RESUMO
Potato virus X (PVX) vectors expressing the Arabidopsis thaliana FLOWERING LOCUS T (FT) or tomato FT ortholog SINGLE-FLOWER TRUSS (SFT) shortened the generation time in tomato due to accelerated tomato flowering and ripening by 14-21 d, and caused a 2-3-fold increase in the number of flowers and fruits, compared with non-infected or empty vector-infected plants. The Arabidopsis FT was more effective than the tomato orthologue SFT and there was no alteration of the flower or fruit morphology. The virus was not transmitted to the next generation; therefore viral vectors with expression of a heterologous FT will be a useful approach to speed breeding in tomato and other species.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Proteínas de Arabidopsis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Melhoramento Vegetal , Flores/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
My research career started with an ambition to work out how genes are regulated in plants. I tried out various experimental systems-artichoke tissue culture in Edinburgh; soybean root nodules in Montreal; soybean hypocotyls in Athens, Georgia; and cereal aleurones in Cambridge-but eventually I discovered plant viruses. Viral satellite RNAs were my first interest, but I then explored transgenic and natural disease resistance and was led by curiosity into topics beyond virology, including RNA silencing, epigenetics, and more recently, genome evolution. On the way, I have learned about approaches to research, finding tractable systems, and taking academic research into the real world. I have always tried to consider the broader significance of our work, and my current projects address the definition of epigenetics, the arms race concept of disease resistance, and Darwin's abominable mystery.
Assuntos
Vírus de Plantas , RNA , Resistência à Doença/genética , Plantas/genética , Vírus de Plantas/genética , Interferência de RNARESUMO
RNA silencing is a well-established antiviral immunity system in plants, in which small RNAs guide Argonaute proteins to targets in viral RNA or DNA, resulting in virus repression. Virus-encoded suppressors of silencing counteract this defence system. In this Review, we discuss recent findings about antiviral RNA silencing, including the movement of RNA through plasmodesmata and the differentiation between plant self and viral RNAs. We also discuss the emerging role of RNA silencing in plant immunity against non-viral pathogens. This immunity is mediated by transkingdom movement of RNA into and out of the infected plant cells in vesicles or as extracellular nucleoproteins and, like antiviral immunity, is influenced by the silencing suppressors encoded in the pathogens' genomes. Another effect of RNA silencing on general immunity involves host-encoded small RNAs, including microRNAs, that regulate NOD-like receptors and defence signalling pathways in the innate immunity system of plants. These RNA silencing pathways form a network of processes with both positive and negative effects on the immune systems of plants.
Assuntos
MicroRNAs , RNA Viral , Antivirais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Resistência à Doença/genética , MicroRNAs/genética , Proteínas NLR/genética , Proteínas NLR/metabolismo , Doenças das Plantas/genética , Imunidade Vegetal/genética , Plantas/genética , Interferência de RNA , RNA de Plantas , RNA Interferente Pequeno/metabolismoRESUMO
Adaptive antiviral immunity in plants is an RNA-based mechanism in which small RNAs derived from both strands of the viral RNA are guides for an Argonaute (AGO) nuclease. The primed AGO specifically targets and silences the viral RNA. In plants this system has diversified to involve mobile small interfering RNAs (siRNAs), an amplification system involving secondary siRNAs and targeting mechanisms involving DNA methylation. Most, if not all, plant viruses encode multifunctional proteins that are suppressors of RNA silencing that may also influence the innate immune system and fine-tune the virus-host interaction. Animal viruses similarly trigger RNA silencing, although it may be masked in differentiated cells by the interferon system and by the action of the virus-encoded suppressor proteins. There is huge potential for RNA silencing to combat viral disease in crops, farm animals, and people, although there are complications associated with the various strategies for siRNA delivery including transgenesis. Alternative approaches could include using breeding or small molecule treatment to enhance the inherent antiviral capacity of infected cells.
Assuntos
Vírus de Plantas , RNA Viral , Animais , Antivirais , Proteínas Argonautas/genética , Interferons/genética , Doenças das Plantas , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Plantas , Interferência de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Hybridization is associated with the activation of transposable elements and changes in the patterns of gene expression leading to phenotypic changes. However, the underlying mechanisms are not well understood. RESULTS: Here, we describe the changes to the gene expression in interspecific Solanum hybrids that are associated with small RNAs derived from endogenous (para)retroviruses (EPRV). There were prominent changes to sRNA profiles in these hybrids involving 22-nt species produced in the DCL2 biogenesis pathway, and the hybridization-induced changes to the gene expression were similar to those in a dcl2 mutant. CONCLUSIONS: These findings indicate that hybridization leads to activation of EPRV, perturbation of small RNA profiles, and, consequently, changes in the gene expression. Such hybridization-induced variation in the gene expression could increase the natural phenotypic variation in natural evolution or in breeding for agriculture.
Assuntos
Solanum lycopersicum , Elementos de DNA Transponíveis , Expressão Gênica , Hibridização Genética , Solanum lycopersicum/genética , Melhoramento Vegetal , RNARESUMO
Loss-of-function analyses are essential to dissect the complex nature of biological processes, including gametogenesis. Virus-induced gene silencing (VIGS) has been widely used in crop species as an amenable and rapid way to generate gene knockdowns. As a transient assay, VIGS circumvents the generation of stable transgenic lines through laborious and time-consuming tissue culture techniques. VIGS involves inoculating plants during early development with genetically manipulated viral constructs carrying an endogenous gene target sequence. The viral infection triggers the host plant gene silencing machinery to process the viral genomic RNA into small RNAs (sRNAs) including the gene complementary region. The sRNAs with complementary sequences to the endogenous gene mediate posttranscriptional gene silencing of the targeted gene. Here, we provide a simple and reproducible VIGS protocol employing the tobacco rattle virus (TRV) in tomato (Solanum lycopersicum cv. M82). As it is stable at later developmental stages this approach is suitable for many traits in tomato including gametogenesis and it can be adapted to other crop species.
Assuntos
Vírus de Plantas , Solanum lycopersicum , Gametogênese Vegetal , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos , Solanum lycopersicum/genética , Vírus de Plantas/genética , Interferência de RNA , RNA Viral , Nicotiana/genéticaRESUMO
SignificanceParamutation involves the transfer of a repressive epigenetic mark between silent and active alleles. It is best known from exceptional non-Mendelian inheritance of conspicuous phenotypes in maize but also in other plants and animals. Recent genomic studies, however, indicate that paramutation may be less exceptional. It may be a consequence of wide-cross hybridization and may contribute to quantitative trait variation or unstable phenotypes in crops. Using the sulfurea (sulf) locus in tomato, we demonstrate that a self-reinforcing feedback loop involving DNA- and histone-methyl transferases CHROMOMETHYLTRANSFERASE3 (CMT3) and KRYPTONITE (KYP) is required for paramutation of sulf and that there is a change in chromatin organization. These findings advance the understanding of non-Mendelian inheritance in plants.
Assuntos
Solanum lycopersicum , Alelos , Animais , Epigênese Genética , Solanum lycopersicum/genética , Mutação , Plantas/genética , Zea mays/genéticaRESUMO
Small (s)RNAs play crucial roles in the regulation of gene expression and genome stability across eukaryotes where they direct epigenetic modifications, post-transcriptional gene silencing, and defense against both endogenous and exogenous viruses. It is known that Chlamydomonas reinhardtii, a well-studied unicellular green algae species, possesses sRNA-based mechanisms that are distinct from those of land plants. However, definition of sRNA loci and further systematic classification is not yet available for this or any other algae. Here, using data-driven machine learning approaches including Multiple Correspondence Analysis (MCA) and clustering, we have generated a comprehensively annotated and classified sRNA locus map for C. reinhardtii. This map shows some common characteristics with higher plants and animals, but it also reveals distinct features. These results are consistent with the idea that there was diversification in sRNA mechanisms after the evolutionary divergence of algae from higher plant lineages.
Assuntos
Chlamydomonas reinhardtii/genética , Loci Gênicos , RNA Antissenso/genética , RNA de Plantas/genética , Composição de Bases , Análise por Conglomerados , Metilação de DNA , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Aprendizado de Máquina , Anotação de Sequência MolecularRESUMO
Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.
Assuntos
Arabidopsis/virologia , Epigênese Genética , Aptidão Genética , Interações Hospedeiro-Patógeno/imunologia , Potyvirus/fisiologia , Adaptação Biológica , Arabidopsis/imunologia , Arabidopsis/metabolismo , Evolução Biológica , Metilação de DNA , TranscriptomaRESUMO
Silencing of transposable elements (TEs) is established by small RNA-directed DNA methylation (RdDM). Maintenance of silencing is then based on a combination of RdDM and RNA-independent mechanisms involving DNA methyltransferase MET1 and chromodomain DNA methyltransferases (CMTs). Involvement of RdDM, according to this model should decrease with TE age but here we show a different pattern in tomato and Arabidopsis. In these species the CMTs silence long terminal repeat (LTR) transposons in the distal chromatin that are younger than those affected by RdDM. To account for these findings we propose that, after establishment of primary RdDM as in the original model, there is an RNA-independent maintenance phase involving CMTs followed by secondary RdDM. This progression of epigenetic silencing in the gene-rich distal chromatin is likely to influence the transcriptome either in cis or in trans depending on whether the mechanisms are RNA-dependent or -independent.
Assuntos
Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Nucleotídeos/genética , Sistemas CRISPR-Cas/genética , Cromatina/metabolismo , Evolução Molecular , Inativação Gênica , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Proteínas de Plantas/metabolismo , RNA Polimerase II/metabolismo , Sequências Repetidas Terminais/genéticaRESUMO
Sugarcane mosaic virus (SCMV) is distributed worldwide and infects three major crops: sugarcane, maize, and sorghum. The impact of SCMV is increased by its interaction with Maize chlorotic mottle virus which causes the synergistic maize disease maize lethal necrosis. Here, we characterised maize lethal necrosis-infected maize from multiple sites in East Africa, and found that SCMV was present in all thirty samples. This distribution pattern indicates that SCMV is a major partner virus in the East African maize lethal necrosis outbreak. Consistent with previous studies, our SCMV isolates were highly variable with several statistically supported recombination hot- and cold-spots across the SCMV genome. The recombination events generate conflicting phylogenetic signals from different fragments of the SCMV genome, so it is not appropriate to group SCMV genomes by simple similarity.
Assuntos
Filogenia , Potyvirus/genética , Recombinação Genética , Genes Virais , Sequenciamento de Nucleotídeos em Larga Escala , Potyvirus/classificação , Saccharum/virologia , Especificidade da EspécieRESUMO
Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Retroelementos/genética , Solanum lycopersicum/genética , Biologia Computacional/métodos , Epigênese Genética/genética , Evolução Molecular , Genes de Plantas/genética , Genoma de Planta/genética , Solanum lycopersicum/crescimento & desenvolvimento , Análise de Sequência de DNA/métodos , Sequências Repetidas Terminais/genéticaRESUMO
The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3'UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.
Assuntos
Proteínas Argonautas/genética , Chlamydomonas reinhardtii/genética , Regulação da Expressão Gênica de Plantas/genética , Expressão Gênica/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Evolução Biológica , Filogenia , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.
Assuntos
Arabidopsis/embriologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Impressão Genômica , Óvulo Vegetal , RNA de Plantas/fisiologia , RNA Interferente Pequeno/genética , Sementes/crescimento & desenvolvimento , Proteínas de Arabidopsis/genéticaRESUMO
MicroRNAs (miRNAs), small RNA molecules of 20-24 nts, have many features that make them useful tools for gene expression regulation-small size, flexible design, target predictability, and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work, we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build tools based on miRNAs, such as synthetic miRNAs, miRNA-responsive 3'UTRs, miRNA decoys, and self-regulatory loops. These tools will facilitate the engineering of gene expression for new applications and improved traits in this alga.
Assuntos
Chlamydomonas reinhardtii/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Chlamydomonas reinhardtii/genética , Regulação da Expressão Gênica , Genes Sintéticos/genética , MicroRNAs/genéticaRESUMO
Nucleotide binding site leucine-rich repeat (NLR) proteins of the plant innate immune system are negatively regulated by the miR482/2118 family miRNAs that are in a distinct 22-nt class of miRNAs with a double mode of action. First, they cleave the target RNA, as with the canonical 21-nt miRNAs, and second, they trigger secondary siRNA production using the target RNA as a template. Here, we address the extent to which the miR482/2118 family affects expression of NLR mRNAs and disease resistance. We show that structural differences of miR482/2118 family members in tomato (Solanum lycopersicum) are functionally significant. The predicted target of the miR482 subfamily is a conserved motif in multiple NLR mRNAs, whereas for miR2118b, it is a noncoding RNA target formed by rearrangement of several different NLR genes. From RNA sequencing and degradome data in lines expressing short tandem target mimic (STTM) RNAs of miR482/2118, we confirm the different targets of these miRNAs. The effect on NLR mRNA accumulation is slight, but nevertheless, the tomato STTM lines display enhanced resistance to infection with the oomycete and bacterial pathogens. These data implicate an RNA cascade of miRNAs and secondary siRNAs in the regulation of NLR RNAs and show that the encoded NLR proteins have a role in quantitative disease resistance in addition to dominant gene resistance that has been well characterized elsewhere. We also illustrate the use of STTM RNA in a biotechnological approach for enhancing quantitative disease resistance in highly bred cultivars.
Assuntos
Bactérias/patogenicidade , Repetições de Microssatélites , Mimetismo Molecular , Oomicetos/patogenicidade , RNA Mensageiro/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Resistência à Doença , MicroRNAs/genética , RNA Mensageiro/genética , RNA de Plantas/genética , RNA de Plantas/metabolismoRESUMO
Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Fotossíntese , Plasmídeos/metabolismo , Biologia Sintética/métodos , Biotecnologia , Chlamydomonas reinhardtii/genética , Expressão Gênica , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Regiões Promotoras GenéticasRESUMO
Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.
Assuntos
MicroRNAs/metabolismo , Vírus de RNA/fisiologia , Ribonuclease III/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Sistemas CRISPR-Cas , Suscetibilidade a Doenças , Solanum lycopersicum/enzimologia , Mutação , Doenças das Plantas/virologia , Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/genéticaRESUMO
Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.