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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.

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

Plastid transformation technology has several attractive features compared with traditional nuclear transformation technology. However, only a handful of species are able to be successfully transformed. Here, we report an efficient and stable plastid transformation protocol for poplar, an economically important tree species grown worldwide. We transformed the Bacillus thuringiensis cry1C gene into the poplar plastid genome, and homoplasmic transplastomic poplar was obtained after two to three rounds of regeneration under antibiotic selection for 7–12 months. The transplastomic poplar expressing Cry1C insecticidal protein showed the highest accumulation level in young leaves, which reached up to 20.7 μg g-1 fresh weight, and comparatively low levels in mature and old leaves, and hardly detectable levels in non-green tissues, such as phloem, xylem and roots. Transplastomic poplar showed high toxicity to Hyphantria cunea and Lymantria dispar, two notorious forest pests worldwide, without affecting plant growth. These results are the first successful examples of insect-resistant poplar generation by plastid genome engineering and provide a new avenue for future genetic improvement of poplar plants.

Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids. Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.

Aim DNA barcoding has been widely applied to species diversity assessment in various ecosystems, including temperate forests, subtropical forests, and tropical rain forests. However, tropical coral islands have never been barcoded before due to the difficulties in field exploring. This study aims at barcoding the flowering plants from a unique ecosystem of the tropical coral islands in the Pacific Ocean and supplying valuable evolutionary information for better understanding plant community assembly of those particular islands in the future. Location Xisha Islands, China. Methods This study built a DNA barcode database for 155 plant species from the Xisha Islands using three DNA markers (ITS, rbcL, and matK). We applied the sequence similarity method and a phylogenetic-based method to assess the barcoding resolution. Results All the three DNA barcodes showed high levels of PCR success (96%-99%) and sequencing success (98%-100%). ITS performed the highest rate of species resolution (>95%) among the three markers, while plastid markers delivered a relatively poor species resolution (85%-90%). Our analyses obtained a marginal increase in species resolution when combining the three DNA barcodes. Main conclusions This study provides the first plant DNA barcode data for the unique ecosystem of tropical coral islands and considerably supplements the DNA barcode library for the flowering plants on the oceanic islands. Based on the PCR and sequencing success rates, and the discriminatory power of the three DNA regions, we recommend ITS as the most successful DNA barcode to identify the flowering plants from Xisha Islands. Due to its high sequence variation and low fungal contamination, ITS could be a preferable candidate of DNA barcode for plants from other tropical coral islands as well. Our results also shed lights on the importance of biodiversity conservation of tropical coral islands.

Fungi have a huge biodiversity and play important roles in soil biogeochemical cycling in island ecosystems. Although island biogeography has been widely studied for macroorganisms, fungal community assembly in true islands and its relationship with island area are less documented. We examined soil fungal communities in 18 oceanic islands of two types (eight non-coral islands and 10 coral islands) using the Illumina MiSeq sequencing technique. Our results showed that fungal α-diversity (species richness) was substantially different among the oceanic islands, with a higher value in non-coral islands than in coral islands. Fungal α-diversity was significantly affected by soil potassium and magnesium (Mg) and plant communities in non-coral islands, whereas only soil Mg significantly affected it in coral islands. Soil fungal community composition was significantly different in the non-coral and coral islands and was influenced by soil property, plant community and spatial distance. The ecological stochasticity model showed that the fungal community assembly was mainly governed by deterministic processes regardless of island type. Fungal β-diversity, but not α-diversity, increased significantly with increasing island area. These findings have implications for the better prediction of soil fungal community dynamics in island systems and biodiversity conservation in fragmented habitats.

International audience; Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'gamma is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'gamma regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'gamma to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'gamma is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'gamma depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'gamma age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

RNA interference (RNAi) has emerged as a novel and feasible strategy for pest management. Methods for cost-effective production and stable delivery of double-stranded RNA (dsRNA) to the target insects are crucial for the wide application of RNAi for pest control. In this study, we tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes, including actin (ACT), signal recognition particle protein 54k (SRP54), heat shock protein 70 (HSC70), shibire (SHI), cactus (CACT), and soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAP), we found that feeding bacteria-expressed dsRNA successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes caused significant mortality. Our results suggest that the oral delivery of bacteria-expressed dsRNA is a potential alternative for the control of P. versicolora, and that ACT and SRP54 genes are the potent targets.

Owing to their sessile life habit, plants are continuously subjected to a broad range of environmental stresses. During periods of (a)biotic stresses, reactive oxygen species (ROS) levels can rise excessively, leading to oxidative stress. Glutathione reductase (GR) plays an important role in scavenging the ROS and maintenance of redox potential of the cell during oxidative stress. To enhance ROS scavenging capacity, and hence stress tolerance, the Arabidopsis thalianaGR2 (AtGR2) gene was expressed from the tobacco plastid (chloroplast) genome, the main source of ROS production in plant photosynthetic tissues, in this study. Leaves of transplastomic tobacco plants had about seven times GR activity and 1.5 times total glutathione levels compared to wild type. These transplastomic tobacco plants showed no discernible phenotype and exhibited more tolerance to methyl viologen-induced oxidative stress than wild-type control plants. The results indicate that introducing AtGR2 in chloroplasts is an efficient approach to increase stress tolerance. This study also provides evidence that increasing antioxidant enzyme via plastid genome engineering is an alternative to enhance plant’s tolerance to stressful conditions.

The Bacillus thuringiensis (Bt) cry3Bb gene was successfully introduced into poplar plastid genome, leading to transplastomic poplar with high mortality to Plagiodera versicolora. Poplar (Populus L.) is one of the main resource of woody industry, but being damaged by insect pests. The feasibility and efficiency of plastid transformation technology for controlling two lepidopteran caterpillars have been demonstrated previously. Here, we introduced B. thuringiensis (Bt) cry3Bb into poplar plastid genome by biolistic bombardment for controlling P. versicolora, a widely distributed forest pest. Chimeric cry3Bb gene is controlled by the tobacco plastid rRNA operon promoter combined with the 5′UTR from gene10 of bacteriophage T7 (NtPrrn:T7g10) and the 3′UTR from the E. coli ribosomal RNA operon rrnB (TrrnB). The integration of transgene and homoplasmy of transplastomic poplar plants was confirmed by Southern blot analysis. Northern blot analysis indicated that cry3Bb was transcribed to both read through and shorter length transcripts in plastid. The transplastomic poplar expressing Cry3Bb insecticidal protein showed the highest accumulation level in young leaves, which reach up to 16.8 μg/g fresh weight, and comparatively low levels in mature and old leaves. Feeding the young leaves from Bt-Cry3Bb plastid lines to P. versicolora caused 100% mortality in the first-instar larvae after only 1 day, in the second-instar larvae after 2 days, and in the third-instar larvae for 3 days. Thus, we report a successful extension of plastid engineering poplar against the chrysomelid beetle.

Plant cells contain three organelles that harbor DNA: the nucleus, plastids, and mitochondria. Plastid transformation has emerged as an attractive platform for the generation of transgenic plants, also referred to as transplastomic plants. Plastid genomes have been genetically engineered to improve crop yield, nutritional quality, and resistance to abiotic and biotic stresses, as well as for recombinant protein production. Despite many promising proof-of-concept applications, transplastomic plants have not been commercialized to date. Sequence-specific nuclease technologies are widely used to precisely modify nuclear genomes, but these tools have not been applied to edit organelle genomes because the efficient homologous recombination system in plastids facilitates plastid genome editing. Unlike plastid transformation, successful genetic transformation of higher plant mitochondrial genome transformation was tested in several research group, but not successful to date. However, stepwise progress has been made in modifying mitochondrial genes and their transcripts, thus enabling the study of their functions. Here, we provide an overview of advances in organelle transformation and genome editing for crop improvement, and we discuss the bottlenecks and future development of these technologies., In addition to the nuclear genome, plants also contain mitochondrial and plastid genomes. This review highlights recent advances in organelle transformation and genome editing for crop improvement and discusses the bottlenecks and future development of these technologies.

Owing to their sessile life habit, plants are continuously subjected to a broad range of environmental stresses. During periods of (a)biotic stresses, reactive oxygen species (ROS) levels can rise excessively, leading to oxidative stress. Glutathione reductase (GR) plays an important role in scavenging the ROS and maintenance of redox potential of the cell during oxidative stress. To enhance ROS scavenging capacity, and hence stress tolerance, the Arabidopsis thalianaGR2 (AtGR2) gene was expressed from the tobacco plastid (chloroplast) genome, the main source of ROS production in plant photosynthetic tissues, in this study. Leaves of transplastomic tobacco plants had about seven times GR activity and 1.5 times total glutathione levels compared to wild type. These transplastomic tobacco plants showed no discernible phenotype and exhibited more tolerance to methyl viologen-induced oxidative stress than wild-type control plants. The results indicate that introducing AtGR2 in chloroplasts is an efficient approach to increase stress tolerance. This study also provides evidence that increasing antioxidant enzyme via plastid genome engineering is an alternative to enhance plant’s tolerance to stressful conditions.

Under natural conditions, plants constantly encounter various fluctuating environmental stresses, which potentially restrict plant growth, plant development and even limit crop productivity. In addition to carbon fixation activity in C4 photosynthesis, NADP-dependent malic enzyme (NADP-ME) has been suggested to play important roles in diverse stress responses in plants. NADP-ME is one of the essential enzymes metabolizing malate, which is important for stabilizing cytoplasmic pH, controlling stomatal aperture, increasing resistance to aluminum excess and pathogen. Pyruvate, another product of NADP-ME reaction, participates in the synthesis of defense compounds such as flavonoids and lignin, which are involved in stresses tolerance such as mechanical wounding and pathogen invasion. Moreover, NADP-ME provides essential reductive coenzyme NADPH in the biosynthesis of flavonoids and lignin. On the other hand, NADPH is crucial for reactive active species (ROS) metabolizing systems such as the ascorbate-glutathione pathway and NADPH-dependent thioredoxin reductase, and is also required by apoplastic oxidative burst in most plant-pathogen interactions. This mini-review is largely focus on the characteristics of gene expression and activity of NADP-ME, as well as its interaction with ROS signaling under a variety of biotic and abiotic stress responses, which will provide a theoretical foundation for breeding of stress resistant crops.

RNA interference (RNAi) has emerged as a novel and feasible strategy for pest management. Methods for cost-effective production and stable delivery of double-stranded RNA (dsRNA) to the target insects are crucial for the wide application of RNAi for pest control. In this study, we tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes, including actin (ACT), signal recognition particle protein 54k (SRP54), heat shock protein 70 (HSC70), shibire (SHI), cactus (CACT), and soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAP), we found that feeding bacteria-expressed dsRNA successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes caused significant mortality. Our results suggest that the oral delivery of bacteria-expressed dsRNA is a potential alternative for the control of P. versicolora, and that ACT and SRP54 genes are the potent targets.

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5’ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3’UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was approximately 9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

To establish a successful in vitro plant regeneration system in Alternanthera philoxeroides (Mart.) Griseb, an orthogonal design was used to investigate the effects of three factors (plant growth regulators, explant types and dark treatment in initial-stage), each having three levels. The effects of these factors and levels on callus induction and shoot regeneration were quantitatively evaluated by analysis of variance. The experimental results showed that the callus induction was significantly affected by 2, 4-dichlorophenoxyacetic acid (2, 4-D), and shoot differentiation from subcultured pieces of callus was enhanced mostly by dark treatment in initial-stage. The optimal conditions for callus induction are obtained from the stem explants cultured on semi-solid Murashige and Skoog (MS) medium plus 2.2 μM BA and 2.2 μM 2, 4-D, with 20 days dark treatment in initial-stage. The highest frequency of shoot regeneration is obtained from the calli cultured on semi-solid MS medium plus 8.8 μM BA, without dark treatment in initial-stage.