Добірка наукової літератури з теми "Plant development, Microproteins, Molecular Biology"

Bluetongue is a disease of domestic and wild ruminants caused by Bluetongue virus (BTV). It has caused several serious outbreaks, the most recent occurring in Northern Europe in 2006 during which high mortality rates of livestock were reported. The only vaccines currently approved and commercially available for use are live-attenuated or inactivated virus strains and although these are effective, there is the risk of reversion in the case of live-attenuated strains to more virulent forms by recombination. Another drawback associated with the use of live-attenuated virus vaccines is that they are not DIVA (differentiate infected from vaccinated animals) compliant, this means that naturally infected animals cannot be distinguished from vaccinated animals. Recombinantly produced vaccines would be preferable to minimize the risks associated with live-attenuated virus vaccines and also enable the development of candidate vaccines that are DIVA-compliant. A number of recombinant vaccine candidates have been developed against BTV, with the most promising vaccine consisting of BTV virus-like particles (VLPs). BTV VLPs were successfully produced in insect cells by the co-expression of the four BTV capsid proteins (VP2, VP3, VP5 and VP7). Sheep vaccinated with insect cell-produced BTV VLPs were shown to be protected against challenge with wild type virus. However, the high costs associated with the production and scale-up of BTV VLPs in insect cells has possibly limited their widespread application. Plants – such as N. benthamiana – provides a safe, efficient and cost effective system for the production of recombinant proteins. In this study the best plant expression vector with which to co-express the four BTV serotype 8 (BTV-8) VPs – which direct formation of BTV-8 VLPs – was identified. Expression and purification of the BTV-8 VLPs was optimised with the aim of producing a VLP-based vaccine for BTV-8. It was further undertaken to develop two novel second generation plant-produced protein body (PB) vaccines that are DIVA compliant. Mice were immunised with the plantproduced VLP and PB vaccines in order to analyse their ability to elicit humoral immune responses.

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades, but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines, initially focusing on the use of insect and mammalian cell expression systems. More recently, several studies have demonstrated the potential for using plant expression systems for the production of virus-like particles (VLPs), which are excellent vaccine candidates, as they do not contain virus genetic material and are DIVA compliant. A vaccine alternative to the currently used live vaccine necessarily needs to provide protection against all nine serotypes of the virus. Cross-protection has been shown to exist between certain serotypes of the virus and as capsid protein VP2 is the protein responsible for AHSV serotype specificity, the idea of a plant-produced VLP vaccine containing a representative VP2 protein from each of the different serotype groups, was conceived. Such a vaccine would potentially provideprotection against all 9 serotypes of the virus and would have DIVA capability. Furthermore, it would address local concerns regarding the use of a live vaccine and would serve as a potentially acceptable prophylactic or rapid response antidote in the wider international context. This work describes two approaches in the development of VLP vaccines in plants. In the first part of this study, the ability of 2 different serotypes of plant-produced AHSV VLPs to safely stimulate an immune response in horses, was investigated. Co-infiltration of Nicotiana benthamiana plants with Agrobacterium constructs encoding the four AHSV serotype 5 structural proteins VP2, VP3, VP5 and VP7, was shown to result in assembly of complete VLPs. Furthermore, co-infiltration with the constructs, encoding VP3 and VP7, together with constructs encoding the two outer capsid proteins VP2 and VP5 of a second serotype, AHSV 4, resulted in assembly of complete AHSV 4 VLPs. Horses vaccinated with plant-produced AHSV 4 and 5 VLPs, all seroconverted after two doses of the vaccine and the virus neutralization titres indicated that the plant-produced VLP vaccines are likely to be at least as effective as the current LAV in protecting against AHSV 4 or AHSV 5. However, they have the added advantage of being free from any of the associated risks of a live vaccine, such as reversion to virulence or genetic re-assortment with field or vaccine strains. In the second part of the study, the use of the so-called SpyTag/SpyCatcher or bacterial “superglue” technology was investigated. This technology is based on the peptide SpyTag irreversibly coupling to the SpyCatcher protein, forming an isopeptide bond when the two are mixed together. The plant-based expression system was used to produce Spy VLPs consisting of either Acinetobacter phage (AP205) VLPs or tobacco mosaic virus (TMV) VLPs displaying a SpyTag or SpyCatcher peptide. In addition, AHSV 5 VP2 displaying SpyTag was expressed in plants and several coupling strategies were tested to determine whether AP205 particles displaying AHSV 5 VP2 could be formed as a result of binding between the SpyTag/SpyCatcher moieties of the recombinant proteins. Although it was not proven that coupling occurred, this research will pave the way towards developing a multivalent vaccine platform whereby VP2 of different AHSV serotypes can be displayed on the Spy VLP surface to allow optimal presentation of these proteins to the animal's immune system. Together, the results obtained in this study show that there is great potential for the production of novel, diverse, efficacious and economically viable AHSV VLP vaccines in plants.

An essential component of development is the accumulation of specific metabolites in a temporal and tissue-specific manner. The growth regulator abscisic acid (ABA), which accumulates at a specific time during seed development, is required for seed maturation and prevents the premature developmental switch from dormancy to germination ABA accumulates differently in two tissues of the seed; levels in the embryo are several-fold higher than in the endosperm and the temporal accumulation of ABA is also different between these tissues. To begin to understand how ABA accumulation is regulated during seed development, the regulation of ABA biosynthesis was investigated. The approach taken was to examine the expression of the biosynthetic enzyme, phytoene desaturase (PDS), which catalyzes a regulated step in ABA synthesis in several other organisms (Bramley, 1985, Sandmann et al., 1989, Hugueney et al., 1992 and Giuliano et al., 1993). Unlike ABA accumulation, PDS transcript and protein levels were higher in the endosperm than in the embryo. The spatial difference in PDS levels did correlate with levels of the pathway intermediate, beta-carotene, suggesting that PDS may control the synthesis of ABA precursors while subsequent enzymes may regulate ABA accumulation. The temporal expression of Pds was also unrelated to ABA accumulation. In the endosperm, transcript levels were initially high and declined during desiccation while protein levels remained high throughout development. In the embryo, transcript levels were low and constant while protein levels declined. There are several maize mutants (viviparous mutants) disrupted in ABA biosynthesis, resulting in decreased levels of ABA and premature germination. Analysis of the Pds allele and transcript in the viviparous-5 mutant showed that the gene contains multiple insertions and deletions, giving rise to a larger transcript. In addition, the 55 kDa PDS protein was not detected in the vp5 mutant by immunoblot analysis, indicating that the vp5 phenotype results from a mutation at the PDS locus. To determine whether the wild type protein encoded by the ABA mutant, vp2, or the pathway intermediate, lycopene, regulate PDS, transcript and protein levels were compared in wild type and mutant (vp2 and vp7, respectively) seeds. The levels of PDS were not significantly different in vp2 or vp7 wild type and mutant seeds, suggesting that neither the VP2 protein nor lycopene regulate PDS at the steady-state transcript or protein level.

Bibliography: leaves 153-185. Vesicular-arbuscular (VA) mycorrhiza formation is a complex process which is under the genetic control of both plant and fungus. This project aims to develop a model infection system in Hordeum vulgare L. (barley) suitable for molecular analysis; to identify host plant genes differentially expressed during the early stages of the infection process; and to screen a mutant barley population for phenotypes which form abnormal mycorrhizas.

There are over a half a million plant species on earth, and we use them in virtually every aspect of our lives. Little or no genomic information exists about the vast majority of these plants. This study investigated the use of Expressed Sequence Tags (ESTs) to locate highly conserved sequences from which to design a set of universal molecular markers for all plant species. Plant species for this study were chosen to representative of the plant kingdom. This was done by sampling several individuals of at least one species from all of the major terrestrial plant groups. Conserved sequences are generally found in a wide range of plants species and often in all plant species. A set of eight degenerate primers was designed specifically to detect Single Nucleotide Polymorphisms (SNPs) using capillary array electrophoresis-single stranded conformational polymorphism (CAE-SSCP). The results of this research confirmed that homologous regions of the genome could be used to design universal molecular markers for all plant species.

The goal of this dissertation was to analyze the regulation and function of cytoplasmic class I small heat shock proteins (sHSPs) during seed development in Arabidopsis thaliana. Results show that two class I sHSPs accumulate in late seed maturation, persist in the dry seed and decline rapidly during germination. HSP17.4 accounts for 90% of total class I sHSP in the dry seed. The temporal pattern of sHSP accumulation during seed development suggests that HSP17.4 may help establish seed properties that are acquired during late seed maturation, such as dormancy or desiccation tolerance. Several mutants with reduced seed dormancy were determined to accumulate wild type levels of HSP17.4, however, all desiccation intolerant seeds analyzed had decreased levels of HSP17.4. Thus, HSP17.4 reduction correlates with desiccation intolerance. In total, these data suggest that HSP17.4 is not sufficient for seed dormancy and that it may be necessary for desiccation tolerance. The localization and regulation of HSP17.4 were examined in developing Arabidopsis seeds by transforming plants with hsp17.4 promoter fused to the β-glucuronidase (GUS) gene. HSP17.4::GUS expression was detected in the cotyledons early in seed development and eventually throughout the embryo. Arabidopsis embryos showed a much different pattern of HSP17.4::GUS expression in response to heat indicating distinct mechanisms regulate sHSP transcription during heat shock and during development. To analyze seed specific transcriptional activator regulation of HSP17.4 transcription, HSP17.4::GUS transgenic plants were crossed to seed transcriptional activator mutants. Results showed aberrant localization of HSP17.4::GUS in fus3-3 and lec1-2 seeds and negligible levels in abi3-6. These results strongly implicate AB13 in the transcriptional regulation of HSP17.4. To analyze more specifically HSP17.4 function, transgenic antisense technology was used to suppress hsp17.4 expression to 30--50% of wild type. These lines exhibited a reduced dormancy phenotype as assayed by reduced sensitivity to germination on ABA and by the ability of fresh seed to germinate. These data provide insight into the localization, regulation and function of HSP17.4 during seed maturation. The seed-specific transcriptional activator ABI3 is implicated in controlling hsp17.4 expression during development. Overall, these results demonstrate the importance of HSP17.4 during seed maturation, and establish a role for sHSPs in dormancy.

Downy mildew of lettuce caused by Bremia lactucae is a serious disease resulting in yield loss. The population structure of the pathogen in the UK is poorly understood. This PhD project concentrated on developing molecular markers to differentiate the genotypic variation of B. lactucae populations, with the aim of improving methods to investigate lettuce - Bremia interactions. Thirty-seven B. lactucae isolates (including single-spore and new field isolates) were collected and characterized for virulence using the conventional International Bremia Evaluation Board (IBEB) differential set. Microsatellite markers (SSR, ISSR) were investigated for Bremia race specific marker development. Three isolates of B. lactucae were characterized by ISSR (inter simple sequence repeat) primers, although the polymorphic DNA could not be cloned in this project due to the highly variable results of the ISSR process. Some microsatellite repeats were found in B. lactucae isolates sequences that amplified by Plasmopara viticola (grape downy mildew) SSR markers. The development of Simple Sequence Repeat (SSR) markers from Bremia genomic DNA was not successful, which might result from the primers used being unsuitable for Bremia microsatellite enrichment. Bremia specific ITS primers were used for quantitative PCR. RxLR primers obtained from UC Davis (USA) were tested using the collection of B. lactucae isolates. RxLR1 primers distinguished between isolates BL801 and BL806. Eight SNPs were identified in three isolates amplified by RxLR5. No polymorphism was observed on the gel for the remaining RxLR primers on single spore races. Unrefined field isolates showed more polymorphisms on the gel than single spore isolates. The phenotypic differences between these two isolates have been identified by the IBEB differential set. Microscopy and qPCR quantification were used to investigate the compatible and incompatible interactions. The results suggest that BL801 is more virulent than BL806, as more infection structures were observed in IBEB resistant cultivars. Results of qPCR and spore count/unit weight of cotyledons showed that BL801 and BL806 were significantly different. The qPCR quantification results from 4 and 5 dpi were correlated with the spore count/unit weight of cotyledons. Although further work is required to develop race specific markers, the methods used in this project demonstrate the potential use of molecular markers to investigate lettuce - Bremia interactions.

Plant mitochondria possess a gene expression system in which post-transcriptional events, such as transcript end maturation and turnover mechanisms play a key role in regulating the transcriptome. In addition, during early developmental stages of embryo germination, differing transcript profiles have been seen. This research focuses on two loci in wheat mitochondria, ccmFn-rps1 and rpl5-Psirps14-cox3, to elucidate the transcription and post-transcriptional events involved in their expression. Northern analysis of the ccmFN-rps1 genes during early seed-to-seedling development reveals a 3.2 kb primary transcript and a 2.7 kb bicistronic mRNA. A 0.7 kb monocistronic rps1 mRNA is detectable up to 2d but there is no detectable monocistronic ccmFN transcript during the stages examined. Transcript ends were mapped using circular-RT-PCR and phosphatase treatment at three different developmental stages and revealed two processing sites as well as a single 3' end common to all three transcripts. The 5' ends of the processed rps1 transcripts are heterogeneous and do not always include the start codon, questioning the rps1 transcript functionality. Gene order varies between plant species due to the high recombination rate in mitochondrial genomes, as is seen for rpl5-Psirps14 in wheat and rice. In both plants, the functional rps14 gene is encoded in the nucleus and the mitochondrial rps14 copy is a pseudogene. In wheat, rpl5-Psirps14 are co-transcribed with cox3 as two RNA species of 3.5 kb and 2.7 kb at 24hr post-imbibition and exhibit developmentally-specific differences in abundance in seedlings. Two promoter regions were mapped in wheat upstream of rpl5 and both transcripts have the same 3' end. In rice 24hr and 6d however, rpl5-Psirps14 are co-transcribed as a 1.4 kb bicistronic mRNA. This presumably reflects the different regulatory signals used in different species. In addition, rpl5 has been subject to several independent gene transfers to the nucleus in the cereal lineages. For example, there is a functional copy of rpl5 in the mitochondria and the nucleus in wheat but it is absent from the mitochondria in rye and maize. In oat mitochondria, rpl5 appears to be a pseudogene and in barley, rearrangements at the 3' end and low transcript levels question its functionality. The characterization of transcription initiation sites, processing sites and 3' ends for these two loci reflect the relaxed nature and flexibility of signals exploited by plant mitochondria. This research supports the significant role of post-transcriptional events in the regulation of gene expression in plant mitochondria.