RESUMEN
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.
Asunto(s)
Control Biológico de Vectores , Plastidios , Interferencia de ARN , ARN de Planta , Thysanoptera , Animales , Control Biológico de Vectores/métodos , Plastidios/genética , ARN Bicatenario , ARN de Planta/genética , Thysanoptera/genética , Nicotiana/genética , Nicotiana/parasitologíaRESUMEN
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.
Asunto(s)
Calor , Plastidios , Regiones Promotoras Genéticas/genética , Transgenes/genética , Expresión Génica , Plastidios/genética , Plastidios/metabolismoRESUMEN
MAIN CONCLUSION: The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR-Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA-gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.
Asunto(s)
Potyvirus , Potyvirus/genética , ARN , Sistemas CRISPR-Cas/genética , Plantas Modificadas Genéticamente/genética , Procesamiento Postranscripcional del ARNRESUMEN
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.
Asunto(s)
Solanum lycopersicum , Tetranychidae , Animales , ARN Bicatenario , Tetranychidae/genética , Solanum lycopersicum/genética , Interferencia de ARN , Plantas Modificadas GenéticamenteRESUMEN
RNA interference (RNAi) technology is a promising and effective approach for pest insect management. Owing to its sequence-guided working mechanism, RNAi has a high degree of species-selectivity, thus minimizing potential adverse effects on nontarget organisms. Recently, engineering plastid (chloroplast) genome, rather than the nuclear genome, to produce double-stranded RNAs has emerged as a powerful way to protect plants from multiple arthropod pests. Here, we review the recent progresses in the plastid-mediated RNAi (PM-RNAi) approach for pest control and the factors influencing its efficacy, and propose the strategies for further efficiency improvement. We also discuss the current challenges and the biosafety-related issues of PM-RNAi technology that need to be addressed for commercial production.
Asunto(s)
Insectos , Control de Plagas , Animales , Interferencia de ARN , Plastidios/genética , ARN BicatenarioRESUMEN
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.
Asunto(s)
Escarabajos , Solanum tuberosum , Animales , ARN Bicatenario/genética , Solanum tuberosum/genética , Actinas , Escarabajos/genética , Larva , Interferencia de ARN , Plastidios/genéticaRESUMEN
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.
Asunto(s)
Mariposas Nocturnas , ARN Bicatenario , Animales , Insectos , Mariposas Nocturnas/genética , Plantas Modificadas Genéticamente/metabolismo , Interferencia de ARN , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN de Planta/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
KEY MESSAGE: Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the future engineering of the plastid genome of kiwifruit. A limitation in the application of plastid biotechnology for molecular farming is the low-level expression of transgenes in non-green plastids compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplasts during ripening of red-fleshed kiwifruit (Actinidia chinensis cv. Hongyang) fruits, which may make kiwifruit an ideal horticultural plant for recombinant protein production by plastid engineering. To identify cis-elements potentially triggering high-level transgene expression in edible tissues of the 'Hongyang' kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in green leaves and fruits at three different developmental stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA, the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulation of plastid gene expression. Altogether, 40 RNA editing sites were verified, and 5 of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression and provides cis-elements for future efforts to engineer the plastid genome of kiwifruit.
Asunto(s)
Actinidia , Actinidia/genética , Actinidia/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/genética , Plastidios/metabolismo , Transcripción GenéticaRESUMEN
MAIN CONCLUSION: Importation of taxadiene synthase into chloroplasts is important for the efficient heterologous production of taxadiene. Taxadiene, the first committed precursor to taxol, is synthesized from geranylgeranyl pyrophosphate (GGPP) by action of taxadiene synthase (TS). Heterologous production of taxadiene could potentially rely on both cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. We suggest the compartmentalized engineering in chloroplast as an efficient approach for taxadiene production. In this study, we directly introduced the TS gene from Taxus brevifolia into the tobacco chloroplast genome and found that the transplastomic plants accumulated a low content of taxadiene, ~ 5.6 µg/g dry weight (DW). Moreover, we tried a combination of MEP and MVA pathways for taxadiene synthesis by nuclear transformation with a truncated version of TS (without encoding a transit peptide) into the transplastomic plants. However, this did not further improve the taxadiene production. In contrast, we found that taxadiene could be produced up to 87.8 µg/g DW in leaves of transgenic plants expressing TS with a chloroplast transit peptide, which was significantly higher than that in leaves of transplastomic plants. Thus, this study highlights the importance of TS importation into chloroplast for production of taxadiene.
Asunto(s)
Isomerasas , Nicotiana , Alquenos , Cloroplastos/genética , Diterpenos , Nicotiana/genéticaRESUMEN
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'γ 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'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ 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'γ 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'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Botrytis/inmunología , Senescencia Celular/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/inmunología , Hojas de la Planta/metabolismo , Proteína Fosfatasa 2/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Senescencia Celular/fisiología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Resistencia a la Enfermedad/inmunología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Genotipo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Mutación , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2/genética , Ácido Salicílico/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Pentosiltransferasa/genética , Enfermedades de las Plantas/genética , Ácido Salicílico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Pentosiltransferasa/metabolismo , Enfermedades de las Plantas/microbiología , Interferencia de ARNRESUMEN
BACKGROUND: HDAC4 is a key regulator of chondrocyte hypertrophy and skeletal development, but it is not clear whether the increase in vascular invasion at growth plates is related to HDAC4 expression. To determine it, we investigated the relationship between HDAC4 and angiogenesis in both in vivo and in vitro models. METHODS: HDAC4 was deleted in Col2α1-Cre; HDAC4fl/fl mice. Growth of the Col2α1-Cre; HDAC4d/d mice was compared with HDAC4fl/fl mice at postnatal days 2, 4, 6, and 8. X-rays were taken to examine skeletal development. At postnatal days 14 and 21, mice were euthanized for specimen collection. Murine chondrocytes were isolated from the ventral parts of rib cages of 6-day-old mice (C57Bl/6) and transfected with a vector expressing HDAC4 as a fusion protein with green fluorescent protein (GFP). Relative expression levels of HDAC4, VEGF, and Hif1α were measured in these cells by Western blot, RT-qPCR, enzyme-linked immunosorbent, histology, and immunohistochemistry assays. RESULTS: The Col2α1-Cre; HDAC4d/d mice were markedly smaller compared with the control mice. At postnatal days 14 and 21, the Col2α1-Cre; HDAC4d/d mice exhibited a shortened growth plate, a larger secondary ossification center, and stronger staining of CD31 and CD34 compared to control mice. The isolated chondrocyte cells exhibited a high transfection efficiency of HDAC4 which resulted in the detection of a significant decrease in VEGF and Hif1α levels compared with the control chondrocytes. CONCLUSIONS: HDAC4 expression in chondrocytes contributes to angiogenesis in the growth plate, and its absence in vivo negatively affects growth plates.
Asunto(s)
Colágeno Tipo II/genética , Eliminación de Gen , Expresión Génica , Histona Desacetilasas/genética , Neovascularización Fisiológica/genética , Animales , Biomarcadores , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno Tipo II/metabolismo , Genes Reporteros , Histona Desacetilasas/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , FenotipoRESUMEN
Photorespiratory hydrogen peroxide (H2 O2 ) plays key roles in pathogenesis responses by triggering the salicylic acid (SA) pathway in Arabidopsis. However, factors linking intracellular H2 O2 to activation of the SA pathway remain elusive. In this work, the catalase-deficient Arabidopsis mutant, cat2, was exploited to elucidate the impact of S-nitrosoglutathione reductase 1 (GSNOR1) on H2 O2 -dependent signalling pathways. Introducing the gsnor1-3 mutation into the cat2 background increased S-nitrosothiol levels and abolished cat2-triggered cell death, SA accumulation, and associated gene expression but had little additional effect on the major components of the ascorbate-glutathione system or glycolate oxidase activities. Differential transcriptome profiles between gsnor1-3 and cat2 gsnor1-3 together with damped ROS-triggered gene expression in cat2 gsnor1-3 further indicated that GSNOR1 acts to mediate the SA pathway downstream of H2 O2 . Up-regulation of GSNOR activity was compromised in cat2 cad2 and cat2 pad2 mutants in which glutathione accumulation was genetically prevented. Experiments with purified recombinant GSNOR revealed that the enzyme is posttranslationally regulated by direct denitrosation in a glutathione-dependent manner. Together, our findings identify GSNOR1-controlled nitrosation as a key factor in activation of the SA pathway by H2 O2 and reveal that glutathione is required to maintain this biological function.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Transducción de Señal , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Microscopía Confocal , Nitrosación , Estrés Oxidativo , Reguladores del Crecimiento de las Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácido Salicílico/metabolismoRESUMEN
Under natural conditions, plants are exposed to various abiotic and biotic stresses that trigger rapid changes in the production and removal of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). The ascorbate-glutathione pathway has been recognized to be a key player in H2O2 metabolism, in which reduced glutathione (GSH) regenerates ascorbate by reducing dehydroascorbate (DHA), either chemically or via DHA reductase (DHAR), an enzyme belonging to the glutathione S-transferase (GST) superfamily. Thus, DHAR has been considered to be important in maintaining the ascorbate pool and its redox state. Although some GSTs and peroxiredoxins may contribute to GSH oxidation, analysis of Arabidopsis dhar mutants has identified the key role of DHAR in coupling H2O2 to GSH oxidation. The reaction of DHAR has been proposed to proceed by a ping-pong mechanism, in which binding of DHA to the free reduced form of the enzyme is followed by binding of GSH. Information from crystal structures has shed light on the formation of sulfenic acid at the catalytic cysteine of DHAR that occurs with the reduction of DHA. In this review, we discuss the molecular properties of DHAR and its importance in coupling the ascorbate and glutathione pools with H2O2 metabolism, together with its functions in plant defense, growth, and development.
Asunto(s)
Glutatión , Peróxido de Hidrógeno , Ácido Ascórbico , Glutatión/metabolismo , Glutatión Reductasa , Homeostasis , Estrés Oxidativo , OxidorreductasasRESUMEN
KEY MESSAGE: 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.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Escarabajos/fisiología , Endotoxinas/metabolismo , Conducta Alimentaria , Proteínas Hemolisinas/metabolismo , Hojas de la Planta/parasitología , Plastidios/metabolismo , Populus/genética , Populus/parasitología , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Escarabajos/efectos de los fármacos , Endotoxinas/toxicidad , Conducta Alimentaria/efectos de los fármacos , Vectores Genéticos/metabolismo , Genoma de Plastidios , Proteínas Hemolisinas/toxicidad , Larva/efectos de los fármacos , Larva/fisiología , Fenotipo , Plantas Modificadas Genéticamente , Transformación GenéticaRESUMEN
MAIN CONCLUSION: 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.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Arthrobacter/enzimología , Betaína/metabolismo , Solanum tuberosum/enzimología , Oxidorreductasas de Alcohol/genética , Arthrobacter/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cloroplastos/enzimología , Cloroplastos/genética , Sequías , Ingeniería Genética , Fotosíntesis , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plastidios/enzimología , Plastidios/genética , Solanum tuberosum/genética , Solanum tuberosum/fisiología , Estrés FisiológicoRESUMEN
Unfortunately, one of the author names has been misspelled in the original publication. The correct spelling is Qiping Song.
RESUMEN
The complexity of plant antioxidative systems gives rise to many unresolved questions. One relates to the functional importance of dehydroascorbate reductases (DHARs) in interactions between ascorbate and glutathione. To investigate this issue, we produced a complete set of loss-of-function mutants for the three annotated Arabidopsis (Arabidopsis thaliana) DHARs. The combined loss of DHAR1 and DHAR3 expression decreased extractable activity to very low levels but had little effect on phenotype or ascorbate and glutathione pools in standard conditions. An analysis of the subcellular localization of the DHARs in Arabidopsis lines stably transformed with GFP fusion proteins revealed that DHAR1 and DHAR2 are cytosolic while DHAR3 is chloroplastic, with no evidence for peroxisomal or mitochondrial localizations. When the mutations were introduced into an oxidative stress genetic background (cat2), the dhar1 dhar2 combination decreased glutathione oxidation and inhibited cat2-triggered induction of the salicylic acid pathway. These effects were reversed in cat2 dhar1 dhar2 dhar3 complemented with any of the three DHARs. The data suggest that (1) DHAR can be decreased to negligible levels without marked effects on ascorbate pools, (2) the cytosolic isoforms are particularly important in coupling intracellular hydrogen peroxide metabolism to glutathione oxidation, and (3) DHAR-dependent glutathione oxidation influences redox-driven salicylic acid accumulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cloroplastos/enzimología , Citosol/enzimología , Estrés Oxidativo , Oxidorreductasas/metabolismo , Ácido Salicílico/metabolismo , Antioxidantes/metabolismo , Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Muerte Celular , ADN Bacteriano/genética , Prueba de Complementación Genética , Glutatión/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Insercional/genética , Mutación/genética , Fenotipo , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Oxidative stress responses are influenced by growth day length, but little is known about how this occurs. A combined reverse genetics, metabolomics and proteomics approach was used to address this question in Arabidopsis thaliana. A catalase-deficient mutant (cat2), in which intracellular oxidative stress drives pathogenesis-related responses in a day length-dependent manner, was crossed with a knockdown mutant for a specific type 2A protein phosphatase subunit (pp2a-b'γ). In long days (LD), the pp2a-b'γ mutation reinforced cat2-triggered pathogenesis responses. In short days (SD), conditions in which pathogenesis-related responses were not activated in cat2, the additional presence of the pp2a-b'γ mutation allowed lesion formation, PATHOGENESIS-RELATED GENE1 (PR1) induction, salicylic acid (SA) and phytoalexin accumulation and the establishment of metabolite profiles that were otherwise observed in cat2 only in LD. Lesion formation in cat2 pp2a-b'γ in SD was genetically dependent on SA synthesis, and was associated with decreased PHYTOCHROME A transcripts. Phosphoproteomic analyses revealed that several potential protein targets accumulated in the double mutant, including recognized players in pathogenesis and key enzymes of primary metabolism. We conclude that the cat2 and pp2a-b'γ mutations interact synergistically, and that PP2A-B'γ is an important player in controlling day length-dependent responses to intracellular oxidative stress, possibly through phytochrome-linked pathways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Espacio Intracelular/metabolismo , Estrés Oxidativo , Fotoperiodo , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Electroforesis en Gel Bidimensional , Flores/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas , Genotipo , Indoles/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Hojas de la Planta/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteoma/metabolismo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ácido Salicílico/metabolismo , Tiazoles/metabolismoRESUMEN
Substantial advancements have been made in recent years in comprehending immune memory, which enhances the secondary response through prior infections. The ability of vertebrate T and B lymphocytes to exhibit classic recall responses has long been regarded as a distinguishing characteristic. However, natural killer (NK) cells have been found to acquire immunological memory in a manner akin to T and B cells. The fundamental principles derived from the investigation of NK cell memory offer novel insights into innate immunity and have the potential to pave the way for innovative strategies to enhance therapeutic interventions against multiple diseases including cancer. Here, we reviewed the fundamental characteristics, memory development and regulatory mechanism of NK cell memory. Moreover, we will conduct a comprehensive evaluation of the accomplishments, obstacles, and future direction pertaining to the utilization of NK cell memory in the field of cancer immunotherapy.