Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of death among children around the world. Responsible for 4 to 6 million deaths per year according to the World Health Organization (WHO), diarrhea is especially dangerous for infants and young children. Globally, it is estimated that 1.4 billion episodes of diarrhea occur in children less than five years of age annually. In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths. And is a major cause of mortality for children in developing countries with approximately one million deaths annually. Rotaviruses belong to the family Reoviridae and are spherical 70-nm particles. The virus genome contains 11 segments of double-stranded RNA, each encoding a viral capsid or nonstructural protein. The identification of a rotavirus nonstructural protein gene (NSP4) encoding a peptide, which functions both as a viral enterotoxin and as a factor involved in the acquisition of host cell membrane during virus budding from cells, provides a new approach for mucosal immunization. Protein expression through chloroplast transformation system offers a number of advantages like high level of transgene expression, transgene containment via maternal inheritance, lack of gene silencing and position effect due to site specific gene integration and also the possibility of multi gene engineering in single transformation event. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen. To achieve an enhanced immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into transgenic chloroplast and was transformed into chloroplast genome of Nicotiana tabacum by homologous recombination. The chloroplast integration of CTB-NSP4(90) fusion gene was confirmed in transgenic tobacco plants by PCR analysis. Southern blot analysis further confirmed site specific gene integration and homoplasmy. Immunoblot analysis of transformed chloroplast confirmed the expression of CTBNSP490 fusion protein both in monomeric and pentameric forms that retained the binding affinity to the enterocytes GM1 ganglioside receptor. Expression levels of CTB-NSP4 protein was quantified by GM1 ganglioside binding ELISA assay; mature leaves expressed CTB-NSP4 fusion protein to upto 2.45 % in total soluble protein, 100-400 fold higher than nuclear expression which was only 0.006%-0.026%. Antibody titration and virus challenge experiments will be performed in mice at Loma Linda University to evaluate the antigenic and protective properties of the chloroplast derived CTB-NSP4 fusion protein.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue´rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes. LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. iv Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development.

Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, and C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in longterm treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low cost platform for oral and injectable delivery of cleavable proinsulin.

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Plastiden enthalten ihr eigenes Genom, das u.a. für Untereinheiten des photosynthetischen Apparates kodiert. Die Expression dieses Apparates wird hauptsächlich posttranskriptionell reguliert. Dafür notwendige Faktoren sind vor allem RNA-Bindeproteine, welche fast ausschließlich kernkodiert und posttranslational in die Plastiden importiert werden. Dazu gehören auch die äußerst abundanten chloroplastidären Ribonukleoproteine (cpRNPs). Die bisher näher untersuchten Mitglieder der cpRNP-Familie aus Arabidopsis thaliana sind an der Prozessierung und Stabilisierung von plastidären Transkripten beteiligt und phylogenetisch eng miteinander verwandt. In dieser Studie wurden zwei noch unbekannte Mitglieder der cpRNP Familie, CP33A und CP33B, näher untersucht. CP33A ist ein essentielles Protein der Chloroplastenbiogenese. Mutanten von CP33A keimen nur in der Gegenwart einer externen Kohlenstoffquelle. Die Blätter sind albinotisch, in ihrer Struktur anomal und das gesamte Wachstum ist stark eingeschränkt. Untersuchungen der RNA-Interaktionspartner von CP33A durch RIP-Chip-Analysen (RNA-Immunopräzipitation und Chip-Hybridisierung) zeigen, dass CP33A mit allen mRNAs assoziiert. Des Weiteren führt der Verlust von CP33A zu einer starken Reduktion vieler Transkripte, vor allem RNAs, die durch die plastidär kodierte RNA Polymerase (PEP) transkribiert werden und unprozessierte Vorläufer-Transkripte. CP33B interagiert ebenfalls mit multiplen plastidären RNAs. Dabei zeigt CP33B eine Präferenz für psbA. Feinkartierung der CP33B-Bindung innerhalb des psbA Leserahmens verdeutlichten, dass CP33B vor allem mit dem 3´Ende des Transkriptes interagiert. Phänotypische und genetische Untersuchungen der cp33b-Nullmutante ließen keinen vom Wildtyp abweichenden Phänotyp identifizieren und zeigten dass CP33B keinen essentiellen Einfluss auf die Proteinakkumulation photosynthetischer Untereinheiten, die Expression plastidärer Transkripte, das Spleißen und die Edierung seiner Ziel-RNAs hat. / Plastids harbour their own genome, which encodes for essential subunits of the photosynthetic apparatus. The expression of these subunits is mainly regulated on the posttranscriptional level. The important factors for posttranscriptional processing are RNA-binding proteins (RBPs), which are almost exclusively nuclear-encoded and imported posttranslational into the plastids. Among them are the chloroplast ribonucleoproteins (cpRNPs). The cpRNPs are a family of highly abundant RNA-binding proteins found in the chloroplast of land plants. Members of the Arabidopsis thaliana cpRNP family, that have been investigated in more detail, are involved in processing and stabilization of plastid transcripts and are phylogenetically closely related. In this study two unknown members of the cpRNPs, CP33A and CP33B, which cluster outside of this phylogenetic group, are investigated. CP33A is essential for chloroplast biogenesis. Null alleles of CP33A only germinate in the presence of an external carbon source. cp33a seedlings are albino, show strong growth inhibition and an abnormal leaf structure. Investigating RNA-ligands of CP33A using RIP-Chip (coimmunoprecipitation coupled to microarray analysis) shows an association with all chloroplast mRNAs. The loss of CP33A leads to a reduction of almost all transcripts, predominantly affecting RNAs transcribed by the plastid-encoded RNA polymerase (PEP) and unspliced and unprocessed precursor mRNAs. CP33B also interacts with multiple plastid RNAs. The main target is the mRNA of psbA. More than 90% of the stromal psbA mRNA is associated with CP33B. Fine mapping efforts suggest that CP33B preferentially interacts with the 3’-end of the psbA reading frame. Phenotypic and genetic analyses of cp33b-null mutants do not show any differences compared to wild-type plants. CP33B has no essential impact on: Protein accumulation of photosynthetic subunits, expression of plastid transcripts, RNA-splicing or RNA-editing of its target RNAs.

The biological control agent Pseudomonas chlororaphis PA23 is effective at protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. Despite the growing importance of biocontrol bacteria in protecting crop plants from fungal pathogens, little is known about how the host plant responds to bacterial priming on the leaf surface and certainly nothing about global changes in gene activity in the presence and absence of S. sclerotiorum. PA23 priming of mature canola plants reduced the number of lesion-forming petals by 90%. Global RNA sequencing of canola tissue at the host-pathogen interface showed a 16-fold reduction in the number of genes uniquely upregulated in response to S. sclerotiorum when pretreated with PA23. Upstream defense-related gene patterns suggest MAMP-triggered immunity via surface receptors detecting PA23 flagellin and peptidoglycans. Although systemic acquired resistance (SAR) was induced in all treatment groups, a response centered around a glycerol-3-phosphate (G3P)-mediated pathway was exclusively observed in canola plants treated with PA23 alone. Activation of these defense mechanisms by PA23 involved production of reactive oxygen species as well as pronounced thylakoid membrane structures and plastoglobule formation in leaf chloroplasts. PA23 therefore primes defense responses in the plant through the induction of unique local and systemic regulatory networks. / October 2016

Le mode vie autotrophique des plantes repose entièrement sur l’intégrité du chloroplaste et notamment l’étape de la biogénèse. La transcription des gènes chloroplastiques, assurée par une PEP (ARN polymérase encodée par le chloroplaste) et deux NEPs (ARN polymérase encodée par le noyau), est l’une des étapes primordiales dans le développement d’un chloroplaste photosynthétique. On distingue trois classes de gènes chloroplastiques : les gènes de classe I, transcrit par la PEP exclusivement; les gènes de classe II, transcrits par la PEP ou les NEPs; et les gènes de classe III, transcrits exclusivement par les NEPs. Pour assurer sa fonction, la PEP doit être associée à des facteurs sigmas. L’un de ceux-ci, la protéine SIG6, est un facteur sigma général et, associé à la PEP, assure la transcription de l’ensemble des gènes de classe I et II lors du développement du chloroplaste photosynthétique. Ainsi, le mutant sig6 présente un phénotype de cotylédons pâles, associé à un retard de biogénèse chloroplastique, ainsi qu’une diminution de la transcription des gènes de classe I, provoquant la diminution de la quantité de protéines de classe I. Dans le laboratoire, nous étudions les deux protéines WHIRLY chloroplastiques (WHY1 et WHY3) pour leur rôle dans le maintien de la stabilité génomique chloroplastique. Toutefois, peu de choses sont encore connues sur leur rôle potentiel dans la transcription ou la biogénèse chloroplastique. Par exemple, lorsque l’on tente de purifier la PEP, on obtient un gros complexe transcriptionnel nommé PTAC (Plastid Transcriptionally Active Chromosome) dans lequel sont retrouvées les deux protéines WHIRLY, suggérant qu’elles pourraient être impliquées dans la transcription chloroplastique. De plus, un possible rôle dans la biogénèse chloroplastique leur a été prêté, notamment chez le maïs. Dans cette étude, nous avons donc cherché à vérifier l’implication des protéines WHIRLY dans la biogénèse chloroplastique par une approche génétique de croisements entre les mutants sig6 et why1why3. Pour cela, nous avons isolé des doubles mutants sig6why1 et sig6why3, ainsi qu’un triple mutant sig6why1why3. À l’aide d’une caractérisation phénotypique et de la quantification de quelques protéines chloroplastiques, nous avons remarqué que la perte d’un des WHIRLY permet de complémenter le phénotype de cotylédons pâles du mutant sig6 et favorise l’expression normale de protéines en principe sous-exprimées dans le mutant sig6. Toutefois, la perte des deux WHIRLY ne permet pas de compenser le phénotype de cotylédons pâles et provoque l’apparition d’un phénotype persistant associé à une expression anormale des protéines chloroplastiques. Ces résultats ne peuvent être expliqués par le rôle des WHIRLY dans le maintien de la stabilité génomique chloroplastique étant donné que le triple mutant sig6why1why3 présente moins de réarrangements que le double mutant why1why3. Finalement, nous montrons que les effets de la perte d’un WHIRLY sur le mutant sig6 peuvent être mimés par l’utilisation de la rifampicine, une drogue inhibant l’ARN polymérase chloroplastique de type bactérienne (PEP). Ensemble, ces résultats démontrent donc l’implication des protéines WHIRLY chloroplastiques dans la biogénèse chloroplastique en association avec la protéine SIG6. Nous proposons un modèle selon lequel les deux protéines WHIRLY permettraient de favoriser l’activité de l’ARN polymérase de type bactérienne, notamment lors du développement du chloroplaste photosynthétique. En cas d’absence d’une des deux protéines, cette diminution partielle d’activité de la PEP favoriserait la mise en place d’un mécanisme de complémentation par le NEPs, permettant finalement de rétablir la biogénèse chloroplastique dans un mutant sig6. En l’absence des deux WHIRLY, le mécanisme de complémentation par les NEPs serait incapable de compenser la forte inhibition de la PEP, se traduisant par une aggravation du retard de développement du chloroplaste dans le mutant sig6. / The autotrophic lifestyle of plants relies entirely on the integrity of chloroplasts and particularly on their biogenesis. Chloroplast gene transcription, performed by a Plastid-Encoded Polymerase (PEP) and two Nuclear-Encoded Polymerases (NEPs), is one of the key steps during the development of photosynthetic chloroplast. There are 3 classes of genes, one transcribed by PEP alone (class I), one by both PEP and NEPs (class II), and the third by NEPs alone (class III). To carry out transcription, PEP associates with plastid sigma factors including the general sigma factor SIG6. sig6 mutants have a pale cotyledon phenotype, a severe decrease in class I gene transcription and a reduction in the level of class I proteins. In our laboratory, we study the role of the two plastid WIHRLY proteins (WHY1 and WHY3) in maintaining plastid genome stability. However, little is known about any role these proteins may play in transcription or chloroplast biogenesis. It seems likely they are involved in plastid gene transcription since they are found in the Plastid Transcriptionally Active Chromosome (PTAC). Moreover, they have been implicated in chloroplast biogenesis in maize. In this study, we verified the implication of these proteins in plastid biogenesis using a genetic approach in which we crossed a sig6 mutant with a why1why3 mutant. We isolated sig6why1 and sig6why3 double mutants and a sig6why1why3 triple mutant. Using a phenotypic characterisation and quantification of some plastid proteins, we show that loss of one of the two Why genes complements the sig6 pale cotyledon phenotype and allows a more normal pattern of expression of plastid proteins that are under-expressed in the sig6 mutant. However, we also show that loss of the two Why genes does not alleviate the sig6 phenotype. Moreover, the triple mutant shows a second pale phenotype on true leaves, and the plastid protein expression pattern is abnormal compared to either sig6 or wild type plants. Those results cannot be explained by the role of WHIRLY proteins in plastid genome stability since the triple mutant shows fewer plastid genome rearrangements than the why1why3 mutant. Finally, we show that inhibition of the PEP polymerase using rifampicin elicits the same complementation of the sig6 phenotype as the loss of one of the two WHIRLY. Together, these results show the implication of WHIRLY proteins in plastid biogenesis in association with SIG6. We propose a model in which WHIRLY act as activators of PEP activity, particularly during the chloroplast biogenesis. Therefore, the absence of one of the WHIRLY would cause a weak inhibition of PEP, facilitating the set-up of a rescue mechanism by NEPs and, consequently, allowing the complementation of plastid biogenesis in the sig6 mutant. However, the absence of the two WHIRLY proteins would cause a strong inhibition of PEP, and the inability of the rescue mechanism by NEPs to compensate for this strong inhibition, resulting in a more severe phenotype in the sig6 mutant.