RESUMEN
We report a sequence of a novel vitivirus from Vitis vinifera obtained using two high-throughput sequencing (HTS) strategies on RNA. The initial discovery from small-RNA sequencing was confirmed by HTS of the total RNA and Sanger sequencing. The new virus has a genome structure similar to the one reported for other vitiviruses, with five open reading frames (ORFs) coding for the conserved domains described for members of that genus. Phylogenetic analysis of the complete genome sequence confirmed its affiliation to the genus Vitivirus, with the closest described viruses being grapevine virus E (GVE) and Agave tequilana leaf virus (ATLV). However, the virus we report is distinct and shares only 51% amino acid sequence identity with GVE in the replicase polyprotein and 66.8% amino acid sequence identity with ATLV in the coat protein. This is well below the threshold determined by the ICTV for species demarcation, and we propose that this virus represents a new species. It is provisionally named "grapevine virus G".
Asunto(s)
Flexiviridae/genética , Vitis/genética , Secuencia de Bases , Flexiviridae/aislamiento & purificación , Nueva Zelanda , Filogenia , ARN Viral/genéticaRESUMEN
A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name "Grapevine virus I".
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Flexiviridae/clasificación , Flexiviridae/aislamiento & purificación , Genoma Viral , Enfermedades de las Plantas/virología , Proteínas Virales/genética , Vitis/virología , Virus ADN/genética , Flexiviridae/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Nueva Zelanda , Sistemas de Lectura Abierta , Filogenia , Análisis de Secuencia de ADNRESUMEN
Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in metabolism, osmosis, hormone response and stress signaling pathways. CPKs are a large multigene family of proteins that are present in all plants studied to date, as well as in protists, oomycetes and green algae, but are not found in animals and fungi. Despite the increasing evidence of the importance of CPKs in developmental and stress responses from various plants, a comprehensive genome-wide analysis of CPKs from algae to higher plants has not been undertaken. This paper describes the evolution of CPKs from green algae to plants using a broadly sampled phylogenetic analysis and demonstrates the functional diversification of CPKs based on expression and functional studies in different plant species. Our findings reveal that CPK sequence diversification into four major groups occurred in parallel with the terrestrial transition of plants. Despite significant expansion of the CPK gene family during evolution from green algae to higher plants, there is a high level of sequence conservation among CPKs in all plant species. This sequence conservation results in very little correlation between CPK evolutionary groupings and functional diversity, making the search for CPK functional orthologs a challenge.
Asunto(s)
Proteínas de Plantas/metabolismo , Plantas/enzimología , Plantas/metabolismo , Proteínas Quinasas/metabolismo , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Proteínas de Plantas/genética , Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevines worldwide resulting in grapevine leafroll disease (GLD), reduced fruit yield, berry quality and vineyard profitability. Being graft transmissible, GLRaV-3 is also transmitted between grapevines by multiple hemipteran insects (mealybugs and soft scale insects). Over the past 20 years, New Zealand has developed and utilized integrated pest management (IPM) solutions that have slowly transitioned to an ecosystem-based biological response to GLD. These IPM solutions and combinations are based on a wealth of research within the temperate climates of New Zealand's nation-wide grape production. To provide context, the grapevine viruses present in the national vineyard estate and how these have been identified are described; the most pathogenic and destructive of these is GLRaV-3. We provide an overview of research on GLRaV-3 genotypes and biology within grapevines and describe the progressive development of GLRaV-3/GLD diagnostics based on molecular, serological, visual, and sensor-based technologies. Research on the ecology and control of the mealybugs Pseudococcus calceolariae and P. longispinus, the main insect vectors of GLRaV-3 in New Zealand, is described together with the implications of mealybug biological control agents and prospects to enhance their abundance and/or fitness in the vineyard. Virus transmission by mealybugs is described, with emphasis on understanding the interactions between GLRaV-3, vectors, and plants (grapevines, alternative hosts, or non-hosts of the virus). Disease management through grapevine removal and the economic influence of different removal strategies is detailed. Overall, the review summarizes research by an interdisciplinary team working in close association with the national industry body, New Zealand Winegrowers. Teamwork and communication across the whole industry has enabled implementation of research for the management of GLD.
Asunto(s)
Closteroviridae , Hemípteros , Vitis , Animales , Ecosistema , Nueva Zelanda , Enfermedades de las Plantas , BiologíaRESUMEN
Mycoviruses can infect many of the major taxa of fungi including yeasts. Mycoviruses in the yeast fungus Geotrichum candidum are not well studied with only three G. candidum-associated viral species characterized to date, all of which belong to the Totiviridae genus Totivirus. In this study, we report the molecular characteristics of another two totiviruses co-infecting isolate Gc6 of G. candidum. The two totiviruses were tentatively named Geotrichum candidum totivirus 2 isolate Gc6 (GcTV2-Gc6) and Geotrichum candidum totivirus 4 isolate Gc6 (GcTV4-Gc6). Both viruses have the typical genome organization of totiviruses comprising two ORFs encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) at the N and C termini, respectively. The genomes of GcTV2-Gc6 and GcTV4-Gc6 are 4592 and 4530 bp long, respectively. Both viruses contain the-frameshifting elements and their proteins could be expressed as a single fusion protein. GcTV2-Gc6 is closely related to a totivirus isolated from the same host whereas GcTV4-Gc6 is related to insect-associated totiviruses. The phylogenetic analysis indicated that GcTV2-Gc6 and GcTV4-Gc6 belong to two different sister clades, I-A and I-B, respectively. It is interesting that all viruses identified from G. candidum belong to the genus Totivirus; however, this might be due to the lack of research reporting the characterization of mycoviruses from this fungal host. It is possible that the RNA interference (RNAi) mechanism cannot actively suppress totivirus accumulation in G. candidum Gc6.
Asunto(s)
Totiviridae , Totivirus , Saccharomyces cerevisiae/genética , Filogenia , Totiviridae/genética , ARN Viral/genéticaRESUMEN
Two co-infecting novel vitiviruses from Actinidia chinensis were identified from mechanically inoculated Nicotiana occidentalis. Both virus genomes were sequenced and share 64% nucleotide identity. Their overall structure is typical of vitiviruses, with five open reading frames (ORFs) and a polyadenylated 3' end. Open reading frame 4 (ORF4) encodes the coat protein, the most conserved gene of the vitiviruses, in which they share 75% amino acid identity, 61-68% with grapevine virus B, 55-59% with grapevine virus A, and 37-42% with grapevine virus E. Based on the molecular criteria for species demarcation in the family Betaflexiviridae, these are two novel viruses, tentatively named Actinidia virus A and Actinidia virus B.
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Actinidia/virología , Flexiviridae/clasificación , Flexiviridae/aislamiento & purificación , Enfermedades de las Plantas/virología , Coinfección , Flexiviridae/genética , Orden Génico , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Nicotiana/virologíaRESUMEN
To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.
RESUMEN
Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.
RESUMEN
Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand, the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD, an integrated management program is used and includes removing (roguing) GLRaV-3-infected vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different grapevine cultivars remains undescribed, especially in cool-climate growing environments, such as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three field sites in New Zealand (Auckland, Hawke's Bay, and Marlborough), each including four cultivars (Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes was observed early in the growing season. In particular, Group X had significantly delayed symptom expression across all three sites compared with Groups I and VI. As the newly infected, young vines matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification by the end of each vintage. The results from this study strongly support the use of VSI for the GLD management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective tool against GLD.
Asunto(s)
Vitis , Closteroviridae , Granjas , Genotipo , Nueva Zelanda , Enfermedades de las PlantasRESUMEN
A new dsRNA virus from the oomycete Phytophthora pluvialis has been characterized and designated as Phytophthora pluvialis RNA virus 1 (PplRV1). The genome of the PplRV1 reference genome is 6742 bp that encodes two predicted open reading frames (ORFs). ORF1 and ORF2 overlap by a 47 nt "slippery" frameshift sequence. ORF1 encodes a putative protein of unknown function. ORF2 shows high similarity to the RNA-dependent RNA polymerase (RdRp) of other dsRNA viruses. Phylogenetic analysis of the putative PplRV1 RdRp and its most closely related viruses showed PplRV1 is distinct from other known viruses (below 33% amino acid similarity), which indicates this virus may belong to a new virus family. Analyses of the geographical distribution of PplRV1 in relation to two genetically distinct classes of its host revealed two corresponding genotypes of the PplRV1 (termed a and b), which share 92.3% nt identity. The reference genome for the second genotype is 6760 bp long and a prediction of its genetic organization shows three ORFs, with ORF2 being split into two ORFs, ORF2a and ORF2b, that is conserved in seven of eleven genotype b isolates. Additionally, a quick and simple diagnostic method using qPCR has been developed, which is suitable for large scale screens to identify PplRV1 in Phytophthora.
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Virus ARN Bicatenario/genética , Phytophthora/virología , Secuencia de Aminoácidos , Virus ARN Bicatenario/clasificación , Genoma Viral/genética , Genotipo , Nueva Zelanda , Sistemas de Lectura Abierta , Filogenia , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Especificidad de la EspecieRESUMEN
Eukaryotic circular single-stranded DNA (ssDNA) viruses were known only to infect plants and vertebrates until the discovery of the isolated DNA mycovirus from the fungus Sclerotinia sclerotiorum. Similar viral sequences were reported from several other sources and classified in ten genera within the Genomoviridae family. The current study reports two circular ssDNA mycoviruses isolated from the phytopathogen Botrytis cinerea, and their assignment to a newly created genus tentatively named Gemydayirivirus. The mycoviruses, tentatively named botrytis gemydayirivirus 1 (BGDaV1) and BGDaV2, are 1701 and 1693 nt long and encode three and two open reading frames (ORFs), respectively. Of the predicted ORFs, only ORF I, which codes for a replication initiation protein (Rep), shared identity with other proteins in GenBank. BGDaV1 is infective as cell-free purified particles and confers hypovirulence on its natural host. Investigation revealed that BGDaV1 is a target for RNA silencing and genomic DNA methylation, keeping the virus at very low titre. The discovery of BGDaV1 expands our knowledge of the diversity of genomoviruses and their interaction with fungal hosts.
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Botrytis/genética , Botrytis/virología , Virus ADN/genética , Virus ADN/aislamiento & purificación , Virus Fúngicos/genética , Virus Fúngicos/aislamiento & purificación , Sistemas de Lectura Abierta/genética , Botrytis/patogenicidad , Virus ADN/clasificación , Virus ADN/patogenicidad , Virus Fúngicos/clasificación , Virus Fúngicos/patogenicidad , Genoma Viral , Interacciones Microbiota-Huesped , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas Virales/genética , VirulenciaRESUMEN
We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country's first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree (Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1-4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Okahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Maori authority over a territory and its associated treasures), we propose the name Karaka Okahu purepure virus in te reo Maori (the Maori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Okahu), and the spotted symptom (purepure, pronounced pooray pooray) that this endemic virus appears to cause.
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Genoma Viral , Enfermedades de las Plantas/virología , Virus de Plantas/clasificación , Virus de Plantas/genética , Virus no Clasificados/clasificación , Virus no Clasificados/genética , Enfermedades Endémicas , Nueva Zelanda , Filogenia , Virus de Plantas/aislamiento & purificación , ARN Viral/genética , Proteínas Virales/genética , Virus no Clasificados/aislamiento & purificaciónRESUMEN
Grapevine leafroll-associated virus 3 (GLRaV-3), an economically significant pathogen of grapevines, is transmitted by Pseudococcus calceolariae, a mealybug commonly found in New Zealand vineyards. To help inform alternative GLRaV-3 control strategies, this study evaluated the three-way interaction between the mealybug, its plant host and the virus. The retention and transmission of GLRaV-3 by P. calceolariae after access to non-Vitis host plants (and a non-GLRaV-3 host) White clover (Trifolium repens L. cv. "Grasslands Huia white clover"), Crimson clover (T. incarnatum), and Nicotiana benthamiana (an alternative GLRaV-3 host) was investigated. For all experiments, P. calceolariae first instars with a 4 or 6 days acquisition access period on GLRaV-3-positive grapevine leaves were used. GLRaV-3 was detected in mealybugs up to 16 days on non-Vitis plant hosts but not after 20 days. GLRaV-3 was retained by second instars (n = 8/45) and exuviae (molted skin, n = 6/6) following a 4 days acquisition period on infected grapevines leaves and an 11 days feeding on non-Vitis plant hosts. Furthermore, GLRaV-3 was transmitted to grapevine (40-60%) by P. calceolariae second instars after access to white clover for up to 11 days; 90% transmission to grapevine was achieved when no alternative host feeding was provided. The 16 days retention period is the longest observed in mealybug vectoring of GLRaV-3. The results suggest that an alternative strategy of using ground-cover plants as a disrupter of virus transmission may be effective if mealybugs settle and continue to feed on them for 20 or more days.
RESUMEN
BACKGROUND: Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. RESULTS: Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. CONCLUSIONS: This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants.
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Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Áfidos/clasificación , Áfidos/metabolismo , Arabidopsis/genética , Secuencia de Bases , Perfilación de la Expresión Génica , Malus/clasificación , Floema/metabolismo , ARN Mensajero/metabolismoRESUMEN
Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.
Asunto(s)
Closteroviridae/fisiología , Regulación de la Expresión Génica de las Plantas , Especificidad del Huésped/genética , Nicotiana/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Vitis/metabolismo , Animales , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/virología , Transcriptoma , Vitis/genética , Vitis/virologíaRESUMEN
Mycoviruses are widely distributed across different phyla of the fungal kingdom. Viruses that share significant sequence similarities have been reported in different fungi, suggesting descent from a common ancestor. In this study, two fungal genera isolated from the same sample, Trichoderma koningiopsis isolate Mg10 and Clonostachys rosea isolate Mg06, were reported to have identical double-stranded RNA (dsRNA) profiles that consist of two virus-like, dsRNA elements (dsRNA-L and dsRNA-S). The complete sequence and genome organization of dsRNA-L from isolate Mg10 was determined. It is 4712 nucleotides (nt) long and contains two non-overlapping open reading frames (ORFs) that code for proteins with similarities to totiviruses. Consequently the virus was given the proposed name Trichoderma koningiopsis totivirus 1 (TkTV1/Mg10). The TkTV1/Mg10 genome structure resembles that of yeast totiviruses in which the region preceding the stop codon of ORF1 contains the elements required for -1 ribosomal frameshifting which may induce the expression of an ORF1-ORF2 (CP-RdRp) fusion protein. Sequence analyses of viral dsRNA-L from C. rosea isolate Mg06 revealed that it is nearly identical with that of TkTV1/Mg10. This relatedness was confirmed by northern blot hybridization and indicates very recent natural horizontal transmission of this virus between unrelated fungi. TkTV1 purified isometric virions were â¼38-40 nm in diameter and were able to transfect T. koningiopsis and C. rosea protoplasts. This is another report of a mycovirus present naturally in two taxonomically distinct fungi.
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Societal and environmental pressures demand high-quality and resilient cropping plants and plant-based foods grown with the use of low or no synthetic chemical inputs. Mild strain cross-protection (MSCP), the pre-immunization of a plant using a mild strain of a virus to protect against subsequent infection by a severe strain of the virus, fits with future-proofing of production systems. New examples of MSCP use have occurred recently. New technologies are converging to support the discovery and mechanism(s) of action of MSCP strains thereby accelerating the popularity of their use.
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Protección Cruzada/inmunología , Interacciones Huésped-Patógeno/inmunología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/prevención & control , Ambiente , Microbiología Ambiental , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/virologíaRESUMEN
BACKGROUND: The discovery that a plant microRNA (miRNAs) from rice (Oryza sativa miR168a) can modify post-transcriptional expression of the mammalian. Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1) gene highlights the potential for cross-kingdom miRNAmRNA interactions. OBJECTIVE: To investigate whether common variants of the conserved miR168a family have the capability for similar cross-kingdom regulatory functions, we selected sequences from three dietary plant sources: rice (Oryza sativa), tomato (Solanum lycopersicum), apple (Malus domestica) and compared their ability to regulate human LDLRAP1 expression. METHODS: Target prediction software intaRNA and RNAhybrid were used to analyze and calculate the energy and alignment score between the miR168a variants and human LDLRAP1 mRNA. An in vitro cell-based Dual-Luciferase® Reporter Assay (pmirGLO, Promega), was then used to validate the miRNA-mRNA interaction experimentally. RESULTS: Computational analyses revealed that a single nucleotide difference at position 14 (from the 5' end of the miRNA) creates a G:U wobble in the miRNA-mRNA duplex formed by tomato and apple miR168a variants. This G:U wobble had only a small effect on the free energy score (-33.8-34.7 kcal/mol). However, despite reasonable hybridization energy scores (<-20 kcal/mol) for all miR168a variants, only the rice miR168a variant lacking a G:U wobble significantly reduced LDLRAP1 transcript expression by 25.8 + 7.3% (p<0.05), as measured by relative luciferase activity. CONCLUSION: In summary, single nucleotide differences at key positions can have a marked influence on regulatory function despite similar predicted energy scores and miRNA-mRNA duplex structures.
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Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica de las Plantas/genética , Malus/genética , MicroARNs/genética , Oryza/genética , Solanum lycopersicum/genética , Biología Computacional , Silenciador del Gen/fisiología , Humanos , ARN Mensajero/genética , ARN de Planta/genéticaRESUMEN
BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.
Asunto(s)
Actinidia/genética , Actinidia/fisiología , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Frutas/crecimiento & desarrollo , Pigmentación/genética , Gusto , Actinidia/crecimiento & desarrollo , Actinidia/metabolismo , Adulto , Alérgenos/genética , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Niño , Codón , Secuencia de Consenso , Ésteres/metabolismo , Frutas/genética , Frutas/metabolismo , Genes de Plantas/genética , Marcadores Genéticos , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Pigmentos Biológicos/biosíntesis , Pigmentos Biológicos/genética , Polimorfismo de Nucleótido Simple , Ácido Quínico/metabolismo , Análisis de Secuencia , Terpenos/metabolismoRESUMEN
This study determined the effects of Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, on A. fumigatus, A. nidulans and A. niger. Protoplasts of virus-free isolates of A. fumigatus, A. nidulans and A. niger were transfected with purified AthCV1 particles and the phenotype, growth and sporulation of the isogenic AthCV1-free and AthCV1-infected lines assessed at 20 °C and 37 °C and gene expression data collected at 37 °C. AthCV1-free and AthCV1-infected A. fumigatus produced only conidia at both temperatures but more than ten-fold reduced compared to the AthCV1-infected line. Conidiation was also significantly reduced in infected lines of A. nidulans and A. niger at 37 °C. AthCV1-infected lines of A. thermomutatus and A. nidulans produced large numbers of ascospores at both temperatures, whereas the AthCV1-free line of the former did not produce ascospores. AthCV1-infected lines of all species developed sectoring phenotypes with sclerotia produced in aconidial sectors of A. niger at 37 °C. AthCV1 was detected in 18% of sclerotia produced by AthCV1-infected A. niger and 31% of ascospores from AthCV1-infected A. nidulans. Transcriptome analysis of the naturally AthCV1-infected A. thermomutatus and the three AthCV1-transfected Aspergillus species showed altered gene expression as a result of AthCV1-infection. The results demonstrate that AthCV1 can infect a range of Aspergillus species resulting in reduced sporulation, a potentially useful attribute for a biological control agent.