RESUMO
MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.
Assuntos
Glycine max , Nematoides , Animais , Glycine max/genética , RNA Guia de Sistemas CRISPR-Cas , Bioensaio , Cotilédone , Nematoides/genéticaRESUMO
Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.
Assuntos
Arachis/genética , Nicotiana/fisiologia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Animais , Ascomicetos/patogenicidade , Fenômenos Biomecânicos , Parede Celular/genética , Parede Celular/metabolismo , Ciclopentanos/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Células Vegetais/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/microbiologia , Tylenchoidea/patogenicidadeRESUMO
Raffinose family oligosaccharides (RFOs) are implicated in plant regulatory mechanisms of abiotic stresses tolerance and, despite their antinutritional proprieties in grain legumes, little information is available about the enzymes involved in RFO metabolism in Fabaceae species. In the present study, the systematic survey of legume proteins belonging to five key enzymes involved in the metabolism of RFOs (galactinol synthase, raffinose synthase, stachyose synthase, alpha-galactosidase, and beta-fructofuranosidase) identified 28 coding-genes in Arachis duranensis and 31 in A. ipaënsis. Their phylogenetic relationships, gene structures, protein domains, and chromosome distribution patterns were also determined. Based on the expression profiling of these genes under water deficit treatments, a galactinol synthase candidate gene (AdGolS3) was identified in A. duranensis. Transgenic Arabidopsis plants overexpressing AdGolS3 exhibited increased levels of raffinose and reduced stress symptoms under drought, osmotic, and salt stresses. Metabolite and expression profiling suggested that AdGolS3 overexpression was associated with fewer metabolic perturbations under drought stress, together with better protection against oxidative damage. Overall, this study enabled the identification of a promising GolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in crops, whilst also contributing to the understanding of RFO metabolism in legume species.
Assuntos
Arachis/genética , Galactosiltransferases/genética , Rafinose/genética , Estresse Fisiológico/genética , Arabidopsis/genética , Dissacarídeos/genética , Secas , Regulação da Expressão Gênica de Plantas/genética , Oligossacarídeos/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , beta-Frutofuranosidase/genéticaRESUMO
Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SIGNIFICANCE: The use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.
Assuntos
Tylenchoidea , Agrobacterium , Animais , Arachis/genética , Cromatografia Líquida , Resistência à Doença/genética , Melhoramento Vegetal , Doenças das Plantas/genética , Raízes de Plantas , Proteômica , América do Sul , Espectrometria de Massas em TandemRESUMO
Peanut wild relatives (Arachis spp.) have high genetic diversity and are important sources of resistance to biotic and abiotic stresses. In this study, proteins were analyzed in root tissues of A. duranensis submitted to a progressive water deficit in soil and the differential abundance was compared to transcript expression profiles obtained by RNA-seq and qRT-PCR. Using a 2-DE approach, a total of 31 proteins were identified, most of which were associated with stress response and drought perception. These comprised a chitinase-2 (unique to stressed condition), an MLP-like protein, a glycine-rich protein DOT1-like, a maturase K and heat shock-related proteins (HSP70 - an isoform unique to the control, and HSP17.3). Other proteins unique to the control condition comprised a transcription initiation factor IIF subunit alpha-like protein, a SRPBCC ligand-binding domain superfamily protein, an Adenine phosphoribosyl transferase, a Leo1-like protein, a Cobalamine-independent methionine synthase and a Transmembrane emp24 domain-containing protein p24delta9-like. Correlation of mRNA expression and corresponding protein abundance was observed for 15 of the identified proteins, with genes encoding the majority of proteins (14) negatively regulated in stressed roots. Proteins identified in this study offer potential for the genetic improvement of cultivated peanut for drought tolerance. SIGNIFICANCE: The comparison of protein abundance and corresponding transcript expression levels (RNA-seq and qRT-PCR) revealed that 15 of the identified proteins showed similar expression behavior, with the majority (14 proteins) negatively regulated in stressed roots. Chitinase-2 (Cht2) was the only protein with an upregulation behavior in all approaches. These proteins appear to play an important role in drought tolerance in A. duranensis and may be further explored in peanut genetic breeding programs.
Assuntos
Arachis/metabolismo , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Raízes de Plantas/metabolismo , Arachis/genética , Desidratação/genética , Desidratação/metabolismo , Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Raízes de Plantas/genética , ProteômicaRESUMO
Peanut, Arachis hypogaea (Linnaeus, 1753) is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species, A. duranensis (Krapovickas & W. C. Gregory, 1994) and A. ipaensis (Krapovickas & W. C. Gregory, 1994), followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes of A. hypogaea, IpaDur1, an induced allotetraploid (A. ipaensis × A. duranensis)4x and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+ bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst for A. hypogaea the number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, although A. hypogaea had additional CMA3+ bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome of A. hypogaea that shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.
RESUMO
Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of â¼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanut's A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanut's subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.
Assuntos
Arachis/genética , Genoma de Planta , Cromossomos de Plantas/genética , Metilação de DNA , Elementos de DNA Transponíveis , Evolução Molecular , Ligação Genética , Anotação de Sequência Molecular , Ploidias , Análise de Sequência de DNA , SinteniaRESUMO
Wild peanut relatives (Arachis spp.) are genetically diverse and were adapted to a range of environments during the evolution course, constituting an important source of allele diversity for resistance to biotic and abiotic stresses. The wild diploid A. stenosperma harbors high levels of resistance to a variety of pathogens, including the root-knot nematode (RKN) Meloidogyne arenaria, through the onset of the Hypersensitive Response (HR). In order to identify genes and regulators triggering this defense response, a comprehensive root transcriptome analysis during the first stages of this incompatible interaction was conducted using Illumina Hi-Seq. Overall, eight cDNA libraries were produced generating 28.2 GB, which were de novo assembled into 44,132 contigs and 37,882 loci. Differentially expressed genes (DEGs) were identified and clustered according to their expression profile, with the majority being downregulated at 6 DAI, which coincides with the onset of the HR. Amongst these DEGs, 27 were selected for further qRT-PCR validation allowing the identification of nematode-responsive candidate genes that are putatively related to the resistance response. Those candidates are engaged in the salycilic (NBS-LRR, lipocalins, resveratrol synthase) and jasmonic (patatin, allene oxidase cyclase) acids pathways, and also related to hormonal balance (auxin responsive protein, GH3) and cellular plasticity and signaling (tetraspanin, integrin, expansin), with some of them showing contrasting expression behavior between Arachis RKN-resistant and susceptible genotypes. As these candidate genes activate different defensive signaling systems, the genetic (HR) and the induced resistance (IR), their pyramidding in one genotype via molecular breeding or transgenic strategy might contribute to a more durable resistance, thus improving the long-term control of RKN in peanut.
Assuntos
Arachis/genética , Resistência à Doença/fisiologia , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Tylenchoidea/imunologia , Animais , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Lipocalinas/metabolismo , Oxilipinas/metabolismo , Raízes de Plantas/genética , Resveratrol , Estilbenos/metabolismoRESUMO
OBJECTIVE: To evaluate psychiatric comorbidities in outpatients receiving care for HIV and Chagas disease at Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil. METHODS: Cross-sectional study with a consecutive sample of 125 patients referred to an outpatient psychiatric clinic from February to December 2010. The Mini International Neuropsychiatric Interview (MINI) was used. Factors associated with more frequent mental disorders were estimated by odds ratios (OR) with 95% confidence intervals (95%CI) by multiple logistic regression. RESULTS: Seventy-six (60.8%) patients with HIV, 40 (32%) patients with Chagas disease, and nine (7.2%) patients with human T-lymphotropic virus were interviewed. The majority were women (64%), with up to 8 years of formal education (56%), and unemployed (81.6%). The median age was 49 years. Suicide risk (n=71) (56%), agoraphobia (n=65) (52%), major depressive episode (n=56) (44.8%), and alcohol/drug abuse (n=43) (34.4%) predominated, the latter being directly associated with lower family income (OR = 2.64; 95%CI 1.03-6.75) and HIV infection (OR = 5.24; 95%CI 1.56-17.61). Suicide risk was associated with non-white skin color (OR = 2.21; 95%CI 1.03-4.75), unemployment (OR = 2.72; 95%CI 1.01-7.34), and diagnosis of major depression (OR = 3.34; 95%CI 1.54-7.44). CONCLUSION: Measures targeting adverse socioeconomic conditions and psychiatric and psychological monitoring and care should be encouraged in this population, considering the association with abuse of alcohol/other psychoactive drugs and suicide risk.
Assuntos
Doença de Chagas/psicologia , Infecções por HIV/psicologia , Infecções por HTLV-I/psicologia , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Ideação Suicida , Adulto , Alcoolismo/epidemiologia , Alcoolismo/psicologia , Brasil/epidemiologia , Comorbidade , Estudos Transversais , Transtorno Depressivo Maior/epidemiologia , Transtorno Depressivo Maior/psicologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais/psicologia , Prevalência , Fatores de Risco , Fatores Socioeconômicos , Transtornos Relacionados ao Uso de Substâncias/psicologiaRESUMO
Objective: To evaluate psychiatric comorbidities in outpatients receiving care for HIV and Chagas disease at Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil. Methods: Cross-sectional study with a consecutive sample of 125 patients referred to an outpatient psychiatric clinic from February to December 2010. The Mini International Neuropsychiatric Interview (MINI) was used. Factors associated with more frequent mental disorders were estimated by odds ratios (OR) with 95% confidence intervals (95%CI) by multiple logistic regression. Results: Seventy-six (60.8%) patients with HIV, 40 (32%) patients with Chagas disease, and nine (7.2%) patients with human T-lymphotropic virus were interviewed. The majority were women (64%), with up to 8 years of formal education (56%), and unemployed (81.6%). The median age was 49 years. Suicide risk (n=71) (56%), agoraphobia (n=65) (52%), major depressive episode (n=56) (44.8%), and alcohol/drug abuse (n=43) (34.4%) predominated, the latter being directly associated with lower family income (OR = 2.64; 95%CI 1.03-6.75) and HIV infection (OR = 5.24; 95%CI 1.56-17.61). Suicide risk was associated with non-white skin color (OR = 2.21; 95%CI 1.03-4.75), unemployment (OR = 2.72; 95%CI 1.01-7.34), and diagnosis of major depression (OR = 3.34; 95%CI 1.54-7.44). Conclusion: Measures targeting adverse socioeconomic conditions and psychiatric and psychological monitoring and care should be encouraged in this population, considering the association with abuse of alcohol/other psychoactive drugs and suicide risk. .