1,720,963 research outputs found
Development of an RNA silencing monitoring system and Pepino mosaic virus resistant transgenic tomato
No full textPepino mosaic virus' (PepMV) is a plus-sense RNA virus, whose proteins are translated from one genomic RNA (gRNA) and three subgenomic RNAs (sgRNAs). gRNA and all sgRNAs share the 3' CP sequence, while only the gRNA contains the 5' RdRp sequence. An siRNA selection software was utilized to choose sequences from the PepMV genome for constructing inverted repeat-structured silencing inducers (INDUCERs). Different INDUCERs were coinfiltrated with their artificial silencing targets containing homologous sequence (REPORTERS). The results suggested that this coinfiltration system can be used to monitor RNA silencing using GUS as reporter. When inoculated with PepMV, leaf tissues infiltrated with INDUCERs showed reduced PepMV quantity. INDUCERs targeting CP appeared to silence PepMV more efficiently than INDUCERs targeting RdRp, probably because the former can degrade gRNA and all sgRNAs while the latter can only degrade gRNA. Tomato was transformed with INDUCER constructs. The resulting INDUCER A2-transgenic tomato was challenged with PepMV and displayed reduced disease symptoms compared with wild-type tomato
Molecular aspects of Vitis CBF gene activation
Get PDFThe 'Vitis' plant genus includes the wild, freezing tolerant ' V. riparia', and a freezing sensitive cultivar widely used in the viticulture industry, 'V. vinifera'. It has been shown that freezing tolerance is controlled through a signal casade known as the ' CBF' ('C-repeat binding factor') pathway in ' Arabidopsis'. The goal of this research was to start investigating how 'Vitis CBF' expression is regulated. 'In silico ' analyses and over expression in 'Arabidopsis' identified putative regulatory promoter elements, in particular for 'Vitis CBF1 ' and '4'. A dual luciferase pCAMBIA-based binary vector that allows a quantitative comparison of different promoters was successfully constructed. Using this vector, it was shown that a cluster of four elements on the 346bp promoter, and a MYC element on the 280 bp promoter appear to be necessary for induction of 'VrCBF4' at ambient temperature. A putative 'CBF'-expression regulating ICE transcription factor was cloned for future transactivation studies with the newly developed vector
Functional analysis of grape CBF genes
No full textFour grape 'CBF' genes had been isolated from both the freezing tolerant wild species 'Vitis riparia' and the freezing susceptible cultivated species 'Vitis vinifera' but the functions and differences amongst these genes were virtually unknown. In this study, it was demonstrated that 'CBF' genes from 'V. riparia ' are functional and target to the nucleus if the predicted NLS sequence is present. Both VrCBF1 and VrCBF4 transiently activated the expression of downstream genes via the CRT elements in their promoters and 'in planta ' they activated the expression of the 'Arabidopsis COR' genes 'AtCOR15a, AtCOR6.6, AtRD29A,' and 'AtCOR47.' VrCBF4 appears to activate CRT-containing genes more efficiently than VrCBF1, perhaps because of its preferential binding to the ACCGACNTNA CRT sequence. Functional differences were observed between VrCBF1 (representing the group of VrCBF1, 2, 3) and VrCBF4. VrCBF1 contributed to cold, freezing, drought and salt stress tolerance in 'Arabidopsis' whereas VrCBF4 contributed only to cold and freezing tolerance. In general, stress tolerant ' Arabidopsis' plants were dwarf in size, flowered late and more leaves were produced prior to flowering. They also produced comparatively small and thicker leaves, with a thick palisade and spongy mesophyll layer of cells, suggesting that the architecture of the leaves might help prevent water loss from the plants, and thus contribute to drought tolerance. 'In silico' analysis revealed the presence of basic and ' CBF'-specific regulatory promoter elements in 'CBF1, 2, 3' and '4' promoters of 'V. riparia' and 'V. vinifera. VrCBF1, 2' and '3' genes have shorter 5' UTR than the 'VrCBF4' gene. Functional analyses showed that 1.6 kb of 'VrCBF1' and ~1 kb of 'VrCBF3 ' and 'VrCBF4' promoters are sufficient to impart cold regulated gene expression and that 'VrCBF1' expresses in the growing region and young leaves whereas 'VrCBF4' expresses mostly in older tissues of transgenic 'Arabidopsis' plants, as expected based on the expression of the endogenous genes in 'V. vinifera' and 'V. riparia.' Deletion analyses with the 'VrCBF4' promoter revealed that cold-induced regulatory elements lie within a 242 by promoter region of 'VrCBF4.
Protein Domains of Vitis CBF1 and Translational Regulation of Cold-Induced Genes via Codon Usage
No full textCBF (C-repeat Binding Factor) transcription factors are vital in ABA-independent signalling during cold acclimation and subsequent freezing tolerance in Vitis. We investigated nuclear localization signals within CBF1; known to be rapidly expressed following exposure to cold; as well as potential alternative translation products of CBF1 stemming from stop codon readthrough. As a result of our interest in suppressor tRNA species and their potential influence on alternative translation products of CBF1, and due to recent developments concerning codon use of genes which demonstrate tissue-specific expression in Vitis, we also examined codon use of genes involved in cold acclimation and freezing tolerance. We demonstrate roles in both activation and localization for two domains of CBF1, while previously each domain was ascribed a single role. We additionally demonstrate that cold regulated genes share a distinct pattern of codon use in Vitis.Natural Sciences and Engineering Research Council of CanadaOntario Graduate ScholarshipGrape Growers of Ontari
Analysis of the Vitis C-Repeat Binding Factor (CBF) genes and their potential roles in both the CBF and stomatal development pathways
No full textVitis vinifera species are adapted for growth in moderate climates, so freezing temperature damage during Ontario winters results in significant revenue loss for producers. Highly conserved Drought Response Element Binding 1/C-Repeat Binding factor (DREB1/CBF) genes have been shown to be crucial in the acquisition of frost tolerance, while Drought Response Element Binding factor 2 (DREB2) genes were thought to function in drought tolerance. Seven CBF genes and two DREB2 genes were cloned from both Vitis vinifera and the more cold hardy Vitis riparia. Vitis CBFs showed differing expression patterns under ambient and cold conditions, with specific subsets expressed in leaf and bud tissues. Transcripts for the two DREB2 genes were not observed in conditions where CBFs had been detected. Transient transactivation assays showed that all Vitis CBFs but one and both DREB2 proteins could induce transcription via the core C-Repeat (CRT)/Drought Response Element (DRE) promoter sequence element. Investigation revealed that the C-terminal hydrophobic domains present in CBF6 but absent from CBF5 contributed to activation. Predicted PEST motifs in both CBF4 and DREB2-3 affect activation by CBF4 only. Further investigation found that CBF and DREB2 proteins had a preference for the sequence surrounding the DRE core based on their classification; CBF proteins preferred an AT-rich sequence, while DREB2 proteins preferred a GC-rich sequence. From the evidence presented in this thesis, we predict that Vitis CBFs and DREB2s have roles in different abiotic stress tolerance pathways, and that the individual members of each of these families have different functions within their respective regulons. Evidence from transient transactivation and overexpression studies suggests that Vitis DREB2-5 and CBF8 may also have a role in the stomatal development pathway. Their overexpression induced a higher stomatal lineage plus pavement cell density, similar to phenotypes previously found for Vitis SPEECHLESS and ICE4. We hypothesize that these four proteins are involved in the first step of the Vitis stomatal development pathway, an increase in stomatal lineage cells. How they do that remains unclear, but the work presented here provides a basis of understanding to help direct forthcoming investigations
Analysis of the Role of Grape ICE Proteins in the CBF Pathway and Stomatal Development
No full textArabidopsis ICE (Inducer of CBF expression) transcription factors play a role in freezing tolerance and stomatal development. This thesis examined the possible functions of ICE genes from grape. Four ICE genes were isolated and sequenced from both the freezing tolerant wild species Vitis riparia and the freezing sensitive cultivated species V. vinifera. All the encoded ICE proteins contain a bHLH domain with an ICE-specific sequence in their highly conserved C-terminus, and only a few amino acid differences between the ICE orthologs. RT-PCR analyses and sequencing showed that all genes produced spliced transcripts in leaves and buds at ambient and low temperature conditions. In addition, the alternative transcripts ICE1i1, ICE2i2 and ICE4i1 were detected in leaves but only ICE1i1 in mature buds. The functions of the various ICE variants in the CBF pathway were analyzed by Agrobacterium-mediated transactivation experiments. The results showed that all ICE proteins can activate the CBF4 promoter, albeit especially ICE2 and ICE3, via a MYC2g element. Cold increased activation, presumably because of cold-induced sumoylation that stabilizes these proteins. ICE1 and ICE4 induced the transcription better from CBF6 promoter, via different MYC elements. The truncated ICE proteins encoded by alternative transcripts were found to give a lower activation compared to their corresponding regular proteins. Homologs of the Arabidopsis stomatal genes SPCH, MUTE and FAMA were isolated and sequenced from wild and wine grape species. Each of these stomatal genes produced regular spliced transcripts, which for FAMA included 2 transcripts with different start sites, early FAMA(E) and late FAMA(L). The sequential presence of SPCH, MUTE and FAMA transcripts in different aged leaves and the effect of transient overexpression of these genes and of ICE genes on the formation of stomata and pavement cells supports their respective functions in three consecutive stages of stomatal development. The role of proteins encoded on alternatively spliced transcripts, SPCHi1, MUTEi1 FAMAi1(E) and FAMAi2(L), is as yet unclear. Transactivation results suggest that grape ICEs interact with grape FAMA(L) to activate VrCBF4, possibly to regulate both stomatal development and the freezing tolerance pathway. A model which suggests a role for all ICE and stomatal genes in either stomatal development or/and the acquisition of freezing tolerance is presented
Investigating Interactions Between Vitis riparia Open Stomata 1 (OST1) and Inducer of CBF Expression (ICE) Proteins
Get PDFFreezing tolerance in plants often increases due to activation of the ICE-CBF pathway. A key activator of this pathway is Inducer of CBF Expression 1 (ICE1), which is positively regulated by Open STomata 1 (OST1) through phosphorylation following cold stress. The phosphorylated ICE1 activates the CBF-COR transcription cascade, resulting in gene expression associated with increased freezing tolerance. While studied extensively in the model plant, Arabidopsis thaliana, much is still unknown about the functions of OST1 homologues in other plant species, such as economically important grape varieties (highly susceptible to freezing injury). Through fluorescence microscopy and co-immunoprecipitation experiments, this thesis aimed to investigate the OST1-ICE association from a highly frost tolerant grape, Vitis riparia. The results of these experiments suggest that VrOST1 interacts with all VrICE proteins regardless of its phosphorylation state and presence of the C-terminal putative interaction domain. These findings can broaden our understanding of the Vitis CBF pathway
Studies on the Stability and Interaction of the E3 Ubiquitin Ligase HOS1 and Proteins Involved with Cold Acclimation and Stomatal Development in Vitis riparia
No full textFrost injury in wine grape (Vitis vinifera) has led to serious losses in grape production every year. Studying the frost tolerance mechanism of the freezing tolerant grape (Vitis riparia) is an important step to solve this problem. ICE (inducer of CBF expression) transcription factors play an important role in preventing freezing injury through the ICE-CBF-COR pathway. On the other hand, ICE proteins are involved in three essential steps of stomatal development by forming dimers with three other bHLH transcription factors SPCH, MUTE and FAMA. In Arabidopsis, the E3 ubiquitin ligase HOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE PROTEIN 1) negatively regulates the ICE-CBF-COR pathway by interacting with and ubiquitinating ICE proteins, which leads to their degradation. It is not yet clear if HOS1 also interacts with and causes degradation of the stomata-specific proteins and any other Vitis ICE protein. We successfully amplified and cloned the cDNA for the single homolog of VrHOS1. BiFC analyses suggests that VrHOS1 can interact with all 4 VrICE proteins, VrSPCH and VrFAMA, but not with VrMUTE. However, the function of Vitis HOS1 protein domains in its localization and protein-protein interaction was not clear. The Co-IP and protein degradation assays were inconclusive due to the low accumulation of VrHOS1 protein extracts and the use of an improper control. Nevertheless, our results indicate a possible connection between cold acclimation and stomatal development via VrHOS1
Gene expression during germination and post-germination of dried and non-dried developing seeds of tomato
No full textIn a number of species, the period of desiccation that terminates seed development is considered essential in effecting the metabolic switch to a germinative program. However, in orthodox seeds developing within fleshy fruits, such as those of tomato, this developmental shift is not only promoted by desiccation, but also by the isolation of undesiccated seeds from the fruit tissues and their continued hydration. In this study, germinative, transcriptional (by northern blotting), and translational (by western blotting) responses to isolation and/or desiccation were examined using Micro-Tom tomato seeds isolated at different stages of development. Desiccation increased the speed of germination of seeds removed from the plant shortly after mass maturity (i.e. 38 days after pollination [DAP]). Similarly, in 38-DAP seeds, the loss of transcripts associated with seed development, as well as the on- or up-regulation of those related to (post)germination, occurred earlier in the dried seeds, underlying the changes occurring at the morphophysiological level. Although the accumulation pattern of few proteins resembled those of the transcripts, overall, the proteins were relatively stable to drying, and no substantial differences were observed between non-dried and dried germinating seeds. Additionally, the transcription profile of germination of non-dried and dried immature seeds of the tomato cultivar CC337 was determined by microarray analysis. Transcriptional changes reflected the progress of germination, which was influenced by desiccation of the seeds. The role of ABA and osmoticum in maintaining seed development and/or preventing germination, and of seed isolation and/or desiccation in terminating development, was also investigated in germinable immature tomato seeds at the transcriptional level. While both ABA and osmoticum prevented the seeds from germinating, the osmoticum was unique in causing the resumption and maintenance of developmental events, and preventing the continuation of the germinative ones. Moreover, desiccation did not terminate irreversibly the developmental program, with both non-dried and dried seeds being equally efficient in inducing developmental events, in response to osmoticum
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