Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

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 ß-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.

Plant organellar genomes: much done, much more to do.

Plastids and mitochondria are the only organelles that possess genomes of endosymbiotic origin. In recent decades, advances in sequencing technologies have contributed to a meteoric rise in the number of published organellar genomes, and have revealed greatly divergent evolutionary trajectories. In this review, we quantify the abundance and distribution of sequenced plant organellar genomes across the plant tree of life. We compare numerous genomic features between the two organellar genomes, with an emphasis on evolutionary trajectories, transfers, the current state of organellar genome editing by transcriptional activator-like effector nucleases (TALENs), transcription activator-like effector (TALE)-mediated deaminase, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), as well as genetic transformation. Finally, we propose future research to understand these different evolutionary trajectories, and genome-editing strategies to promote functional studies and eventually improve organellar genomes.

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

Gut Microbiota Accelerate the Insecticidal Activity of Plastid-Expressed Bacillus thuringiensis Cry3Bb to a Leaf Beetle, Plagiodera versicolora.

Bacillus thuringiensis is widely used as a biopesticide, and its crystal protein expressed in transgenic crops has been successfully used for the management of insect pests. However, whether the midgut microbiota contribute to the Bt insecticidal mechanism remains controversial. We previously demonstrated that transplastomic poplar plants expressing Bt Cry3Bb are highly lethal to willow leaf beetle (Plagiodera versicolora), one of the major pests causing severe damage to Salicaceae plants such as willows and poplars. Here, we demonstrate that feeding poplar leaves expressing Cry3Bb to nonaxenic P. versicolora larvae leads to significantly accelerated mortality, and overgrowth and dysbiosis of the gut microbiota, compared with axenic larvae. Corroborating work done with Lepidopteran insects, plastid-expressed Cry3Bb can cause the lysis of the beetle's intestinal cells, lead to the entry of intestinal bacteria into the body cavity, and thus cause the dynamic changes in the flora of the midgut and blood cavity in P. versicolora. Reintroduction of Pseudomonas putida, a gut bacterium of P. versicolora, into axenic P. versicolora larvae further enhances mortality upon feeding on Cry3Bb-expressing poplar. Our results indicate the important contribution of host gut microbiota in promoting the B. thuringiensis crystal protein insecticidal activity and provide new insights into the mechanism of pest control by Bt-transplastomic approaches. IMPORTANCE The contribution of gut microbiota to Bacillus thuringiensis Cry3Bb insecticidal activity in a leaf beetle was demonstrated using transplastomic poplar plants, providing a potential new approach to improve the efficiency of plastid transformation technology for pest control by expression of Bt toxins.

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

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 (ß-Actin, SRP54, and SNAP) for RNAi using in vitro double-stranded RNAs (dsRNAs) fed to HV, and found that dsRNAs targeting ß-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.

Transplastomic tomatoes expressing double-stranded RNA against a conserved gene are efficiently protected from multiple spider mites.

Spider mites are serious pests and have evolved significant resistance to many chemical pesticides, thus making their control challenging. Several insect pests can be combated by plastid-mediated RNA interference (PM-RNAi), but whether PM-RNAi can be utilized to control noninsect pests is unknown. Here, we show that three species of spider mites (Tetranychus evansi, Tetranychus truncatus, and Tetranychus cinnabarinus) take up plastid RNA upon feeding. We generated transplastomic tomato plants expressing double-stranded RNA (dsRNA) targeted against a conserved region of the spider mite ß-Actin mRNA. Transplastomic plants exhibited high levels of resistance to all three spider mite species, as evidenced by increased mortality and suppression of target gene expression. Notably, transplastomic plants induced a more robust RNAi response, caused higher mortality, and were overall better protected from spider mites than dsRNA-expressing nuclear transgenic plants. Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for spider mites and extend the application range of the technology to noninsect pests.

A multiple shoot induction system for peptide-mediated gene delivery into plastids in Arabidopsis thaliana, Nicotiana benthamiana, and Fragaria×ananassa.

The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.

Functional peptide-mediated plastid transformation in tobacco, rice, and kenaf.

In plant engineering, plastid transformation is more advantageous than nuclear transformation because it results in high levels of protein expression from multiple genome copies per cell and is unaffected by gene silencing. The common plastid transformation methods are biolistic bombardment that requires special instruments and PEG-mediated transformation that is only applicable to protoplast cells. Here, we aimed to establish a new plastid transformation method in tobacco, rice, and kenaf using a biocompatible fusion peptide as a carrier to deliver DNA into plastids. We used a fusion peptide, KH-AtOEP34, comprising a polycationic DNA-binding peptide (KH) and a plastid-targeting peptide (AtOEP34) to successfully deliver and integrate construct DNA into plastid DNA (ptDNA) via homologous recombination. We obtained transformants in each species using selection with spectinomycin/streptomycin and the corresponding resistance gene aadA. The constructs remained in ptDNA for several months after introduction even under non-selective condition. The transformants normally flowered and are fertile in most cases. The offspring of the transformants (the T1 generation) retained the integrated construct DNA in their ptDNA, as indicated by PCR and DNA blotting, and expressed GFP in plastids from the integrated construct DNA. In summary, we successfully used the fusion peptide method for integration of foreign DNA in tobacco, rice, and kenaf ptDNA, and the integrated DNA was transmitted to the next generations. Whereas optimization is necessary to obtain homoplasmic plastid transformants that enable stable heterologous expression of genes, the plastid transformation method shown here is a novel nanomaterial-based approach distinct from the conventional methods, and we propose that this easy method could be used to target a wide variety of plants.

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.

Application progress of plant-mediated RNAi in pest control.

RNA interference (RNAi)-based biopesticides are novel biologic products, developed using RNAi principles. They are engineered to target genes of agricultural diseases, insects, and weeds, interfering with their target gene expression so as to hinder their growth and alleviate their damaging effects on crops. RNAi-based biopesticides are broadly classified into resistant plant-based plant-incorporated protectants (PIPs) and non-plant-incorporated protectants. PIP RNAi-based biopesticides are novel biopesticides that combine the advantages of RNAi and resistant transgenic crops. Such RNAi-based biopesticides are developed through nuclear or plastid transformation to breed resistant plants, i.e., dsRNA-expressing transgenic plants. The dsRNA of target genes is expressed in the plant cell, with pest and disease control being achieved through plant-target organism interactions. Here, we review the action mechanism and strategies of RNAi for pest management, the development of RNAi-based transgenic plant, and the current status and advantages of deploying these products for pest control, as well as the future research directions and problems in production and commercialization. Overall, this study aims to elucidate the current development status of RNAi-based biopesticides and provide guidelines for future research.

[High-level expression of anti-interleukin-6 single chain variable fragment through plastid transformation technology].

Interleukin-6 (IL-6) is a pleiotropic cytokine which participates in the pathogenesis of a variety of clinical disorders, including many kinds of cancers. Anti-IL-6 antibody was proved to be useful for the immunotherapy of various inflammatory diseases. Plants are low-cost platforms for producing specific proteins of therapeutic interest. Two dependent transplastomic tobacco lines expressing murine anti-IL-6 single chain variable fragment (scFv) were generated after bombardment and regeneration, homoplasmy was then verified by Southern blotting analysis. The anti-IL-6 scFv gene was successfully expressed at both transcriptional and translational levels in transplastomic tobacco plants. Functional anti-IL-6 scFv accumulated to 1% of total soluble proteins, namely 41 mg/kg fresh weight. There was no obvious phenotypic difference between the wild-type and the transplastomic tobacco plants, including the growth rate, the height of mature plants and the number of siliques. The high-level expression of anti-IL-6 scFv indicates the potential for cost-effective production of scFV using transplastomic plants.

Modification of Chloroplast Antioxidant Capacity by Plastid Transformation.

As immobile organisms, green plants must be frequently challenged by a broad range of environmental stresses. During these constantly adverse conditions, reactive oxygen species (ROS) levels can rise extremely in plants, leading to cellular dysfunction and cell death presumably due to irreversible protein overoxidation. Once considered merely as deleterious molecules, cells seek to remove them as efficiently as possible. To enhance ROS scavenging capacity, genes encoding antioxidative enzymes can be directly expressed from the genome of plastid (chloroplast), a major compartment for ROS production in photosynthetic organisms. Thus, overexpression of antioxidant enzymes by plastid engineering may provide an alternative to enhance plant's tolerance to stressful conditions specifically related with chloroplast-derived ROS. Here, we describe basic procedures for expressing glutathione reductase, a vital component of ascorbate-glutathione pathway, in tobacco via plastid transformation technology.

Control of a sap-sucking insect pest by plastid-mediated 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.

Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6.

Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2-3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities.

Efficient control of western flower thrips by plastid-mediated RNA interference.

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Resistance to RNA interference by plant-derived double-stranded RNAs but not plant-derived short interfering RNAs in Helicoverpa armigera.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Transplastomic approaches for metabolic engineering.

The plastid (chloroplast) genome of seed plants represents an attractive target of metabolic pathway engineering by genetic transformation. Although the plastid genome is relatively small, it can accommodate large amounts of foreign DNA that precisely integrates via homologous recombination, and is largely excluded from pollen transmission due to the maternal mode of plastid inheritance. Since the engineering of metabolic pathways often requires the expression of multiple transgenes, the possibility to conveniently stack transgenes in synthetic operons makes the transplastomic technology particularly appealing in the area of metabolic engineering. Absence of epigenetic gene silencing mechanisms from plastids and the possibility to achieve high transgene expression levels further add to the attractiveness of plastid genome transformation. This review focuses on engineering principles and available tools for the transplastomic expression of enzymes and pathways, and highlights selected recent applications in metabolic engineering.