Дисертації 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.

Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). An outbreak of the disease has been reported in wild endangered Cape parrots (Poicephalus robustus), which is endemic to South Africa. No treatment or vaccine is commercially available. In this study, an investigation into the outbreak was undertaken. BFDV diversity was assessed and viral load and clinical signs correlated. A plant-produced BFDV subunit vaccine was produced in parallel with a corresponding challenge model. Cape parrots were assessed and 53 blood samples collected. Viral load was determined using quantitative real-time PCR (qPCR), and 22 BFDV full-length genome sequences acquired to infer phylogenetic relatedness. The capsid gene (cp) was optimised for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N. benthamiana). Virus-like particles (VLPs) were purified and analysed using transmission electron microscopy. Virions from a Palm cockatoo (Probosciger aterrimus) were purified and a BFDV dsDNA molecular clone was synthesised and replication assessed in 293TT mammalian cells and N. benthamiana using rolling circle replication and qPCR. Two distinct BFDV phylogenetic clusters were reported for Cape parrots, and a direct correlation was seen between viral load in the blood and clinical signs in PBFD-afflicted birds. The CP was successfully expressed in N. benthamiana, and increased through optimisation of Agrobacterium infiltration density and the inclusion of the NSs silencing suppressor. The CP formed VLPs, which were shown to be morphologically similar to infectious virions. The dsDNA molecular clone was shown to replicate autonomously in mammalian 293TT cells, and in plants with the assistance of the Bean yellow dwarf virus replication associated protein (Rep). BFDV genetic diversity in Cape parrots highlights the importance of ensuring new strains are not inadvertently introduced into the wild. This is the first systematic investigation of virus diversity in Cape parrots and assessment of BFDV viral load in a wild psittacine population. The CP was successfully produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is the first reported replication of BFDV in tissue culture, and will greatly expand the scope of available research.

Porcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirusassociated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication-associated protein. Recombinantly expressed CP can selfassemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLPbased and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study was to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. Production of PCV-2 CP in Nicotiana benthamiana via transient Agrobacterium-mediated expression was optimised by comparing two expression vectors, pEAQ-HT and pCBP2, and VLPs were also expressed in Escherichia coli. VLPs produced in plants and in E. coli were used to set up a lateral flow device. In addition, various purification methods of VLPs such as ion exchange chromatography (IEC) and sucrose gradient ultracentrifugation were explored to obtain pure VLPs free of bacterial contamination. The VLPs were successfully expressed in N. benthamiana with both pEAQ-HT and pCBP2, and VLPs were subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs. To further purify the VLPs IEC was used, and fully assembled VLPs which were free of contamination were prepared. Purified VLPs expressed in plants and E. coli were successfully used as coating antigen in lateral flow devices, which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera. E. coli-made VLPs showed higher affinity to PCV-2 antibodies compared to plant-made VLPs. In conclusion, this study has successfully demonstrated the potential to use a plantbased transient expression system to produce affordable diagnostic reagent, especially for developing countries. This is the first study that expressed PCV-2 VLPs using a pCBP-2 expression vector and used PCV-2 VLPs as a coating reagent in the development of a lateral flow test as a proof of concept.

Legumes include some of the most economically important crops and are characterised by their ability to form a symbiotic relationship with nitrogen fixing rhizobia bacteria. Effective legume-rhizobia symbioses result in the formation of a wart-like structure on the root called the nodule. In this nodule, the rhizobia provide nitrogen to the host plant in exchange for energy and shelter. Nodulation is induced by the recognition of a rhizobia derived, strain specific signal, called Nod factor. Recognition of Nod factor from compatible rhizobia leads to complex signalling within and between the epidermis and the cortical cells of the root. Concomitantly, the rhizobia infect the young root hair cells, which are strictly located on the part of the root where the root hairs emerge, a region called the Zone Of Nodulation (ZON). Only when the plant’s Nod factor signalling and the rhizobia infection event in the ZON are in complete synchronicity, do nodules form. Nodulation research in the last few decades has identified a large number of genes associated with, or required for, the formation and development of nodules. These genes today form the foundation of our understanding of the early nodulation signalling pathway. However, there are a number of novel nodulation signalling components still to be discovered, which may hold the key to the missing links in our current knowledge on this signalling pathway. The work reported in this thesis aimed to identify novel components of the early nodulation signalling pathway using modern RNA-seq technology. Approximately 3,000 genes were identified as differentially regulated between roots inoculated with compatible, or a Nod factor-deficient mutant, rhizobia (nodC-). Targeting only the ZON to isolate the transcriptome allowed for the identification of genes having little and/or transient expression in the ZON, which would otherwise have been masked by other dominating transcripts. Gene ontology analysis identified both individual genes and molecular pathways that are likely regulated during early nodulation. Detailed expression analysis of selected candidate genes revealed that some of these genes, including two gibberellin (GA) biosynthesis genes and a TIR-NBS-LRR encoding a putative resistance gene, are specifically expressed in tissue responding to compatible rhizobia inoculation (ZON and nodules). Previous studies utilising mutant and pharmacological analyses have indicated that GA plays an important role in nodule development. However, its precise action, including the location and timing in which GA acts during nodulation, is yet to be determined. To better understand the role of GA in nodulation, follow up studies were conducted for two candidate GA biosynthesis genes identified here, GmGA20ox a and GmGA3ox 1a. Histochemical analysis revealed cell-type specific activation of the GmGA20ox a promoter during nodulation, which changed in localisation at different stages of nodule organogenesis. However, silencing of this gene had no effect on nodule number or appearance. Similarly, follow-up studies were conducted for the TIR-NBS-LRR coding gene, GmTIR-NBS-LRR. Unlike many TIR-NBS-LRR genes which are associated with pathogen-related effector molecule perception, this gene was up-regulated by the symbiotic bacteria, specifically in response to rhizobia-derived Nod factor. Silencing and over-expression of GmTIR-NBS-LRR both resulted in reduced, although not significantly, nodule numbers. However, the complex regulation of the TIR-NBS-LRR gene family makes the interpretation of these results difficult. Based on recent findings, including those reported in this thesis, updated models and predictions relating to the early nodulation pathway are presented and discussed. This includes outlining potential role(s) for the candidate genes identified here in the ZON transcriptome. The mechanistic action of GA and the TIR-NBS-LRR candidate as they relate to nodule formation are also discussed.

The post-genomics era has put at the disposal of modern plant breeders an endless list of genetic building blocks for the design of new biotechnological crops. After a first wave of single-gene transgenic with controversial public acceptance, genomic information and technology is paving the way for increasingly complex designs based in multiple gene engineering. Those designs aiming at the production of inexpensive health-promoting compounds are most likely to be welcomed by consumers. In this project we plan to develop new multigene assembling tools. During this PhD, a standardized collection of interchangeable genetic parts (including promoters, CDS, P-DNAs, etc) and vectors will be developed. The collection, inspired in Synthetic Biology standards, will be made easy-to-assemble in an interchangeable, semi-idempotent and seamless fashion by the addition of flanking recognition sites of type IIS Restriction endonucleases. The construction of the collection will facilitate multigene engineering and will constitute a first step towards enabling Synthetic Biology in plants.
Sarrión Perdigones, MA. (2014). Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35399
TESIS

RNA silencing is a major regulator of gene expression in plants, controlling from development to transposable element silencing and stress responses. As part of the silencing machinery, micro (mi)RNAs orchestrate silencing of their targets, either directly or through cascades of secondary small interfering (si)RNAs. To investigate the role of RNA silencing in plant immunity, I chose to focus on the miR482/2118 family, because of its diversity and presence in many plant species since the appearance of seed plants, with most genomes containing several copies, and because its members target sequences conserved in a family of disease resistance genes known Nucleotide biding site leucine-rich repeat (NLR) genes. In this dissertation, I wanted to address the extent to which the miRNA family and its derived phasiRNAs regulate expression of defence genes as well as contribute to quantitative resistance in crops. I explore the structural differences of miR482/2118 members in Solanum lycopersicum and show that they are functionally significant and affect their target preferences. My approach was based on small RNA sequencing and degradome data to characterize targets of these miRNAs, including the recently discovered tomato TAS5 locus. I also generated transgenic tomatoes constitutively expressing target mimic RNAs that sequester different miR482/2118 members. These tomato mimic RNA lines were less susceptible than their non-transgenic precursors to pathogens Phytophthora infestans and Pseudomonas syringae. Additionally, I investigated the role of small RNAs and their effector proteins during vegetative and reproductive development in tomato. I employed transcript and small RNA sequencing and CRISPR-Cas9 techniques of gene editing to investigate the impact of these factors in gamete viability and transposable element silencing in vegetative meristems. The results presented here provide new evidence about the extent that RNA silencing contributes to the regulation of vital processes in plants. My study primarily explores the extent to which structural differences between the members of the miR482/2118 family affect their range of action, and the use of target mimics against these miRNAs as biotechnological approach for enhancing disease resistance in highly bred cultivars.

Receptor-like kinases (RLKs) are encoded for by one of the largest gene families in Arabidopsis and represent the predominant form of cell surface receptors in plants. RLKs mediate signal transduction in diverse processes including steroid-mediated growth pathways, pathogen-triggered innate immune responses. Here I present characterization of mutant phenotypes, expression patterns, and genetic interactions for the BAK1 INTERACTING RECEPTOR (BIR) family of Leucine-rich Repeat-RLKs, three members of which have had no previous characterization. Furthermore, I show that cell death, aerial growth, and lateral root development defects in bir1-1 are suppressed by mutations of the LRR-RLK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1); I identify a novel primary root growth phenotype in bir1-1 mutants, as well as a lateral root development phenotype for bir3 mutants; and primary root growth and aerial defects in bir3.bir4;bak1 triple mutants. Using an allelic series of bak1 mutations I show that bir phenotypes are dependent upon particular functions of BAK1, and propose that the BIR family exhibits a novel function, previously undescribed for LRR-RLKs, as regulators of co-receptor/ligand-binding receptor complex specificity.

Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

El desarrollo de la flor ha sido un importante campo de investigación por muchos años y el análisis extensivo de interacciones genéticas resultó en un marco de referencia que permitió entender cómo se produce la especificación de los órganos florales. Recientemente, la introducción de tecnologías de nivel genómico permitió confrimar y expandir los modelos existentes sobre el establecimiento de la identidad de los órganos florales y otros eventos importantes que ocurren durante la formación de la flor. A pesar de esto, hay otros aspectos del desarrollo floral para los cuales no existen modelos generales, como el caso de la formación de los límites entre los órganos de la flor. El gen CUP-SHAPED COTYLEDON1 (CUC1) Arabidopsis thaliana es un factor de transcripción clave involucrado en la regulación del desarrollo floral mediante el control de la formación de estos límites o fronteras. En las plantas, la separación correcta entre distintos tejidos es fundamental para el mantenimiento de los meristemos y para coordinar la formación de nuevos órganos. Esto ocurre a través de todo el desarrollo de la planta, desde la separación temprana de cotiledones en las dicotiledóneas, hasta la fromación de límites entre óvulos durante la fase reproductiva. CUC1 reprime el crecimiento en la zona de los límites que contribuye a establecer y se propuso que lo hace afectando la división celular. A pesar de este rol crucial de CUC1, los mecanismos moleculares a través de los cuales controla la formación de límites no están descriptos de manera extensiva. Aquí, la red regulatoria de CUC1 es caracterizada a nivel genómico, usando tecnologías de última generación. En este trabajo, varios aspectos de la función de CUC1 fueron analizados por primera vez mediante la combinación de enfoques complementarios que incluyen la transcriptómica, el perfil de unión al DNA del factor de transcripción y el análisis de interacción entre proteínas. Los resultados obtenidos por estas técnicas permitieron dilucidar un set de dianas transcripcionales, vías moleculares e interactores proteicos de CUC1 que ayudan a delinear los mecanismos por los cuales este factor de transcipción del tipo NAC contribuye al establecimiento de los límites entre órganos florales. Estos resultados representan un avance sustancial para la comprensión de los eventos moleculares controlados por CUC1 en esta etapa fundamental del desarrollo de una planta. En este sentido, esta Tesis provee un cuerpo de trabajo fundacional que puede utilizarse para explorar la red regulatoria de CUC1 más profundamente.
Flower development has been an active field of research for many years and the thorough analysis of genetic interactions provided a general framework to understand how floral organs are specified. More recently, the introduction of genome-wide technologies helped confirm and expand the existing models about organ identity establishment and other important events during flower formation. Still, there are other aspects of flower development for which general models are lacking, such as the formation of floral organ boundaries. The Arabidopsis thaliana CUP-SHAPED COTYLEDON1 (CUC1) gene is a key transcription factor involved in the regulation of flower development by controlling boundary formation. In plants, proper boundaries are fundamental for meristem maintenance and to coordinate organogenesis. This occurs throughout plant development, from the early separation of cotyledons in dicots to the formation of boundaries between ovules during the reproductive phase. CUC1 suppresses growth in the boundary regions that it helps to delimit, and it has been proposed that it does so by affecting cell division. Despite the crucial role of CUC1, the molecular mechanisms by which it controls boundary formation are still poorly understood. Here, CUC1 regulatory network is characterized at the genome-wide level, using state-of-the-art genomic technologies. In this work, several aspects of CUC1 function were analyzed for the first time through the combination of complementary genome-wide approaches including transcriptomics, transcription factor binding profiles and protein interactome analyses. The results obtained from such techniques allowed to elucidate a set of transcriptional targets, molecular pathways and CUC1 interactors that help delineate the mechanisms by which this NAC transcription factor contributes to the establishment of floral organ boundaries. These results represent a substantial advance in the understanding of the molecular events that are controlled by CUC1 in this key developmental stage of plant development. In this regard, this Thesis provides a foundational body of work that can be used to further explore CUC1's regulatory network.

East Tennessee State University has taken several approaches to offering professional development for graduate students over the past several years. This includes graduate student research grants, thesis and dissertation awards, teaching awards, awards for service for the public good, Graduate Student Success Specialist service, Thesis/Dissertation/Capstone Boot Camp, Add-on Fellowships, GA Fee Scholarship, Thesis/Dissertation Scholarships, formal courses (GRAD), graduate student research magazine, and workshops. These will be briefly described, including funding sources supporting the initiatives. A mention of professional development for faculty will also be presented. There will be ample time for discussion of strategies and sharing of ideas by participants.

Specialization on insect and animal pollinators is thought to be the driving force for the evolution of floral traits. Specifically in the New World (NW), the oil-bee pollination syndrome has led to the convergence of floral characters in two distantly related families of core eudicots, Malpighiaceae and Krameriaceae. Both families display a flag-like structure that establishes a zygomorphic flower and floral oil rewards in epithelial elaiophores. These traits work concomitantly to attract and reward female oil-bees that help fertilize these flowers and in return receive oils. The underlying genetics of floral zygomorphy were studied in several clades of core eudicots, which implicated CYCLOIDEA2-(CYC2-)like genes of the TCP gene family to play a role in the establishment and maintenance of this trait. In Malpighiaceae, previous work demonstrated that two CYC2-like genes, CYC2A and CYC2B, are expressed during development correlated with establishing zygomorphy in flowers of NW Malpighiaceae. In this thesis work, I investigated the underlying developmental and genetic basis for the establishment of non-homologous and yet functionally similar traits in the oil-bee pollinated flowers of Malpighiaceae and Krameriaceae. In Chapter 1, I investigated the modification of a conserved CYC2 genetic program in the Old World (OW) acridocarpoids of Malpighiaceae following a major biogeographic disjunction. And in Chapter 2, I studied the floral ontogeny and genetic basis of floral zygomorphy development in Krameria lanceolata Torrey of Krameriaceae. This thesis work sheds light on the significance of the interdisciplinary study of floral symmetry evolution through comparative pollination ecology, development, and genetics.

Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.

PART 1: Thirty-one medicinal plant species from Hawaii, Morocco, and the Sonoran Desert, USA have been shown in past studies to be highly inhibitory to pathogenic bacteria, fungi, and certain cancer cell lines. However, none were tested for antiviral activity. Acetone and methanol extracts from these species were bio-assayed for antiviral activity against herpes simplex virus types 1 and 2 (HSV 1 and HSV 2) and for cytotoxicity to the Vero C1008 cell line. Extracts from these species were tested in vitro for antiviral activity using an immunoperoxidase mini-plaque reduction assay to detect viral structural protein synthesis. Sulforhodamine B and neutral red assays were used to qualitatively and quantitatively assess the cytotoxicity of extracts to C1008 cells, and to compute a 50% cytotoxic concentration (CC50) using a dose response curve. Eight of the 31 plant species assayed showed significant antiviral activity against herpes simplex virus types 1 and 2. The acetone extract of Kalanchoe pinnata Pers. (Crassulaceae) produced the most promising results with an IC50 of 0.025 mg/ml and a CC50 of 1.25 mg/ml yielding a therapeutic index of 50. Additionally, this extract reduced plaque numbers to zero or near zero at a concentration of 0.1 mg/ml when added 30 min before and up to 8 h post infection. Further tests were performed on the K. pinnata extract in pursuit of the mechanisms of observed antiviral properties. Quantitative PCR was used to determine HSV susceptibility to the acetone extract. Antiviral mechanisms were investigated by measuring the reduction of viral DNA at different time points post infection and by measuring the reduction of viral RNA transcripts for five specific genes: alpha gene UL54, beta genes UL23 and UL30, and gamma genes US4 and UL17. Examination of transcript number found a significant decrease in viral DNA replication and early and late gene transcription when infected cells were exposed to K. pinnata suggesting post entry events were blocked by extract.PART 2: The professional development curriculum was written for the Alpine School District and will offer teachers the opportunity to develop and enhance skills for effective science teaching emphasizing molecular biology and microbiology disciplines. The course begins with four assumptions about the nature of secondary science in-service. First, the understandings and abilities required to be a masterful teacher of science are not static. Second, science content increases and changes, and a teacher's understanding in science must keep pace. Third, knowledge about the process of learning is continually developing, requiring teachers to stay informed. And fourth, we live in a changing society that deeply influences events in schools; social changes affect students as they come to school and affect what they need to carry away with them. While the main intent of this course is to improve the knowledge base for secondary life science teachers in the microbiology and molecular biology disciplines, it is expected that teachers will return to their own classroom and use the materials and ideas they have acquired from this course. Included in the concepts stressed are: 1) the historical routes of molecular biology, (2) biotechnology and its influence in society, (3) the relationship between viruses and evolution, order and organization, (4) the immune system and examples that stress structure and function, change and constancy, and (5) the personal and social impact of pathogens. Teachers are introduced to new information in virology, molecular biology and immunology using case studies, practicing scientific inquiry, and recognizing the unifying themes of biology in microbiology and molecular biology.

Cystine-knot peptides are members of the large family of Cysteine-rich proteins with a dimension typically less than 50 amino acids in their mature form, characterized by the presence of six conserved cysteine (Cys) residues linked by three disulfide bonds in a knotted arrangement (Rees & Lipscomb, 1980). Peptides containing the knot motif are widespread in various species such as fungi, insects, mollusks, and mammals, where they mainly play a defense role against microorganisms, acting as toxins, or as signals in cell defense pathways (Craik et al., 2001; Iyer & Acharya, 2011; Schwarz, 2017; Vitt et al., 2001). In plants, cystine-knot peptides (also referred to as cysteine-knot miniproteins) are often involved in resistance to pathogens with the function of protease inhibitors, namely metallocarboxypeptidases and serine proteases (Daly & Craik, 2011; Molesini et al., 2017). A class of cystine-knot protease inhibitors specific to the Solanaceae family was described for the first time in the 1980s (Hass & Hermodson, 1981a; Rees & Lipscomb, 1980). This class includes the tomato cystine-knot miniproteins 1 and 2 (TCMP-1 and TCMP-2), of 37 and 44 amino acids, respectively. TCMP-1 and TCMP-2 display a sequential expression pattern, which is highly modulated during flower and fruit development. TCMP-1 is expressed at a very high level in flower buds before anthesis, then its expression decreases rapidly after anthesis and increases again during fruit development (Cavallini et al., 2011). TCMP-1 mRNA level is very low in leaves, although its expression is induced by wounding and elicitors of responses to biotic stress (Díez-Díaz et al., 2004; Martineau et al., 1991). The expression of TCMP-2 is low in flower buds before anthesis, and gradually increases after fertilization, reaching a maximum in green and ripe fruits, whereas it is apparently absent in leaves, roots, and stems (Cavallini et al., 2011; Pear et al., 1989; Treggiari et al., 2015). Indeed, the TCMP-2 promoter (also named 2A11; X13743; [Pear et al., 1989]) has been successfully used to improve qualitative trait in tomato fruit (Davuluri et al., 2005). Although the biological activity of metallocarboxypeptidase inhibitors supports a role for Solanaceous cystine-knot proteins in plant defense, it has recently been demonstrated that tomato TCMPs are implicated in fruit development (Molesini et al., 2018). In the paper by Molesini et al. (2018), tomato plants transformed with a chimeric gene containing the entire TCMP-1 coding region under the control of the TCMP-2 promoter (pTCMP-2::TCMP-1) showed a marked increase in the expression of TCMP-2 before anthesis, associated with anticipated fruit production. This evidence suggests that TCMPs are regulators of fruiting time and that the maintenance of a correct TCMP-1/TCMP-2 ratio is required for proper initial fruit growth. The mode of action of TCMPs remains largely unexplored also due to the absence of homologous genes in other model species, including Arabidopsis thaliana. In several cases, Cysteine-rich peptides act as signaling molecules in plant development, by interfering with receptors or modifying the activity of multimeric complexes (de Coninck & de Smet, 2016; Tavormina et al., 2015). The general aim of this thesis was a further investigation of the functional role played by TCMP-2 during the first phases of reproductive development. Specific aims were: 1) phenotypic and molecular analyses of pTCMP-2::TCMP-1 and 35S::TCMP-2 plants, during the transition from vegetative growth to reproductive development; 2) identification of TCMP-2 interacting partners by Yeast Two-Hybrid (Y2H) screening using a tomato cDNA library; 3) functional study of one of these interactors (i.e., a B-BOX motif-containing protein) in both Arabidopsis and tomato. A detailed analysis of pTCMP-2::TCMP-1 plants during the transition from the vegetative to the reproductive stage showed an anticipated termination of the sympodial units linked with an induced expression of the florigen gene SINGLE FLOWER TRUSS (SFT), which is the main inducer of flowering. Moreover, MicroTom plants over-expressing TCMP-2 under the control of the CaMV35S constitutive promoter exhibited a reduction of the primary shoot length, very often accompanied by a decreased number of leaves before the first inflorescence, which are indicators of early flowering. The Y2H screen permitted the identification of 47 potential interactors of TCMP-2. Among them, we focused on the B-Box domain-containing protein 16 (SlBBX16). The interaction between TCMP-2 and SlBBX16 was validated in vivo in plant cells by bimolecular fluorescence complementation (BIFC) analysis. We demonstrated that TCMP-2 is also able to interact with SlBBX17, which is the closest tomato homolog of SlBBX16, and with the SlBBX16 Arabidopsis homolog, AtBBX31. A recent study showed the involvement of AtBBX30 and AtBBX31 microproteins (also referred to as miP1b and miP1a) in a multiprotein complex which regulates flowering time in Arabidopsis (Graeff et al., 2016). These proteins interact with the flowering regulator CONSTANS (AtBBX1) and additionally engage in a larger protein complex involving the co-repressor protein TOPLESS through a specific amino acid motif (PFVFL). These interactions suppress the CO-mediated induction of FT expression, causing the severe late flowering phenotype observed in plants over-expressing AtBBX30/31. The implication of AtBBX30/31 in flowering control may indicate that the TCMP-2-SlBBX16/SlBBX17 interaction could play a role in the regulation of flowering. To test this hypothesis, we ectopically overexpressed TCMP-2, SlBBX16 and SlBBX17 in Arabidopsis. The overexpression of TCMP-2 led to an anticipated flowering phenotype linked to an increased FT mRNA level, whereas the overexpression of SlBBX16 and SlBBX17 in Arabidopsis WT and AtBBX30/31 KO mutant resulted in a slight delay in flowering time, suggesting that tomato BBXs were unable to fully phenocopy AtBBX30/31 overexpression. One of the possible reasons for the weak phenotype displayed by A. thaliana over-expressing SlBBX16 and SlBBX17 could be attributed to their inability to interact with AtCO and AtTPL, since the interaction between AtBBX30/31 with AtCO and AtTPL is due to the PFVLF motif (Graeff et al., 2016), which is absent in SlBBX16 and SlBBX17. Indeed, the Y2H experiments revealed no interaction between the tomato BBXs and AtCO and AtTPL, even when using a mutated version of SlBBXs containing the PFVLF motif. The functional study in tomato was conducted on SlBBX17, which presents a peculiar expression pattern in the floral organs. MicroTom plants over-expressing SlBBX17 showed a number of leaves at the first inflorescence similar to that of WT plants, but a delay in the time to reach the flower anthesis stage and a reduced number of ripe fruits. To investigate whether in tomato TCMP-2 and SlBBX17 may participate in a multiprotein complex similar to that described in Arabidopsis (AtBBX30/31-CO-TPL), we carried out ad hoc Y2H analyses to test the interaction between TCMP-2 and SlCO1 and SlTPL1 and the interaction between SlBBX17 and SlCO1 and SlTPL1. Our data indicate that neither TCMP-2 nor SlBBX17 can directly bind to SlCO1 and SlTPL1. Further investigation of the role in flowering and fruiting of SlBBX16, the homologue of SlBBX17, may provide additional insight into the function of BBXs microproteins in tomato.

Bibliography: leaves 153-185.
[ix], 200, [29] leaves, [6] leaves of plates : ill. (chiefly col.) ; 30 cm.
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.
Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1995

[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31.
[CAT] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31.
[EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch.
Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055
TESIS