RESUMO
Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation 'Candidatus Phytoplasma mali' and its host plant. Here, we report that the 'Ca. P. mali' strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.
Assuntos
Phytoplasma , Doenças das Plantas , Ubiquitina-Proteína Ligases , Arabidopsis/enzimologia , Arabidopsis/parasitologia , Malus/parasitologia , Phytoplasma/enzimologia , Phytoplasma/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
MAIN CONCLUSION: A coordinated regulation of different metabolic pathways was highlighted leading to the accumulation of important compounds that may contribute to the final quality of strawberry fruit. Strawberry fruit development and ripening involve complex physiological and biochemical changes, ranging from sugar accumulation to the production of important volatiles compounds that contribute to the final fruit flavor. To better understand the mechanisms controlling fruit growth and ripening in cultivated strawberry (Fragaria × ananassa), we applied a molecular approach combining suppression subtractive hybridization and next generation sequencing to identify genes regulating developmental stages going from fruit set to full ripening. The results clearly indicated coordinated regulation of several metabolic processes such as the biosynthesis of flavonoid, phenylpropanoid and branched-chain amino acids, together with glycerolipid metabolism and pentose and glucuronate interconversion. In particular, genes belonging to the flavonoid pathway were activated in two distinct phases, the first one at the very early stages of fruit development and the second during ripening. The combination of expression analysis with metabolomic data revealed that the functional meaning of these two inductions is different, as during the early stages gene activation of flavonoid pathway leads to the production of proanthocyanidins and ellagic acid-derived tannins, while during ripening anthocyanins are the main product of flavonoid pathway activation. Moreover, the subtractive approach allowed the identification of different members of the same gene family coding for the same or very similar enzymes that in some cases showed opposite regulation during strawberry fruit development. Such regulation is an important trait that can help to understand how plants specifically channel metabolic intermediates towards separate branches of a biosynthetic pathway or use different isoforms of the same enzyme in different organs or developmental stages.
Assuntos
Fragaria/metabolismo , Frutas/metabolismo , Flavonoides/metabolismo , Fragaria/genética , Fragaria/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Redes e Vias Metabólicas , Metabolômica , Análise de Sequência de DNA , Técnicas de Hibridização Subtrativa , TranscriptomaRESUMO
Small RNAs are involved in a plethora of functions in plant genomes. In general, transcriptional gene silencing is mediated by 24-nucleotide siRNAs and is required for maintaining transposable elements in a silenced state. However, microRNAs are not commonly associated with transposon silencing. In this study, we performed small RNA transcriptome and degradome analyses of the Rosaceae model plant Fragaria vesca (the woodland strawberry) at the genome-wide level, and identified miRNA families and their targets. We report a highly specific mechanism of LTR retrotransposon silencing mediated by an abundant, ubiquitously expressed miRNA (fve-miR1511) generated from a single locus. This miRNA specifically targets LTR retroelements, silencing them post-transcriptionally by perfectly pairing to the highly conserved primer binding site for methionyl initiator tRNA that is essential for reverse transcription. We investigated the possible origins of this miRNA, and present evidence that the pre-miR1511 hairpin structure probably derived from a locus coding for tRNA(iM) (et) through a single microinversion event. Our study shows that this miRNA targets retrotransposons specifically and constitutively, and contributes to features such as genome stability, size and architecture in a far more direct way than previously thought.
Assuntos
Endorribonucleases , Fragaria/genética , Genoma de Planta/genética , MicroRNAs/genética , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , RNA Helicases , Retroelementos/genética , Transcriptoma , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Sequências Repetidas Terminais/genéticaRESUMO
MAIN CONCLUSION: A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early stages of apple fruit development. Apple (Malus × domestica Borkh.) is an economically important fruit appreciated for its organoleptic characteristics and its benefits for human health. The first stages after fruit set represent a very important and still poorly characterized developmental process. To enable the profiling of genes involved in apple early fruit development, we combined the suppression subtractive hybridization (SSH) protocol to next-generation sequencing. We identified and characterized genes induced and repressed during fruit development in the apple cultivar 'Golden Delicious'. Our results showed an opposite regulation of genes coding for enzymes belonging to flavonoid and monolignol pathways, with a strong induction of the former and a simultaneous repression of the latter. Two isoforms of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, key enzymes located at the branching point between flavonoid and monolignol pathways, showed opposite expression patterns during the period in analysis, suggesting a possible regulation mechanism. A targeted metabolomic analysis supported the SSH results and revealed an accumulation of the monomers catechin and epicatechin as well as several forms of procyanidin oligomers in apple fruitlets starting early after anthesis, together with a decreased production of other classes of flavonoids such as some flavonols and the dihydrochalcone phlorizin. Moreover, gene expression and metabolites accumulation of 'Golden Delicious' were compared to a wild apple genotype of Manchurian crabapple (Malus mandshurica (Maxim.) Kom.). Significant differences in both gene expression and metabolites accumulation were found between the two genotypes.
Assuntos
Biflavonoides/metabolismo , Catequina/metabolismo , Flavonoides/metabolismo , Malus/enzimologia , Proantocianidinas/metabolismo , Biflavonoides/genética , Catequina/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Flavonoides/genética , Sequenciamento de Nucleotídeos em Larga Escala , Malus/genética , Malus/crescimento & desenvolvimento , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Proantocianidinas/genética , Isoformas de Proteínas , Análise de Sequência de DNA , Técnicas de Hibridização SubtrativaRESUMO
Over the course of the year, temperate trees experience extremes in temperature and day length. In order to protect themselves from frost damage in winter, they enter a dormant state with no visible growth where all leaves are shed and buds are dormant. Also the young floral tissues need to withstand harsh winter conditions, as temperature fruit trees like apple develop their flower buds in the previous year of fruit development. So far, the genetic control of induction and release of dormancy is not fully understood. However, the transcription factor family of DORMANCY-Associated MADS-box (DAM) genes plays a major role in the control of winter dormancy. One of these genes is MdDAM4. This gene is expressed in the early phase of bud dormancy, but little is known about its function. Six transgenic apple lines were produced to study the function of MdDAM4 in apple. For plant transformation, the binary plasmid vector p9oN-35s-MdDAM4 was used that contains the coding sequence of MdDAM4 driven by the 35S promoter. Transgenicity of the lines was proven by PCR and southern hybridization. Based on siRNA sequencing and phenotypic observations, it was concluded that line M2024 overexpresses MdDAM4 whereas the gene is silenced in all other lines. Phenotyping of the transgenic lines provided evidence that the overexpression of MdDAM4 leads to an earlier induction and a later release of dormancy. Silencing this gene had exactly the opposite effects and thereby led to an increased duration of the vegetation period. Expression experiments revealed genes that were either potentially repressed or activated by MdDAM4. Among the potentially suppressed genes were several homologs of the cytokinin oxidase 5 (CKX5), five LOX homologs, and several expansins, which may indicate a link between MdDAM4 and the control of leaf senescence. Among the potentially activated genes is MdDAM1, which is in line with observed expression patterns during winter dormancy. MdDAM2, which shows little expression during endodormancy also appears to be activated by MdDAM4. Overall, this study provides experimental evidence with transgenic apple trees for MdDAM4 being an important regulator of the onset of bud dormancy in apple.
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A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.
Assuntos
Closteroviridae , Viroides , Vitis , Irã (Geográfico) , Viroma , Viroides/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças das PlantasRESUMO
Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'.
Assuntos
Malus/enzimologia , Malus/crescimento & desenvolvimento , Oxigenases/metabolismo , Arabidopsis/genética , Cromossomos Artificiais Bacterianos/metabolismo , Regulação da Expressão Gênica de Plantas , Loci Gênicos/genética , Malus/genética , Oxigenases/genética , Fenótipo , Plantas Geneticamente Modificadas , Análise de Sequência de DNARESUMO
The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas F-Box/genética , MicroRNAs , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Proteínas de Arabidopsis/metabolismo , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Regulação para Baixo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Mutação , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Apple trees require a long exposure to chilling temperature during winter to acquire competency to flower and grow in the following spring. Climate change or adverse meteorological conditions can impair release of dormancy and delay bud break, hence jeopardizing fruit production and causing substantial economic losses. In order to characterize the molecular mechanisms controlling bud dormancy in apple we focused our work on the MADS-box transcription factor gene MdDAM1. We show that MdDAM1 silencing is required for the release of dormancy and bud break in spring. MdDAM1 transcript levels are drastically reduced in the low-chill varieties 'Anna' and 'Dorsett Golden' compared to 'Golden Delicious' corroborating its role as a key genetic factor controlling the release of bud dormancy in Malus species. The functional characterization of MdDAM1 using RNA silencing resulted in trees unable to cease growth in winter and that displayed an evergrowing, or evergreen, phenotype several years after transgenesis. These trees lost their capacity to enter in dormancy and produced leaves and shoots regardless of the season. A transcriptome study revealed that apple evergrowing lines are a genocopy of 'Golden Delicious' trees at the onset of the bud break with the significant gene repression of the related MADS-box gene MdDAM4 as a major feature. We provide the first functional evidence that MADS-box transcriptional factors are key regulators of bud dormancy in pome fruit trees and demonstrate that their silencing results in a defect of growth cessation in autumn. Our findings will help producing low-chill apple variants from the elite commercial cultivars that will withstand climate change.
RESUMO
DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5' end phosphorylated, as shown by phosphatase treatments, and methylated at the 3'-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to beta-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.
Assuntos
Geminiviridae/genética , Plantas/virologia , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Metilação , Fosforilação , Interferência de RNA , Vírus de RNA/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/classificação , RNA Viral/química , RNA Viral/classificação , Nicotiana/virologiaRESUMO
Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.
Assuntos
Proteínas de Arabidopsis/metabolismo , Inativação Gênica , Doenças das Plantas/virologia , Vírus de Plantas/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Caulimovirus/genética , Geminiviridae/genética , MicroRNAs/metabolismo , Mutação , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/classificação , RNA Viral/classificação , RNA Viral/metabolismo , Ribonuclease III/genética , Tobamovirus/genéticaRESUMO
Fungicides are applied intensively to prevent downy mildew infections of grapevines (Vitis vinifera) with high impact on the environment. In order to develop alternative strategies we sequenced the genome of the oomycete pathogen Plasmopara viticola causing this disease. We show that it derives from a Phytophthora-like ancestor that switched to obligate biotrophy by losing genes involved in nitrogen metabolism and γ-Aminobutyric acid catabolism. By combining multiple omics approaches we characterized the pathosystem and identified a RxLR effector that trigger an immune response in the wild species V. riparia. This effector is an ideal marker to screen novel grape resistant varieties. Our study reveals an unprecedented bidirectional noncoding RNA-based mechanism that, in one direction might be fundamental for P. viticola to proficiently infect its host, and in the other might reduce the effects of the infection on the plant.
Assuntos
Interações Hospedeiro-Patógeno , Oomicetos/crescimento & desenvolvimento , Oomicetos/genética , Doenças das Plantas/microbiologia , Vitis/microbiologia , Inativação Gênica , Doenças das Plantas/imunologia , Análise de Sequência de DNA , Vitis/imunologiaRESUMO
An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases.
Assuntos
Fragaria/genética , Fatores Imunológicos/uso terapêutico , MicroRNAs/uso terapêutico , RNA de Plantas/uso terapêutico , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Fatores Imunológicos/farmacologia , Inflamação/patologia , Metilação , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Receptor 3 Toll-Like/metabolismoRESUMO
We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.
Assuntos
Duplicação Gênica , Genes de Plantas/genética , Genoma de Planta , Malus/genética , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Ligação Genética , Estudo de Associação Genômica Ampla , Malus/crescimento & desenvolvimento , FilogeniaRESUMO
The proper number and distribution of stomata are essential for the efficient exchange of gases between the atmosphere and the aerial parts of plants. We show that the density and development of stomatal complexes on the epidermis of Arabidopsis thaliana leaves depend, in part, on the microRNA-mediated regulation of Agamous-like16 (AGL16), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. Primary stomatal complexes can give rise to higher-order complexes derived from satellite meristemoids. Expression of a miR824-resistant AGL16 mRNA, but not the wild-type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher-order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines overexpressing miR824 decreased the incidence of stomata in higher-order complexes. These findings and the nonoverlapping patterns of AGL16 mRNA and miR824 localization led us to propose that the miR824/AGL16 pathway functions in the satellite meristemoid lineage of stomatal development.
Assuntos
Arabidopsis/embriologia , MicroRNAs/metabolismo , Epiderme Vegetal/embriologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Poliadenilação , Capuzes de RNA/metabolismo , Splicing de RNA/genética , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. PRINCIPAL FINDINGS: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). CONCLUSIONS: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.
Assuntos
Sequência Consenso , Genoma de Planta , Heterozigoto , Vitis/genética , Cromossomos de Plantas , DNA de Plantas/genética , Evolução Molecular , Fenóis/metabolismo , Doenças das Plantas/genética , Polimorfismo de Nucleotídeo Único , Terpenos/metabolismo , Fatores de Transcrição/metabolismo , Vitis/metabolismoRESUMO
RNA silencing refers to a broad range of phenomena sharing the common feature that large, double-stranded RNAs or stem-loop precursors are processed to ca. 21-26 nucleotide small RNAs, which then guide the cleavage of cognate RNAs, block productive translation of these RNAs, or induce methylation of specific target DNAs. Although the core mechanisms are evolutionarily conserved, epigenetic maintenance of silencing by amplification of small RNAs and the elaboration of mobile, RNA-based silencing signals occur predominantly in plants. Plant RNA silencing systems are organized into a network with shared components and overlapping functions. MicroRNAs, and probably trans-acting small RNAs, help regulate development at the posttranscriptional level. Small interfering RNAs associated with transgene- and virus-induced silencing function primarily in defending against foreign nucleic acids. Another system, which is concerned with RNA-directed methylation of DNA repeats, seems to have roles in epigenetic silencing of certain transposable elements and genes under their control.
Assuntos
Inativação Gênica , Desenvolvimento Vegetal , Plantas/genética , Interferência de RNA , RNA de Plantas/genética , Metilação de DNA , Modelos Genéticos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologiaRESUMO
SUMMARY Induced resistance was studied in the model pathosystem Arabidopsis-Phytophthora brassicae (formerly P. porri) in comparison with the agronomically important late blight disease of potato caused by Phytophthora infestans. For the quantification of disease progress, both Phytophthora species were transformed with the vector p34GFN carrying the selectable marker gene neomycine phosphotransferase (nptII) and the reporter gene green fluorescent protein (gfp). Eighty five per cent of the transformants of P. brassicae and P. infestans constitutively expressed GFP at high levels at all developmental stages both in vitro and in planta. Transformants with high GFP expression and normal in vitro growth and virulence were selected to quantify pathogen growth by measuring the in planta emitted GFP fluorescence. This non-destructive monitoring of the infection process was applied to analyse the efficacy of two chemical inducers of disease resistance, a functional SA-analogue, benzothiadiazole (BTH), and beta-aminobutyric acid (BABA) which is involved in priming mechanisms of unknown nature. BABA pre-treatment (300 microm) via soil drench applied 24 h before inoculation completely protected the susceptible Arabidopsis accession Landsberg erecta (Ler) from infection with P. brassicae. A similar treatment with BTH (330 microm) did not induce resistance. Spraying the susceptible potato cultivar Bintje with BABA (1 mm) 2 days before inoculation resulted in a phenocopy of the incompatible interaction shown by the resistant potato cultivar Matilda while BTH (1.5 mm) did not protect Bintje from severe infection. Thus, in both pathosystems, the mechanisms of induced resistance appeared to be similar, suggesting that the Arabidopsis-P. brassicae pathosystem is a promising model for the molecular analysis of induced resistance mechanisms of potato against the late blight disease.