RESUMO
Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Células Vegetais , Temperatura , Melhoramento Vegetal , Plantas Geneticamente Modificadas/genética , Genoma de PlantaRESUMO
Chrysanthemum morifolium is one of the most popular ornamental plants in the world. However, as C. morifolium is a segmental hexaploid, self-incompatible, and has a sizable heterologous genome, it is difficult to modify its trait systematically. Genome editing technology is one of the attractive methods for modifying traits systematically. For the commercial use of genetically modified C. morifolium, rigorous stabilization of its quality is essential. This trait stability can be achieved by avoiding further genome modification after suitable trait modification by genome editing. Since C. morifolium is a vegetatively propagated plant, an approach for removing genome editing tools is required. In this study, we attempted to use the piggyBac transposon system to remove specific DNA sequences from the C. morifolium genome. Using the luminescence as a visible marker, we demonstrated that inoculation of Agrobacterium harboring hyperactive piggyBac transposase removes inserted 2.6 kb DNA, which harbors piggyBac recognition sequences, from the modified Eluc sequence.
RESUMO
Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.
RESUMO
Insect pests cause serious damage in crop production, and various attempts have been made to produce insect-resistant crops, including the expression of genes for proteins with anti-herbivory activity, such as Bt (Bacillus thuringiensis) toxins. However, the number of available genes with sufficient anti-herbivory activity is limited. MLX56 is an anti-herbivory protein isolated from the latex of mulberry plants, and has been shown to have strong growth-suppressing activity against the larvae of a variety of lepidopteran species. As a model of herbivore-resistant plants, we produced transgenic tomato lines expressing the gene for MLX56. The transgenic tomato lines showed strong anti-herbivory activities against the larvae of the common cutworm, Spodoptera litura. Surprisingly, the transgenic tomato lines also exhibited strong activity against the attack of western flower thrips, Frankliniera occidentalis. Further, growth of the hadda beetle, Henosepilachna vigintioctopunctata, fed on leaves of transgenic tomato was significantly retarded. The levels of damage caused by both western flower thrips and hadda beetles were negligible in the high-MLX56-expressing tomato line. These results indicate that introduction of the gene for MLX56 into crops can enhance crop resistance against a wide range of pest insects, and that MLX56 can be utilized in developing genetically modified (GM) pest-resistant crops.
Assuntos
Expressão Gênica , Látex , Morus/genética , Proteínas de Plantas , Plantas Geneticamente Modificadas , Solanum lycopersicum , Animais , Bacillus thuringiensis , Insetos , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/parasitologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/parasitologiaRESUMO
Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.
RESUMO
Here, we report the complete genome sequences of two strains of Xanthomonas campestris pv. campestris (MAFF106712 and MAFF302021), which cause black rot in crucifer crops, isolated from Chinese cabbage and cauliflower, respectively, in Japan. The MAFF106712 chromosome was 5,002,720 bp, with a G+C content of 65.2%, and harbored one plasmid of 78,747 bp. The MAFF302021 chromosome was 5,048,651 bp, with a G+C content of 65.1%.
RESUMO
Jasmonic acid (JA) plays an important role in the induction of herbivore resistance in many plants. However, JA-independent herbivore resistance has been suggested. An herbivore-resistance-inducing substance was isolated from Tobacco mosaic virus-infected tobacco (Nicotiana tabacum) leaves in which a hypersensitive response (HR) was induced and identified as loliolide, which has been identified as a ß-carotene metabolite. When applied to tomato (Solanum lycopersicum) leaves, loliolide decreased the survival rate of the two-spotted spider mite, Tetranychus urticae, egg deposition by the same pest, and the survival rate of larvae of the common cutworm Spodoptera litura without exhibiting toxicity against these herbivores. Endogenous loliolide levels increased not only with an infestation by S litura larvae, but also with the exogenous application of their oral secretions in tomato. A microarray analysis identified cell-wall-associated defense genes as loliolide-responsive tomato genes, and exogenous JA application did not induce the expression of these genes. Suppressor of zeaxanthin-less (szl), an Arabidopsis (Arabidopsis thaliana) mutant with a point mutation in a key gene of the ß-carotene metabolic pathway, exhibited the decreased accumulation of endogenous loliolide and increased susceptibility to infestation by the western flower thrip (Frankliniella occidentalis). A pretreatment with loliolide decreased susceptibility to thrips in the JA-insensitive Arabidopsis mutant coronatine-insensitive1 Exogenous loliolide did not restore reduced electrolyte leakage in szl in response to a HR-inducing bacterial strain. These results suggest that loliolide functions as an endogenous signal that mediates defense responses to herbivores, possibly independently of JA, at least in tomato and Arabidopsis plants.
Assuntos
Benzofuranos/metabolismo , Herbivoria , Nicotiana/química , Animais , Arabidopsis/efeitos dos fármacos , Benzofuranos/química , Benzofuranos/isolamento & purificação , Carotenoides/metabolismo , Morte Celular , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/parasitologia , Spodoptera/fisiologia , Tetranychidae/fisiologia , Nicotiana/virologia , Vírus do Mosaico do TabacoRESUMO
The phylloplane yeast Pseudozyma antarctica secretes an esterase, named PaE, and xylanase when cultivated with xylose. We previously observed that the lipophilic layer of Micro-Tom tomato leaves became thinner after the culture filtrate treatment. The leaves developed reduced water-holding ability and became wilted. In this study, the purified enzymes were spotted on Micro-Tom leaves. PaE, but not xylanase, thinned the lipophilic layer of leaves and decreased leaf resistance to the phytopathogenic fungus Botrytis cinerea. Disease severity increased significantly in detached leaves and potted plants treated with the culture filtrate and B. cinerea spores compared with those treated with inactivated enzyme and B. cinerea alone. Spore germination ratios, numbers of penetrating fungal hyphae in the leaves, and fungal DNA contents also increased significantly on the detached leaves. Japanese knotweed (Fallopia japonica), a serious invasive alien weed in Europe and North America, also became susceptible to infection by the rust pathogen Puccinia polygoni-amphibii var. tovariae following the culture filtrate treatment. The culture filtrate treatment increased disease development in plants induced by both phytopathogenic fungi. Our results suggest that P. antarctica culture filtrate could be used as an adjuvant for sustainable biological weed control using phytopathogenic fungi.
Assuntos
Agentes de Controle Biológico , Esterases/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/prevenção & controle , Ustilaginales/fisiologia , Agentes de Controle Biológico/administração & dosagem , Esterases/administração & dosagem , Esterases/isolamento & purificação , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/isolamento & purificação , Solanum lycopersicum , Fenótipo , Desenvolvimento Vegetal/efeitos dos fármacos , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologiaRESUMO
Protein introduction into cells is more difficult in plants than in mammalian cells, although it was reported that protein introduction was successful in shoot apical meristem and leaves only together with a cell-penetrating peptide. In this study, we tried to introduce superfolder green fluorescent protein (sGFP)-fused to adenylate cyclase as a reporter protein without a cell-penetrating peptide into the cells of tobacco leaves by treatment with atmospheric non-thermal plasmas. For this purpose, CO2 or N2 plasma was generated using a multi-gas plasma jet. Confocal microscopy indicated that sGFP signals were observed inside of leaf cells after treatment with CO2 or N2 plasma without substantial damage. In addition, the amount of cyclic adenosine monophosphate (cAMP) formed by the catalytic enzyme adenylate cyclase, which requires cellular calmodulin for its activity, was significantly increased in leaves treated with CO2 or N2 plasma, also indicating the introduction of sGFP-fused adenylate cyclase into the cells. These results suggested that treatment with CO2 or N2 plasma could be a useful technique for protein introduction into plant tissues.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Meristema/metabolismo , Folhas de Planta/metabolismo , Gases em Plasma , Pressão , Adenilil Ciclases/genética , Dióxido de Carbono/química , Proteínas de Fluorescência Verde/genética , Nitrogênio/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Wilt disease in plants, which is caused by the soil-borne bacterial pathogen Ralstonia solanacearum, is one of the most devastating plant diseases. We previously detected bacterial wilt disease-inhibiting activity in an extract from yeast cells. In the present study, we purified this activity and identified one of the substances responsible for the activity as the amino acid histidine. The exogenous application of l-histidine, but not d-histidine, inhibited wilt disease in tomato and Arabidopsis plants without exhibiting any antibacterial activity. l-Histidine induced the expression of genes related to ethylene (ET) biosynthesis and signaling as well as the production of ET in tomato and Arabidopsis plants. l-Histidine-induced resistance to R. solanacearum was partially abolished in ein3-1, an ET-insensitive Arabidopsis mutant line. Resistance to the fungal pathogen Botrytis cinerea, which is known to require ET biosynthesis or signaling, was also induced by exogenously applied l-histidine. These results suggest that l-histidine induces resistance to R. solanacearum and B. cinerea partially through the activation of ET signaling in plants.
Assuntos
Etilenos/metabolismo , Histidina/farmacologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/patogenicidade , Solanum lycopersicum/microbiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/microbiologia , Resistência à Doença/efeitos dos fármacos , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/metabolismo , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Transdução de Sinais/efeitos dos fármacos , Leveduras/químicaRESUMO
The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/genética , Nicotiana/genética , Ozônio/metabolismo , Proteínas de Plantas/genética , Estômatos de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Nicotiana/enzimologia , Nicotiana/metabolismoRESUMO
Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.
Assuntos
Esterases/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Ustilaginales/enzimologia , Arabidopsis , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas , Solanum lycopersicum , Folhas de Planta/química , Ustilaginales/crescimento & desenvolvimentoRESUMO
To understand the role of the rice flavonoid phytoalexin (PA) sakuranetin for blast resistance, the fungus-responsive characteristics were studied. Young rice leaves in a resistant line exhibited hypersensitive reaction (HR) within 3 days post inoculation (dpi) of a spore suspension, and an increase in sakuranetin was detected at 3 dpi, increasing to 4-fold at 4 dpi. In the susceptible line, increased sakuranetin was detected at 4 dpi, but not at 3 dpi, by which a large fungus mass has accumulated without HR. Induced expression of a PA biosynthesis gene OsNOMT for naringenin 7-O-methyltransferase was found before accumulation of sakuranetin in both cultivars. The antifungal activity of sakuranetin was considerably higher than that of the major rice diterpenoid PA momilactone A in vitro and in vivo under similar experimental conditions. The decrease and detoxification of sakuranetin were detected in both solid and liquid mycelium cultures, and they took place slower than those of momilactone A. Estimated local concentration of sakuranetin at HR lesions was thought to be effective for fungus restriction, while that at enlarged lesions in susceptible rice was insufficient. These results indicate possible involvement of sakuranetin in blast resistance and its specific relation to blast fungus.
Assuntos
Antifúngicos/metabolismo , Flavonoides/metabolismo , Fungos/metabolismo , Interações Hospedeiro-Patógeno , Oryza/metabolismo , Oryza/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Antifúngicos/farmacologia , Resistência à Doença , Flavonoides/farmacologia , Fungos/efeitos dos fármacos , Inativação Metabólica , Testes de Sensibilidade Microbiana , Oryza/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Transcrição GênicaRESUMO
Nicotiana tabacum (tobacco) cultivars possessing the N resistance gene to Tobacco mosaic virus (TMV) induce a hypersensitive response, which is accompanied by the production of phytohormones such as salicylic acid (SA) and jasmonic acid (JA), to enclose the invaded virus at the initial site of infection, which inhibits viral multiplication and spread. SA functions as a positive regulator of TMV resistance. However, the role of JA in TMV resistance has not been fully elucidated. Exogenously applied methyl jasmonate, a methyl ester of JA, reduced local resistance to TMV and permitted systemic viral movement. Furthermore, in contrast to a previous finding, we demonstrated that silencing of CORONATINE-INSENSITIVE 1 (COI1), a JA receptor, reduced viral accumulation in a tobacco cultivar possessing the N gene, as did that of allene oxide synthase, a JA biosynthetic enzyme. The reduction in viral accumulation in COI1-silenced tobacco plants was correlated with an increase in SA, and lowering SA levels by introducing an SA hydroxylase gene attenuated this reduction. Viral susceptibility did not change in a COI1-silenced tobacco cultivar lacking the N gene. These results suggest that JA signaling is not directly responsible for susceptibility to TMV, but is indirectly responsible for viral resistance through the partial inhibition of SA-mediated resistance conferred by the N gene, and that a balance between endogenous JA and SA levels is important for determining the degree of resistance.
Assuntos
Ciclopentanos/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/virologia , Oxilipinas/farmacologia , Proteínas de Plantas/metabolismo , Vírus do Mosaico do Tabaco/patogenicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Nicotiana/genéticaRESUMO
Salicylic acid (SA) plays a key role in plant resistance to pathogens. Accumulation of SA is induced by wounding in tobacco plants in which the expression of WIPK and SIPK, two mitogen-activated protein kinases, is suppressed. Here, the mechanisms underlying the abnormal accumulation of SA in WIPK/SIPK-suppressed plants have been characterized. SA accumulation started around 12 h after wounding and was inhibited by cycloheximide (CHX), a protein synthesis inhibitor. SA accumulation, however, was enhanced several fold when leaf discs were transferred onto CHX after floating on water for ≥6 h. Temporal and spatial analyses of wound-induced and CHX-enhanced SA accumulation suggested that wounding induces activators for SA accumulation followed by the generation of repressors, and late CHX treatment inhibits the production of repressors more efficiently than that of activators. Microarray analysis revealed that the expression of many disease resistance-related genes, including N, a Resistance (R) gene for Tobacco mosaic virus and R gene-like genes, was up-regulated in wounded WIPK/SIPK-suppressed plants. Expression of the N gene and R gene-like genes peaked earlier than that of most other genes as well as SA accumulation, and was mainly induced in those parts of leaf discs where SA was highly accumulated. Moreover, wound-induced SA accumulation was decreased by the treatments which compromise the function of R proteins. These results indicate that signaling leading to the expression of disease resistance-related genes is activated by wounding in WIPK/SIPK-suppressed plants, and induction of R gene and R gene-like genes might lead to the biosynthesis of SA.
Assuntos
Perfilação da Expressão Gênica , Genes de Plantas/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Ácido Salicílico/metabolismo , Supressão Genética , Benzoquinonas/farmacologia , Cicloeximida/farmacologia , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Temperatura Alta , Lactamas Macrocíclicas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Supressão Genética/efeitos dos fármacos , Nicotiana/efeitos dos fármacos , Nicotiana/enzimologia , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction pathways in eukaryotic cells. In tobacco, two MAPK, wound-induced protein kinase (WIPK) and salicylic acid (SA)-induced protein kinase (SIPK), are activated by biotic and abiotic stresses. Both WIPK and SIPK positively regulate the biosynthesis of jasmonic acid (JA) or ethylene (ET) while negatively regulating SA accumulation. We showed previously that recombinant tobacco MAPK phosphatase (NtMKP1) protein dephosphorylates and inactivates SIPK in vitro, and overexpression of NtMKP1 repressed wound-induced activation of both SIPK and WIPK. To elucidate the role of NtMKP1 in response to biotic and abiotic stresses, we generated transgenic tobacco plants in which NtMKP1 expression was suppressed. Suppression of NtMKP1 expression resulted in enhanced activation of WIPK and SIPK and production of both JA and ET upon wounding. Wound-induced expression of JA- or ET-inducible genes, basic PR-1 and PI-II, was also significantly enhanced in these plants. Furthermore, NtMKP1-suppressed plants exhibited enhanced resistance against a necrotrophic pathogen, Botrytis cinerea, and lepidopteran herbivores, Mamestra brassicae and Spodoptera litura. These results suggest that NtMKP1 negatively regulates wound response and resistance against both necrotrophic pathogens and herbivorous insects through suppression of JA or ET pathways via inactivation of MAPK.
Assuntos
Botrytis/fisiologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Regulação da Expressão Gênica de Plantas , Lepidópteros/fisiologia , Nicotiana/enzimologia , Doenças das Plantas/imunologia , Animais , Ciclopentanos/análise , Ciclopentanos/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Etilenos/análise , Etilenos/metabolismo , Herbivoria , Larva , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxilipinas/análise , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/análise , Reguladores de Crescimento de Plantas/metabolismo , Imunidade Vegetal , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Ácido Salicílico/metabolismo , Estresse Fisiológico , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/fisiologiaRESUMO
Transcriptional gene silencing (TGS)--a phenomenon observed in endogenous genes/transgenes in eukaryotes--is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions-ASRs), based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume); the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements.
Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Arabidopsis/genética , Metilação de DNA , Elementos Facilitadores Genéticos , Dosagem de Genes , Estudos de Associação Genética , Fenótipo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Retroelementos , Nicotiana/genética , TransgenesRESUMO
Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of ß-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato.
Assuntos
Resistência à Doença/genética , Ralstonia solanacearum/fisiologia , Solanum lycopersicum/genética , Transcriptoma , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Interações Hospedeiro-Patógeno , Lignina/metabolismo , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Caules de Planta/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ralstonia solanacearum/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.
Assuntos
Arabidopsis/microbiologia , Diterpenos/farmacologia , Naftóis/farmacologia , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/patogenicidade , Solanum lycopersicum/microbiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Abscísico/metabolismo , Antibacterianos/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diterpenos/química , Diterpenos/isolamento & purificação , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Solanum lycopersicum/efeitos dos fármacos , Análise em Microsséries , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Naftóis/isolamento & purificação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Transdução de Sinais , Relação Estrutura-Atividade , Nicotiana/efeitos dos fármacos , Nicotiana/genéticaRESUMO
We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9ß-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.