No description available.

Anthrax, a fatal bacterial infection is caused by Bacillus anthracis, a gram-positive, spore forming, capsulated, rod shaped organism. Centers for Disease Control (CDC) lists anthrax as Category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of the disease and potential for delivery as a biological weapon. The currently available human vaccine in the United States (AVA anthrax vaccine adsorbed) is prepared from Alum adsorbed formalin treated supernatant culture of toxigenic, non-encapsulated strain of Bacillus anthracis with the principle component being protective antigen (PA83). Evaluation of anthrax vaccine given to nearly 400,000 US military personnel by Vaccine Adverse Event Reporting System (VAERS) showed adverse effects such as flu-like symptoms, local pain, large degree of inflammation, edema, malaise, rash, arthralgia, and headache following vaccination. All the adverse reactions are attributed to the composition of vaccine components. These vaccine preparations contain trace contaminants of lethal and edema factors that contribute to the adverse side effects. Also, the current method of vaccine manufacture has limited production capacity.The production of PA83, in plants through chloroplast genetic engineering might eliminate the concerns of adverse side effects and the levels of expression would ensure the availability of vaccine for the human population in an environmentally friendly approach. The primary concern is whether the PA83 purified from transgenic chloroplasts is as immunogenic as the PA83 in the AVA. For this, PA83 has been expressed in transgenic chloroplasts of Nicotiana tabacum var. petit Havana, by inserting the pag (2205 bp) with the N-terminal 6X histidine tag, into the chloroplast genome by homologous recombination. Chloroplast integration of the pag was confirmed by PCR and Southern analysis. The PA83 protein was detected in transgenic chloroplasts by immunoblot analysis using anti-PA83 antibodies. Maximum expression levels of PA83 (14.17% TSP) were observed in mature leaves upon continuous illumination, due to the presence psbA 5'UTR, a light and developmentally regulated translation enhancer sequence. The PA83 has been purified by affinity chromatography using Ni resin columns. Chloroplast derived PA83 was functional in vitro and was able to lyse the mouse macrophages when combined with the lethal factor. The in vitro assays showed that the crude extracts contained up to 20ug/ml of functional PA83.The immunization studies of PA83 on Balb/c mice, revealed highly immunogenic IgG titers. Subcutaneous immunization with purified chloroplast derived PA83 with adjuvant yielded IgG titers up to 1:320,000, similar to that of the group immunized with PA83 derived from Bacillus anthracis. Immunization of groups with PA83 combined with alhydrogel adjuvant showed four - eight times higher immune response than the groups without adjuvant. The higher expression levels of PA83 in transgenic chloroplasts might ensure the availability of anthrax vaccine to the general public and the high immune response observed in the mouse model would enable the replacement of the current AVA with a cleaner and safer vaccine.

Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.

Antigen expression through the Chloroplast Transformation Technology (CTT) produces bioencapsulated subunit-vaccines, capable of eliciting immune responses when delivered orally. Considerable challenges to effective plant-based vaccines are the normalization of dosage and preservation of accumulated antigen, which is complicated by variable high water content and protease activity. This study critically examines the efficacy of lyophilization in dehydrating plant-tissues and preserving plant-derived antigens with vaccine potential. Lyophilization was optimized through gravimetric analysis using lettuce expressing Protective Antigen (PA) of Bacillus anthracis (LS-HPAG) and the human autoantigen Proinsulin (Pins) fused to Cholera toxin subunit B (LS-CTB-Pins). Lyophilization for 48-hours was sufficient treatment to reduce lettuce to 4.57% of its original weight, which retained .058% water content in the bound state; these levels corresponded with oven-dried controls while antigen was stabilized for over a year of storage at room temperature. A simulated gastric fluid assay was applied to evaluate stability of plant derived antigens during digestion. It was observed that lettuce plant cells conferred protection through antigen bioencapsulation for up to an hour under enzymatic digestive conditions. LS-HPAG immunogenicity was then demonstrated through the induction of a PA-specific IgG response by through oral boosting of C57/BL6 test mice. Survival during toxin challenge demonstrated a protective immune response if 40% of animal immunized by plant-derived PA. Lastly, the inclusion of excipient and adjuvant additives will be considered and utilized for the development of prototype vaccine capsule formulations.

No description available.

No description available.

Pelargonium peltatum, ivy geranium, experiences foliar bleaching at temperatures exceeding 30° C. Contessa™ Red (heat tolerant) and Temprano™ Lavender (heat susceptible) were compared. Established plants underwent temperature treatments of 15/20° C or 25/30° C night/day with moisture treatments of 80% or 30% substrate volumetric water content (VWC). Photosynthesis, leaf greenness and growth data were collected at days 0, 7 and 11. No differences in photosynthetic rate nor a decrease in greenness in developed leaves occured in either cultivar due to high temperature or drought. Contessa™ Red had overall greater growth and leaf greenness than Temprano™ Lavender. Greenness and growth increased similarly for both cultivars at 80% VWC. Any decrease in foliar bleaching due to drought was likely due to a decrease in growth. A second study using Temprano™ Lavender indicated foliar bleaching occurs in newly emerging, developing leaves.

Die Transkription in Plastiden höherer Pflanzen erfolgt durch die plastidärkodierte, bakterienähnliche RNA-Polymerase PEP und die kernkodierten Phagentyp-RNA-Polymerasen RpoTp und RpoTmp (NEP). Da für NEP bislang keine Transkriptionsfaktoren identifiziert wurden, wurden die entsprechenden Enzyme aus A. thaliana und N. tabacum mit verschiedenen, N-terminalen Epitopen in E. coli exprimiert und für pulldown assays zur Identifikation interagierender Proteine eingesetzt. Des Weiteren wurden Epitop-markierte Tabak RpoTp und Arabidopsis RpoTp und -Tmp in vivo exprimiert und zur Co-IP verwendet. In diesen Studien wurden als potentielle Interaktionspartner von RpoTp Ycf1 und Ycf2 gefunden. Des Weiteren konnte mit 3xFLAG-RpoT-exprimierenden Arabidopsis-Mutanten gezeigt werden, dass RpoTp und -Tmp teilweise membranassoziiert sind. Außerdem wurde die duale Lokalisation der Arabidopsis RpoTmp in den Chloroplasten und Mitochondrien nachgewiesen. Mittels RIP-Chip wurden mit RpoTp assoziierte RNAs analysiert und mögliche, bisher unbekannte NEP-Transkripte gefunden. Plastidäre Haushaltsgene besitzen meist sowohl PEP- als auch NEP-Promotoren. Anhand transplastomischer Tabakpflanzen, in denen NEP-Promotoren von accD , rpoB und rrn16 gegen einen PEP-Promotor ausgetauscht bzw. durch Mutagenese ausgeschaltet wurden, sollte die Arbeitsteilung von NEP und PEP in Abhängigkeit vom Entwicklungsstadium beleuchtet werden. Dabei wurde gezeigt, dass die Transkription durch PEP für accD zu einer leichten Überexpression, für rpoB hingegen zu einer verzögerten Entwicklung und verringerten Transkriptmengen führte. Zudem wurden durch RNA-Seq die Aktivierung zusätzlicher TSSs in den Mutanten gezeigt, welche die Effekte auf RNA- und Proteinebene erklärte, und der alternative Promotor PaccD-158 identifiziert, welcher auch im Wildtyp genutzt wird. Es wird diskutiert, inwiefern die Rolle von NEP und PEP individuell für einzelne Gene in Abhängigkeit ihrer jeweiligen Funktion betrachtet werden muss. / The transcription in plastids of higher plants is accomplished by the plastid encoded, bacterial-type RNA polymerase PEP and by the nuclear encoded, phagetype RNA polymerases RpoTp and RpoTmp (NEP). As the identification of transcription factors for NEP failed so far, in this work the corresponding enzymes from A. thaliana and N. tabacum containing different, N-terminally fused epitope tags were expressed in E. coli and used for pulldown assays to identify interacting proteins. Furthermore epitope-tagged tobacco RpoTp and Arabidopsis RpoTp and -Tmp were expressed in vivo and applied for co-immunoprecipitation. In these studies Ycf1 and Ycf2 were found as potential interaction partners of RpoTp. In addition, the 3xFLAG-RpoT-expressing Arabidopsis mutants were used to show, that RpoTp and -Tmp are partly associated with the thylakoid membrane. Further, immunoblot assays confirmed the dual localization of the Arabidopsis RpoTmp in chloroplasts as well as in mitochondria. Moreover, via RIP-Chip analyses RNAs associated with RpoTp were analysed and potential new NEP transcripts were found. Most plastidial housekeeping genes possess PEP as well as NEP promoters. The division of labor between NEP and PEP according to the developmental stage was studied on the basis of transplastomic tobacco plants, in which NEP promoters of accD, rpoB and rrn16 have been exchanged with a PEP promoter or knocked out by mutagenesis. It was shown, that transcription of accD by PEP lead to a slight overexpression, but PEP-dependent transcription of rpoB led to a delayed development and decreased transcript levels. Via RNA-seq an activation of additional TSSs could be shown in the mutants, which explains the effects on RNA and protein level, and the alternative promoter PaccD-158 was identified, that is also used in the wildtype. It is discussed, how the roles and the division of labor of NEP and PEP should be considered individually for each gene according to its function.

Die Transkription der Chloroplasten ist erstaunlich komplex. Ihr relativ kleines Genom kodiert für Komponenten der Photosynthese und der eigenen Genexpressionsmaschine. Es wird mindestens durch zwei RNA Polymerasen transkribiert. Neben der plastidär kodierten RNA Polymerase (PEP), existiert mindestens eine weitere kernkodierte RNA Polymerase (NEP). Die PEP spielt eine wichtige Rolle bei der Expression der Photosynthesegene und ist essentiell für die Biogenese der Chloroplasten und schließlich für das Überleben der Pflanzen. In der vorliegenden Arbeit wird erstmals die spezifische Interaktion der PEP mit ihren Transkriptionseinheiten unter verschiedenen Lichtbedingungen in vivo auf plastomweiter Ebene gezeigt. Darunter befinden sich hauptsächlich DNA Fragmente, die Photosynthesegene repräsentieren. Außerdem zeigt die PEP eine eindeutige Präferenz zu den rRNA Genen und einigen tRNA Genen. Eine Reduktion dieser Assoziation der PEP in Dunkelperioden lässt einen lichtabhängigen Prozess bei der PEP-DNA Assoziation vermuten. Außerdem wurde die PEP Aktivität an den bestimmten DNA Regionen in vivo ermittelt durch die Analyse naszierender Transkripte. Ein Großteil der plastidären RNA Spezies kopräzipitiert mit der PEP, aber sie scheint präferentiell mit tRNAs zu interagieren. Die Analyse der Verteilung der PEP bewies erstmals, dass sie hauptsächlich mit den Membranen assoziiert. Dort befindet sich auch das transkriptionsaktive Chromosom (TAC). Im TAC wurden vor kurzem neben der PEP auch Mitglieder von RNA Prozessierungsfaktoren identifiziert, was auf eine mögliche Kopplung von Transkription und posttranskriptionellen Prozessen hindeutet. Für einen Einblick in dieses Interaktionsnetzwerk wurden Transkriptanalysen des TACs ausgeführt. Mit wenigen Ausnahmen assoziieren alle plastidären RNA Spezies mit dem TAC. Einige RNAs liegen bereits prozessiert vor, was eine Verbindung zwischen Transkription und posttranskriptionellen Prozessen noch im TAC vermuten lässt. / Chloroplast transcription is astonishingly complex. The relative small genome of plastids, which codes for components of the photosynthetic machinery as well as for components of their own gene expression machinery, is transcribed by at least two different RNA polymerases. Beside the plastid-encoded plastid RNA polymerase (PEP) a nuclear-encoded plastid RNA polymerase (NEP) exists as well. PEP plays a major role in the expression of photosynthesis genes and is essential for chloroplast biogenesis and thus for plant survival. This work shows the specific in vivo interaction between PEP and its transcription units under different light conditions on a plastome-wide scale. Among them are DNA fragments that represent photosynthetic genes. In addition, PEP shows clear preferences to rRNA and tRNA genes. Furthermore, the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that PEP-DNA association is a light-dependent process. To survey PEP activity in vivo on plastid DNA regions, the association to nascent transcripts was analyzed. The majority of plastid RNA species could be found in PEP precipitates, but PEP seems to interact more strongly with tRNAs. Analysis of the suborganellar distribution of PEP shows that PEP is preferably associated with chloroplastmembranes. The transcriptionally active chromosome (TAC) was also found to be membrane-attached. Beside PEP different RNA processing factors were identified within the TAC and related purifications, indicating a possible coupling of transcription and posttranscriptional processes. To gain more insights into this interaction network, transcripts of TAC were analyzed. It is shown that nearly all plastid RNA species with only a few exceptions are associated to the TAC and that at least selected transcripts are already processed. This indicates that there is a link between transcription and posttranscriptional processes already within the TAC.

cpRNPs (chloroplastidäre Ribonukleoproteine) sind kernkodierte RNA-Bindeproteine, die jeweils zwei RRM (RNA recognition motif)-Domänen besitzen und in die Chloroplasten höherer Landpflanzen importiert werden. Die Mitwirkung dieser Proteine an der Reifung plastidärer mRNAs in vitro, ihre bemerkenswert hohe Abundanz sowie die Beeinflussung ihrer Funktion durch lichtabhängige posttranslationale Modifikationen weisen auf eine bedeutende Rolle der cpRNPs in der Regulierung der plastidären Genexpression hin. In dieser Arbeit erfolgte erstmals eine Analyse der in vivo bestehenden Interaktionen zwischen cpRNPs und RNA im Stroma von Chloroplasten mittels RIP-Chip-Analysen. Es konnte gezeigt werden, dass ein Großteil der plastidären mRNA-Spezies mit CP31A und CP29A kopräzipitiert. Neben der mehrheitlichen Überlappung der RNA-Interaktionen konnten spezifische Präferenzen von CP29A und CP31A für bestimmte Transkripte identifiziert werden. Um Einblicke in die molekularen Funktionen der cpRNPs zu erhalten, wurden cp29a- und cp31a- Einzel- und Doppelmutanten hinsichtlich der Akkumulation, sowie des Spleiß- und Edierungsstatus plastidärer mRNAs untersucht. Da unter Standard-Wachstumsbedingungen lediglich wenige milde Defekte innerhalb der mRNA-Reifung in den Mutanten detektiert wurden, erfolgten weitere Analysen unter Kältestress-Bedingungen, die zu einer Induktion der CP29A-Expression und zu dem Ausbleichen junger Blattgewebe in cp29a- und cp31a-Mutanten führten. In diesen chlorotischen Geweben wurden reduzierte Akkumulationen plastidärer Proteine sowie PEP-abhängig transkribierter mRNAs detektiert. Darüber hinaus Prozessierungsdefekte für einige Transkripte beobachtet: Die Reifung der 23S-rRNA findet in cp29a- und cp31a-Mutanten nur eingeschränkt statt. In cp31a-Mutanten sind zudem die Prozessierung der ycf3-RNA sowie die Spaltung eines polycistronischen rpl33-Vorläufers gestört. In cp29a-Mutanten ist unter anderem die Prozessierung der rpoC1- und der ycf3-mRNA defizitär. / cpRNPs (chloroplast ribonucleoproteins) are nuclear encoded RNA-binding proteins, which contain two RRM (RNA recognition motif) domains and reside within the chloroplasts of higher land plant species. They are involved in plastid mRNA accumulation and processing in vitro. Their involvement in multiple steps of mRNA maturation and their remarkable abundance together with their regulation via posttranslational modifications identified the cpRNPs as potential central regulators of chloroplast gene expression. This work investigated the in vivo interactions of CP29A and CP31A from Arabidopsis with RNA in isolated chloroplasts via RIP-Chip. In this approach the association of both proteins with the majority of plastid mRNA species could be shown. While the interaction profiles of CP29A and CP31A were largely overlapping, also specific preferences for some transcripts were detected. To gain insights in the molecular functions of CP29A and CP31A, null mutants for one or both cpRNPs were analyzed for mRNA accumulation, splicing and editing. Since only few mild mRNA maturation defects were detected at standard growth conditions, analyses at cold stress were performed. Cold stress leads to an increase in CP29A expression and in both cp29a- and cp31a single- and double mutants a bleaching of young leaf tissue was observed. The chlorotic tissues showed a strong decrease in plastid protein accumulation accompanied by reduced levels of PEP-dependent transcripts. In addition some processing defects were observed: The maturation of the 23S-rRNA was reduced in cp29a- and cp31a mutants. In addition the processing of the ycf3-mRNA and a polycistronic rpl33 transcript were defective in cp31a mutants. In cp29a mutants processing defects within the rpoC1- and the ycf3-mRNA were detected.