Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation.

In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/µg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells.

Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts.

Expression of fungal cutinase and swollenin in tobacco chloroplasts reveals novel enzyme functions and/or substrates.

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.

Among 12billion injections administered annually, unsafe delivery leads to >20million infections and >100million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1 diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections.

Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery.

Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10(+) T cell but not Foxp3(+) regulatory T cells, suppression of interferon-gamma and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.

Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars.

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

Plant-made vaccine antigens and biopharmaceuticals.

Plant cells are ideal bioreactors for the production and oral delivery of vaccines and biopharmaceuticals, eliminating the need for expensive fermentation, purification, cold storage, transportation and sterile delivery. Plant-made vaccines have been developed for two decades but none has advanced beyond Phase I. However, two plant-made biopharmaceuticals are now advancing through Phase II and Phase III human clinical trials. In this review, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear and chloroplast or transient viral) and their current limitations or challenges. We provide suggestions for advancing this valuable concept for clinical applications and conclude that greater research emphasis is needed on large-scale production, purification, functional characterization, oral delivery and preclinical evaluation.

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms.

BACKGROUND: The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms. RESULTS: The complete chloroplast genome sequence of Citrus sinensis is 160,129 bp in length, and contains 133 genes (89 protein-coding, 4 rRNAs and 30 distinct tRNAs). Genome organization is very similar to the inferred ancestral angiosperm chloroplast genome. However, in Citrus the infA gene is absent. The inverted repeat region has expanded to duplicate rps19 and the first 84 amino acids of rpl22. The rpl22 gene in the IRb region has a nonsense mutation resulting in 9 stop codons. This was confirmed by PCR amplification and sequencing using primers that flank the IR/LSC boundaries. Repeat analysis identified 29 direct and inverted repeats 30 bp or longer with a sequence identity > or = 90%. Comparison of protein-coding sequences with expressed sequence tags revealed six putative RNA edits, five of which resulted in non-synonymous modifications in petL, psbH, ycf2 and ndhA. Phylogenetic analyses using maximum parsimony (MP) and maximum likelihood (ML) methods of a dataset composed of 61 protein-coding genes for 30 taxa provide strong support for the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids and asterids. The MP and ML trees are incongruent in three areas: the position of Amborella and Nymphaeales, relationship of the magnoliid genus Calycanthus, and the monophyly of the eurosid I clade. Both MP and ML trees provide strong support for the monophyly of eurosids II and for the placement of Citrus (Sapindales) sister to a clade including the Malvales/Brassicales. CONCLUSION: This is the first complete chloroplast genome sequence for a member of the Rutaceae and Sapindales. Expansion of the inverted repeat region to include rps19 and part of rpl22 and presence of two truncated copies of rpl22 is unusual among sequenced chloroplast genomes. Availability of a complete Citrus chloroplast genome sequence provides valuable information on intergenic spacer regions and endogenous regulatory sequences for chloroplast genetic engineering. Phylogenetic analyses resolve relationships among several major clades of angiosperms and provide strong support for the monophyly of the eurosid II clade and the position of the Sapindales sister to the Brassicales/Malvales.