhanced defense in plants. For example, in Arabidopsis, priming of the SA-dependent defense pathway is linked to histone lysine methylation. Such modifications could create a memory of the primary infection that is associated with an amplified gene response upon exposure to a second stress-stimulus. In addition, the priming status of a plant for induced resistance can be inherited to its offspring. However, analyses on the molecular mechanisms of generational and transgenerational priming in the common bean (Phaseolus vulagris L.), an economically important crop, are absent. Here, we provide evidence that resistance to P. syringae pv. phaseolicola infection was induced in the common bean with the synthetic priming activators BABA and INA. Resistance was assessed by evaluating symptom appearance, pathogen accumulation, changes in gene expression of defense genes, as well as changes in the H3K4me3 and H3K36me3 marks at the promoter-exon regions of defense-associated genes. We conclude that defense priming in the common bean occurred in response to BABA and INA and that these synthetic activators primed distinct genes for enhanced disease resistance. We hope that an understanding of the molecular changes leading to defense priming and pathogen resistance will provide valuable knowledge for producing disease-resistant crop varieties by exposing parental plants to priming activators, as well as to the development of novel plant protection chemicals that stimulate the plant's inherent disease resistance mechanisms.
RESUMO
BACKGROUND: Conserved domains are recognized as the building blocks of eukaryotic proteins. Domains showing a tendency to occur in diverse combinations ('promiscuous' domains) are involved in versatile architectures in proteins with different functions. Current models, based on global-level analyses of domain combinations in multiple genomes, have suggested that the propensity of some domains to associate with other domains in high-level architectures increases with organismal complexity. Alternative models using domain-based phylogenetic trees propose that domains have become promiscuous independently in different lineages through convergent evolution and are, thus, random with no functional or structural preferences. Here we test whether complex protein architectures have occurred by accretion from simpler systems and whether the appearance of multidomain combinations parallels organismal complexity. As a model, we analyze the modular evolution of the PWWP domain and ask whether its appearance in combinations with other domains into multidomain architectures is linked with the occurrence of more complex life-forms. Whether high-level combinations of domains are conserved and transmitted as stable units (cassettes) through evolution is examined in the genomes of plant or metazoan species selected for their established position in the evolution of the respective lineages. RESULTS: Using the domain-tree approach, we analyze the evolutionary origins and distribution patterns of the promiscuous PWWP domain to understand the principles of its modular evolution and its existence in combination with other domains in higher-level protein architectures. We found that as a single module the PWWP domain occurs only in proteins with a limited, mainly, species-specific distribution. Earlier, it was suggested that domain promiscuity is a fast-changing (volatile) feature shaped by natural selection and that only a few domains retain their promiscuity status throughout evolution. In contrast, our data show that most of the multidomain PWWP combinations in extant multicellular organisms (humans or land plants) are present in their unicellular ancestral relatives suggesting they have been transmitted through evolution as conserved linear arrangements ('cassettes'). Among the most interesting biologically relevant results is the finding that the genes of the two plant Trithorax family subgroups (ATX1/2 and ATX3/4/5) have different phylogenetic origins. The two subgroups occur together in the earliest land plants Physcomitrella patens and Selaginella moellendorffii. CONCLUSION: Gain/loss of a single PWWP domain is observed throughout evolution reflecting dynamic lineage- or species-specific events. In contrast, higher-level protein architectures involving the PWWP domain have survived as stable arrangements driven by evolutionary descent. The association of PWWP domains with the DNA methyltransferases in O. tauri and in the metazoan lineage seems to have occurred independently consistent with convergent evolution. Our results do not support models wherein more complex protein architectures involving the PWWP domain occur with the appearance of more evolutionarily advanced life forms.
Assuntos
Metiltransferases/genética , Proteínas Nucleares/genética , Plantas/genética , Estrutura Terciária de Proteína , Anêmonas-do-Mar/genética , Animais , Arabidopsis/genética , Clorófitas/genética , Humanos , Proteínas Nucleares/químicaRESUMO
Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have central roles in cell proliferation, growth and development. In animals, PcG and trxG proteins form higher order protein complexes that contain SET domain proteins with histone methyltransferase activity, and are responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. However, whether H3K4 methyltransferase complexes exist in Arabidopsis thaliana remains unknown. Here, we make use of the yeast two-hybrid system and the bimolecular fluorescence complementation assay to provide evidence for the self-association of the Arabidopsis thaliana SET-domain-containing protein SET DOMAIN GROUP 26 (SDG26), also known as ABSENT, SMALL, OR HOMEOTIC DISCS 1 HOMOLOG 1 (ASHH1). In addition, we show that the ASHH1 protein associates with SET-domain-containing sequences from two distinct histone lysine methyltransferases, the ARABIDOPSIS HOMOLOG OF TRITHORAX-1 (ATX1) and ASHH2 proteins. Furthermore, after screening a cDNA library we found that ASHH1 interacts with two proteins from the heat shock protein 40 kDa (Hsp40/DnaJ) superfamily, thus connecting the epigenetic network with a system sensing external cues. Our findings suggest that trxG complexes in Arabidopsis thaliana could involve different sets of histone lysine methyltransferases, and that these complexes may be engaged in multiple developmental processes in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Choque Térmico/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Genes Homeobox , Histona-Lisina N-Metiltransferase/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Genes in eukaryotic organisms function within the context of chromatin, and the mechanisms that modulate the structure of chromatin are defined as epigenetic. In Arabidopsis, pathogen infection induces the expression of at least one histone deacetylase, suggesting that histone acetylation/deacetylation has an important role in the pathogenic response in plants. How/whether histone methylation affects gene response to pathogen infection is unknown. To gain a better understanding of the epigenetic mechanisms regulating the interaction between Pseudomonas syringae and Arabidopsis thaliana, we analysed three different Arabidopsis ash1-related (absent, small or homeotic discs 1) mutants. We found that the loss of function of ASHH2 and ASHR1 resulted in faster hypersensitive responses (HRs) to both mutant (hrpA) and pathogenic (DC3000) strains of P. syringae, whereas control (Col-0) and ashr3 mutants appeared to be more resistant to the infection after 2 days. Furthermore, we showed that, in the ashr3 background, the PR1 gene (PATHOGENESIS-RELATED GENE 1) displayed the highest expression levels on infection with DC3000, correlating with increased resistance against this pathogen. Our results show that, in both the ashr1 and ashh2 backgrounds, the histone H3 lysine 4 dimethylation (H3K4me2) levels decreased at the promoter region of PR1 on infection with the DC3000 strain, suggesting that an epigenetically regulated PR1 expression is involved in the plant defence. Our results suggest that histone methylation in response to pathogen infection may be a critical component in the signalling and defence processes occurring between plants and microbes.
Assuntos
Arabidopsis/genética , Arabidopsis/microbiologia , Epigênese Genética , Genes de Plantas/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Pseudomonas syringae/fisiologia , Arabidopsis/imunologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Imunoprecipitação da Cromatina , DNA Bacteriano/genética , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Heterocromatina/metabolismo , Histonas/metabolismo , Metilação , Mutagênese Insercional/genética , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Estrutura Terciária de ProteínaRESUMO
A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Limoninas/análise , Carne/análise , Resíduos de Praguicidas/análise , Animais , Bovinos , Cromatografia de Fase Reversa , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
Este estudio tuvo por objetivo la evaluación de los efectos de germinación sobre la calidad nutricional en dos variedades comerciales de sorgo bajo en taninos: sorgo rojo sin testa (ICSY-LM 89513) y sorgo blanco (ISIAO Dorado). Después de 24 horas de germinación la concentración de taninos condensados (equivalentes de catequina) se redujo en 60 por ciento y 40 por ciento para el sorgo rojo y blanco respectivamente. Sin embargo los niveles de taninos se incrementaron a 100 por ciento a las 96 horas de germinación. La concentración de ácido fítico disminuyó cerca del 90 por ciento a las 96 horas para ambas variedades. El contenido de lisina se incrementó en 110 por ciento (72 horas de germinación) y 129 por ciento (48 horas) para el sorgo blanco y rojo respectivamente. Los contenidos de tiamina, niacina y riboflavina se incrementaron en 73,200 y 353 por ciento respectivamente para el sorgo rojo a las 72 h. de germinación y 15,44 y 93 por ciento respectivamente para el sorgo blanco a las 48 horas de germinación. La digestibilidad enzimática "in vitro" se incrementó a las 72 h. en 39.3 por ciento para el sorgo blanco. La concentración de albúminas fue incrementada a las 72 horas en 80 por ciento y 74 por ciento para el sorgo rojo y blanco respectivamente. La Relación de Eficiencia Proteica Calculada indica mejorías nutricionales con la germinación, resultando este proceso un método práctico y sencillo que prevee mejores propiedades nutricionales a las semillas de sorgo para ser usado como alimento humano