Advances in plastid transformation for metabolic engineering in higher plants

The plastid(chloroplast)genome of higher plants is an appealing target for metabolic engineering via genetic transformation.Although the bacterial-type plastid genome is small compared with the nuclear genome,it can accommodate large quantities of foreign genes that precisely integrate through homologous recombination.Engineering complex metabolic pathways in plants often requires simultaneous and concerted expression of multiple transgenes,the possibility of stacking several transgenes in synthetic operons makes the transplastomic approach amazing.The potential for extraordinarily high-level transgene expression,absence of epigenetic gene silencing and transgene containment due to the exclusion of plastids from pollen transmission in most angiosperm species further add to the attractiveness of plastid transformation technology.This minireview describes recent advances in expanding the toolboxes for plastid genome engineering,and highlights selected high-value metabolites produced using transplastomic plants,including artemisinin,astaxanthin and paclitaxel.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

The expression of double-stranded RNAs(dsRNAs)from the plastid genome has been proven to be an effective method for controlling herbivorous pests by targeting essential insect genes.However,there are limitations to the efficiency of plastid-mediated RNA interference(PM-RNAi)due to the initial damage caused by the insects and their slow response to RNA interference.In this study,we developed transplastomic poplar plants that express dsRNAs targeting the b-Actin(dsACT)and Srp54k(dsSRP54K)genes of Plagiodera versicolora.Feeding experiments showed that transplastomic poplar plants can cause significantly higher mortality in P.versicolora larvae compared with nuclear transgenic or wild-type poplar plants.The efficient killing effect of PM-RNAi on P.versicolora larvae was found to be dependent on the presence of gut bacteria.Importantly,foliar application of a gut bacterial strain,Pseudomonas putida,will induce dysbiosis in the gut bacteria of P.versicolora larvae,leading to a significant acceleration in the speed of killing by PM-RNAi.Overall,our findings suggest that interfering with gut bacteria could be a promising strategy to enhance the effectiveness of PM-RNAi for insect pest control,offering a novel and effective approach for crop protection based on RNAi technology.

The Translational Apparatus of Plastids and Its Role in Plant Development

Chloroplasts （plastids） possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco （plastid-encoded components） and Arabidopsis （nucleus-encoded components）. This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant develop- ment at various levels, presumably via retrograde signaling pathway（s）. In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.

Control of a sap-sucking insect pest by plastidmediated RNA interference

Expression of double-stranded RNAs in plastids offers great potential for the efficient control of chewing insects.However,many insect pests do not consume plant tissue but rather feed on the host plant by sucking sap from the vascular system.Whether or not plastid-mediated RNA interference(RNAi)can be employed to control sap-sucking insects is unknown.Here,we show that five species of sap-sucking hemipteran insects acquire plastid RNA upon feeding on plants.We generated both nuclear transgenic and transplastomic tobacco plants expressing double-stranded RNAs targeting the MpDhc64C gene,a newly identified efficient target gene of RNAi whose silencing causes lethality to the green peach aphid Myzus persicae.In a whole-plant bioassay,transplastomic plants exhibited significant resistance to aphids,as evidenced by reduced insect survival,impaired fecundity,and decreased weight of survivors.The protective effect was comparable with that conferred by the best-performing nuclear transgenic plants.We found that the proportion of aphids on mature leaves of transplastomic plants was significantly lower compared with that of nuclear transgenic plants.When aphids were allowed to infest.only the mature leaves,transplastomic plants grew significantly faster and were overall better protected from the pest compared with nuclear transgenic plants.When monitored by electrical-penetration-graph analyses and aphid avoidance response experiments,the insects displayed remarkable alterations in feeding behavior,which was different in nuclear transgenic and transplastomic plants,likely reflecting specific avoidance strategies to toxic RNA molecules.Taken together,our study demonstrates that plastid-mediated RNAi provides an efficient strategy for controlling at least some sap-sucking insect pests,even though there is most likely no or only very little chloroplast RNA in the sap.

Complete protection from Henosepilachna vigintioctopunctata by expressing long double-stranded RNAs in potato plastids

RNA interference(RNAi)has emerged as a powerful technology for pest management.Previously,we have shown that plastid-mediated RNAi(PM-RNAi)can be utilized to control the Colorado potato beetle,an insect pest in the Chrysomelidae family;however,whether this technology is suitable for controlling pests in the Coccinellidae remained unknown.The coccinellid 28-spotted potato ladybird(Henosepilachna vigintioctopunctata;HV)is a serious pest of solanaceous crops.In this study,we identified three efficient target genes(β-Actin,SRP54,and SNAP)for RNAi using in vitro double-stranded RNAs(dsRNAs)fed to HV,and found that dsRNAs targetingβ-Actin messenger RNA(dsACT)induced more potent RNAi than those targeting the other two genes.We next generated transplastomic and nuclear transgenic potato(Solanum tuberosum)plants expressing HV dsACT.Long dsACT stably accumulated to up to 0.7%of the total cellular RNA in the transplastomic plants,at least three orders of magnitude higher than in the nuclear transgenic plants.Notably,the transplastomic plants also exhibited a significantly stronger resistance to HV,killing all larvae within 6 d.Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for HV,extending the application range of this technology to Coccinellidae pests.

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.