Showing papers on "Cauliflower mosaic virus published in 2016"
TL;DR: It is found that Arabidopsis plants infected with Cauliflower mosaic virus or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity.
Abstract: Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.
92 citations
TL;DR: MtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants and may act in a similar fashion to RDR6.
Abstract: Host RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1. In MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level. MtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.
60 citations
TL;DR: This approach provides opportunities for the high-throughput transformation of S. viridis and potentially facilitates translational research in a monocot model plant.
Abstract: Traditional method of Agrobacterium-mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium-mediated genetic transformation of S. viridis using spike dip. Pre-anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5-day-old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025% Silwet L-77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β-glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron-interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high-throughput transformation and potentially facilitates translational research in a monocot model plant.
56 citations
TL;DR: This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P 6 in intracellular movement.
Abstract: The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.
42 citations
TL;DR: The results presented here demonstrate the feasibility of improving high temperature and oxidative stress tolerance in plants through the expression of LeHSP21 gene.
Abstract: It is well established that small heat shock proteins (sHSPs) play an important role in thermotolerance in various organisms due to their abundance and diversity. In the present study, a chloroplast small heat shock protein gene (LeHSP21) from tomato (Lycopersicon esculentum cv PKM-1) was constitutively expressed in tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants via Agrobacterium-mediated transformation. When compared to wild-type control plants, transgenic tobacco plants constitutively expressing LeHSP21, driven by the cauliflower mosaic virus 35S promoter, exhibited improved tolerance to both high temperature and oxidative stress. Furthermore, when the seedlings were subjected to high temperature treatment, the activities of anti-oxidative enzymes and the content of proline were significantly higher in transgenic plants than those in the wild-type plants. Our results presented here demonstrate the feasibility of improving high temperature and oxidative stress tolerance in plants through the expression of LeHSP21 gene.
31 citations
TL;DR: Results indicate the potential for engineering the trafficking properties of a transporter to produce plants that are tolerant to mineral stress and suggest that BOR1(K590A)-GFP-HPT confers tolerance to excess-B by excluding B from the cytosol of shoot cells.
Abstract: Boron (B) is a metalloid that is essential for plant growth but is toxic when present in excess. Arabidopsis BOR1 is a borate exporter, facilitating B translocation from root to shoot under limited-B conditions. BOR1 shows stele side polar localization in the plasma membrane of various root cells, presumably to support B translocation toward the stele. BOR1 is degraded under high-B supply through vacuolar sorting via ubiquitination at the K590 residue to prevent the accumulation of B to a toxic level in shoots. A previous study showed that overexpression of BOR1 under control of the cauliflower mosaic virus 35S RNA promoter improved the growth of Arabidopsis under limited-B conditions without affecting the growth under sufficient-to-excess-B conditions. In this study, we unexpectedly found that ubiquitous expression of a stabilized BOR1 variant improved tolerance to excess-B in Arabidopsis. We established transgenic plants expressing BOR1-GFP fused with hygromycin phosphotransferase (HPT) and BOR1(K590A)-GFP-HPT under control of the ubiquitin 10 promoter. BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were expressed in various cell types in leaves and roots and showed weak polar localization in root tip cells. BOR1-GFP-HPT, but not BOR1(K590A)-GFP-HPT, was degraded through an endocytic pathway under high-B conditions. Transgenic plants with the stabilized variant BOR1(K590A)-GFP-HPT showed improved root and shoot growth under excess-B conditions. The concentration of B was greater in the shoots of plants with BOR1(K590A)-GFP-HPT or BOR1-GFP-HPT than in those of untransformed wild-type plants. These results suggest that BOR1(K590A)-GFP-HPT confers tolerance to excess-B by excluding B from the cytosol of shoot cells. Results from this study indicate the potential for engineering the trafficking properties of a transporter to produce plants that are tolerant to mineral stress.
22 citations
TL;DR: Gene expression of SlAKR4B was induced by NaCl, H2O2, and plant hormones such as salicylic acid and jasmonic acid, suggesting that SlAKr4B is involved in the stress response.
21 citations
TL;DR: Transgenic lettuce plants with a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.
Abstract: We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery.
We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.
19 citations
TL;DR: The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself.
16 citations
TL;DR: It is suggested that a physical interaction between MPs andPDLPs is not required for assembly of the movement tubule and that the beneficial role of PDLPs in virus movement is confined to the structural context of the PD.
Abstract: Intercellular spread of plant viruses involves passage of the viral genome or virion through a plasmodesma (PD). Some viruses severely modify the PD structure, as they assemble a virion carrying tubule composed of the viral movement protein (MP) inside the PD channel. Successful modulation of the host plant to allow infection requires an intimate interaction between viral proteins and both structural and regulatory host proteins. To date, however, very few host proteins are known to promote virus spread. Plasmodesmata-located proteins (PDLPs) localised in the PD have been shown to contribute to tubule formation in cauliflower mosaic virus and grapevine fanleaf virus infections. In this study, we have investigated the role of PDLPs in intercellular transport of another tubule-forming virus, cowpea mosaic virus. The MP of this virus was found to interact with PDLPs in the PD, as was shown for other tubule-forming viruses. Expression of PDLPs and MPs in protoplasts in the absence of a PD revealed that these proteins do not co-localise at the site of tubule initiation. Furthermore, we show that tubule assembly in protoplasts does not require an interaction with PDLPs at the base of the tubule, as has been observed in planta. These results suggest that a physical interaction between MPs and PDLPs is not required for assembly of the movement tubule and that the beneficial role of PDLPs in virus movement is confined to the structural context of the PD.
16 citations
TL;DR: The PeNAC1 promoter is a non-tissue-specific and stress-inducible promoter containing a GA-responsive element and a MYB recognition sequence that are responsible for induced expression patterns and the presence of a putative NAC protein-binding motif in the PeN AC1 promoter indicates that PeNac1 is either regulated by other NAC transcription factors or is self-regulated.
Abstract: The PeNAC1
promoter is a non-tissue-specific and stress-inducible promoter containing a GA-responsive element and a MYB recognition sequence that are responsible for induced expression patterns.
NAC transcription factors play vital roles in complex signaling networks during plant stress responses. Promoters as crucial molecular switches are involved in the transcriptional regulation of gene activities dynamic network controlling a variety of biological processes, such as developmental processes, responses to hormone and abiotic stress. In this study, a 1217-bp flanking fragment of the stress-responsive NAC gene PeNAC1 was isolated from Populus euphratica. In transgenic Arabidopsis, this promoter fragment was found to have a higher activity than the cauliflower mosaic virus 35S promoter and remained active throughout the plant life cycle, particularly in the spiral vessels and cortical cells of vascular tissues of various organs. We identified a gibberellic acid-responsive element, required for response to gibberellic acid and involved in the salt-stress signaling pathway, and a MYB recognition sequence, which has an important role in promoter response to drought stress, in the PeNAC1 promoter. These results suggest that the PeNAC1 promoter is more effective, non-tissue-specific, and inducible. In addition, the presence of a putative NAC protein-binding motif in the PeNAC1 promoter indicates that PeNAC1 is either regulated by other NAC transcription factors or is self-regulated. Our research will help reveal the regulatory mechanism of the upstream region of the PeNAC1 gene and provide a foundation for the use of the PeNAC1 promoter in molecular breeding.
TL;DR: Full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter are constructed, which will greatly enhance the reverse genetic studies ofCCYV gene functions.
Abstract: Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.
TL;DR: The histochemical and fluorometric measurement of GUS activity and the gene expression quantification by RT-qPCR analysis demonstrate that the DMMV promoter is able to direct gene expression in citrus as strongly as the D35S promoter and represents great application potential in citrus biotechnology.
Abstract: Four caulimovirus-derived constitutive promoters were evaluated for gene expression in citrus and their expression levels were compared with a 35S promoter. Chimeric promoters made with duplicated enhancer sequences from the cauliflower mosaic virus (D35S), the cassava vein mosaic virus (DCsVMV), the figwort mosaic virus (DFMV), the mirabilis mosaic virus (DMMV) and the peanut chlorotic streak virus (DPCLSV) were inserted into a transformation vector fused to the gus reporter gene. Gene expression patterns driven by these promoters were analyzed in the transgenic citrus cultivar Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.). The histochemical and fluorometric measurement of GUS activity and the gene expression quantification by RT-qPCR analysis demonstrate that the DMMV promoter is able to direct gene expression in citrus as strongly as the D35S promoter and represents great application potential in citrus biotechnology. The DFMV, DCsVMV and DPCLSV constitutive promoters were weaker compared to the D35S promoter but can be considered for use in gene stacking strategies for the development of transgenic citrus. Our results also reveal the importance of the evaluation of specific promoter fragments for a particular crop cultivar due to the availability of species-specific transcription factors that can define the strength and tissue specificity of a determinate promoter.
TL;DR: The results implicate PtrCesA4 promoter as a good genetic tool for controlling gene function in wood development and tree molecular breeding.
Abstract: An efficient shoot regeneration method was developed using leaf explants of Populus alba × P. glandulosa, and the optimized shoot regeneration medium contained 0.1 mg L−1 NAA, 0.5 mg L−1 6-BA and 0.002 mg L−1 TDZ. The factors for Agrobacterium-mediated transformation of this hybrid were further optimized as follows: no preculture step, Agrobacterium cell suspension with an OD600 of 0.6, 20 min infection time and 2d co-cultivation duration. To investigate Populus trichocarpa cellulose synthase A4 promoter (PtrCesA4pro), we constructed the PtrCesA4pro:: β-glucuronidase (GUS) binary vector, in which the cauliflower mosaic virus 35S (CaMV35S) promoter of pBI121was replaced with the PtrCesA4pro to drive the expression of a GUS reporter gene. Forty-eight kanamycin-resistant plantlets were obtained through Agrobacterium-mediated transformation of this hybrid. Genomic DNA PCR and Southern blot analyses confirmed the integration of PtrCesA4pro::GUS into this hybrid and the expression of GUS gene was driven by the PtrCesA4 promoter. A histochemical GUS staining presented the activity of PtrCesA4 promoter in the transgenic lines. No GUS signal was observed in leaves, including the veins and petioles, and notably weak staining was detected in the secondary xylems of the roots. The PtrCesA4 promoter was highly active in the fibers, the vessels of developing xylems, and the fibers of mature phloems. Surprisingly, GUS staining was detected in the cambial cells of the highly lignified stems of these transgenic trees. Our results implicate PtrCesA4 promoter as a good genetic tool for controlling gene function in wood development and tree molecular breeding.
TL;DR: Results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes.
TL;DR: Transgenic lines expressing a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces, suggesting an inhibitory effect of Ph PH4 repressor on anthocyanin synthesis.
Abstract: The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3'-hydroxylase (F3'H) and Flavonoid 3'5'-hydroxylase (F3'5'H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis.
23 Feb 2016
TL;DR: Results demonstrate that RsMYB1 has potential to enhance anthocyanin content in the chrysanthemum cultivars under the control of the cauliflower mosaic virus 35S promoter.
Abstract: A MYB transcription factor gene, RsMYB1, from radish was introduced into the chrysanthemum cultivars 'Peach ND', 'Peach Red', and 'Vivid Scarlet' under the control of the cauliflower mosaic virus 35S promoter. Presence of RsMYB1 in transgenic lines was confirmed using polymerase chain reaction (PCR). Results of reverse-transcription-PCR analysis revealed that the expression of RsMYB1 was stable in all transgenic lines and could enhance the expression levels of three key biosynthetic genes (F3H, DFR, and ANS) involved in anthocyanin production. Accordingly, anthocyanin content was significantly higher in transgenic lines than in control of all the cultivars, although the increasement was not visually observed in any of the transgenic lines. Therefore, these results demonstrate that RsMYB1 has potential to enhance anthocyanin content in the chrysanthemums.
TL;DR: This is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum, and there was no variation in ploidy level between transgenic plants and donor plants (non-transformants).
Abstract: This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the mornin...
TL;DR: It is reported that SODIUM POTASSIUM ROOT DEFECTIVE3 (NaKR3), which encodes a heavy metal-associated domain protein, is involved in salt tolerance in Arabidopsis.
Abstract: Salinity is a major abiotic stress in agriculture. Here, we report that SODIUM POTASSIUM ROOT DEFECTIVE3 (NaKR3), which encodes a heavy metal-associated domain protein, is involved in salt tolerance in Arabidopsis. The results of quantitative reverse transcription-polymerase chain reaction analysis revealed that NaKR3 was induced by high salinity and osmotic stresses, but not by Cu(2+) stress. Transient expression of NaKR3-GFP in Arabidopsis protoplasts showed that the NaKR3 protein was localized in the cytosol. Transgenic Arabidopsis plants constitutively expressing NaKR3 under the control of the cauliflower mosaic virus 35S promoter exhibited increased tolerance to salt treatment. Furthermore, overexpression of NaKR3 increased the expression of SOS1 and SOS3, but decreased the accumulation of salt-induced proline. Taken together, our results indicate that NaKR3 is involved in the salt stress response in Arabidopsis.
TL;DR: The marker-free transformed plants produced displayed increased resistance against bacterial and fungus phytopathogens, while their extracts were characterized by antimicrobial activity for human and animal pathogens, and may be used as producers of cecropin P1 in pharmaceutics.
Abstract: Kalanchoe pinnate (Kalanchoe pinnata L. ) plants with synthetic gene of antimicrobial peptide cecropin P1 (CP1) under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) were produced. For transformation, a modified binary vector not containing selective genes of tolerance against antibiotics and herbicides was used. Screening of the marker-free transformed plants was conducted on the medium without selective antibiotics by revealing antibacterial activity of plant extracts and cecropin P1. The marker-free plants produced displayed increased resistance against bacterial and fungus phytopathogens, while their extracts were characterized by antimicrobial activity for human and animal pathogens. These plants meet the requirements of biosafety and may be used as producers of cecropin P1 in pharmaceutics.
TL;DR: Generation of infectious clones of PSV-P enables studies on virus-host interaction as well as revealing viral genes functions, and results obtained resulted in analogous results.
TL;DR: In this paper, the expression traits of the cotton ( Gossypium hirsutum ) translation elongation factor 1A gene family were analyzed and fused to the β-glucuronidase (GUS) reporter gene to characterize its expression pattern in tobacco.
TL;DR: This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11, a cytokine that plays a key regulatory role in the immune system.
Abstract: Interleukin-11 (IL-11) is a cytokine that plays a key regulatory role in the immune system. Recombinant human IL-11 (rhIL-11) exerts a preventative effect against apoptotic cell death and inhibits preadipocyte differentiation. IL-11 also is used to stimulate the bone marrow to produce platelets in order to prevent low platelets that may be caused by chemotherapy. Unfortunately, the high production cost of IL-11 associated. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-11. Production of rhIL-11 proteins in whole-plant expression system will be more economical when compared to the current E. coli based expression system. The human rhIL-11 gene was codon optimized to maximize plant host system expression. IL-11 expression vector under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into tobacco by Agrobacteriummediated transformation. The 5′-leader sequence (called O) of tobacco mosaic virus (TMV) as a translational enhancer was added to construct. Transgenic tobacco plants expressing various levels of rhIL-11 protein were generated. Western blotting of the stably transformed lines demonstrated accumulation of the appropriately sized rhIL-11 protein in leaves. This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11. © Korean Society for Plant Biotechnology.
TL;DR: This is the first comprehensive study of viruses on Brassica crops in the Black Sea Region of Turkey, the most important centre for cabbage production in Turkey, using virus-specific polyclonal antiserum to determine the distribution of viruses.
Abstract: In Turkey, cabbage is the most economically important member of the genus Brassica. Samsun, located in the Black Sea Region is the greatest cabbage producing area in Turkey. Surveys were done to determine the distribution of viruses in cabbage cultivated areas in 2012–2014. According to the results of field observations, characteristic virus symptoms were observed on some cabbage plants. The average percentages of symptoms-affected plants were recorded as 2–20% in the visited fields. A total of 535 leaf samples were collected from cabbage fields and tested for the presence of Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Turnip yellow mosaic virus (TYMV), and Beet western yellows virus (BWYV) by double-antibody sandwich Enzyme-linked immunosorbent assay (DAS-ELISA) using virus-specific polyclonal antiserum. The ELISA results revealed that 8.1% and 6.9% of these samples were infected with TuMV and CaMV, respectively. The TuMV+CaMV mixed infection ratio wa...
TL;DR: The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.
Abstract: A method was developed for the production and purification of biologically active recombinant human interferon α-2b (rhIFN α-2b) synthesized by expression in Nicotiana benthamiana plants. A gene construct containing a modified hIFN α-2b gene was cloned in two vectors based on tobacco mosaic virus driven by an actin promoter from Arabidopsis thaliana (pA-IFN-A) and cauliflower mosaic virus driven by a 35S promoter (pA-IFN-S). The expression vectors were introduced into the plant cells by agroinfiltration. The maximum rates of synthesis achieved in the case of pA-IFN-A and pA-IFN-S 5 days after agroinfiltration were determined to be 200 and 20 mg per 1 kg of fresh leaves, respectively. The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.
TL;DR: The feasibility of controlling peanut stem necrosis disease caused by Tobacco streak virus (TSV) in groundnut (Arachis hypogaea L.) was explored by expressing double-stranded RNA of the replicase (Rep) gene of TSV inGroundnut through genetic engineering.
Abstract: The feasibility of controlling peanut stem necrosis disease caused by Tobacco streak virus (TSV) in groundnut (Arachis hypogaea L.) was explored by expressing double-stranded RNA of the replicase (Rep) gene of TSV in groundnut through genetic engineering. A hairpin (hp) RNAi construct containing 535-bp sense and antisense TSV-Rep sequences flanking a 742-bp spacer sequence (Pdk intron) under the control of the constitutive Cauliflower mosaic virus 35S promoter was made in the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Cotyledon explants of groundnut cultivar TMV-7 were transformed with A. tumefaciens harboring the hpRNA cassette. The presence of the transgene in the transgenic plants was confirmed up to T3 generation by PCR amplification of the 535-bp fragment of TSV-Rep gene. The bioassay results indicated that necrotic lesions were observed on the leaves of the wild-type plants 7–9 days after inoculation with TSV and stem necrosis appeared 16–20 days after inoculation, whereas the transgenic plants did not develop symptoms until harvest. ELISA results indicated that the wild-type plants inoculated with TSV recorded the highest virus concentration as compared to the transgenic lines.
01 Jan 2016
TL;DR: The authors analyzed the constitutive expression of the s-Glucuronidase (GUS) reporter gene fused to three promoters: the cauliflower mosaic virus (CaMV) 35S, the chimerical A9 promoter which contains rice Act1, and the Ubiquitine-1 promoter from maize.
Abstract: This work analyzed the constitutive expression of the s-Glucuronidase (GUS) reporter gene fused to three promoters: the cauliflower mosaic virus (CaMV) 35S, the chimerical A9 promoter which contains rice Act1, and the Ubiquitine-1 promoter from maize. The activity of the promoters was qualitative and quantitatively obtained in different tissues and various growth stages of rice plants (cv J-104) transformed by biolistic. All the promoters were found to be active, with distinct patterns of relative activity in leaves, stem and roots from in vitro and ex vitro plants, and in plants of T1 progeny. The chimerical A9 promoter increased significantly levels of GUS expression in all the tissues and at all growth stages of the plants.
TL;DR: Results showed that organ specific expression in potato led to nearly 3 fold higher hCT accumulation than constitutive expression and Kardal cultivar expressed hCT about 2 times higher than Marfona cultivar.
Abstract: Calcitonin (CT), a 32 amino acid polypeptide hormone is a powerful and specific inhibitor of bone resorption and is used to treat several human diseases like hypercalcemia and osteoporosis. To date, many pharmaceutical proteins of mammalian origin have been synthesized in plants. To increase the production level of heterologous proteins in plants, strategies such as choice of stronger promoters and optimization of codon usage are of major concern. In this study, a human calcitonin (hCT) gene, driven by two different promoters (Patatin Class I and Cauliflower mosaic virus 35S) was expressed in two types of potato's cultivars (cvs) Kardal and Marfona plants, using Agrobacterium-mediated transformation. The transgenic plants were analyzed by molecular methods and hCT concentration was determined by quantitative EASIA. The results showed the localization of hCT production in Kardal potato tubers led to 0.7% of total soluble proteins whiles total soluble protein was 0.2% when CaMV 35S promoter was deployed, these results of Marfona when used Patatin Class I and Cauliflower mosaic virus 35S promoters were 0.3% of the total soluble protein and 0.1% of the total soluble protein in the respect. Data were analyzed by SPSS software using analytical statistics. There was significant difference in mean score of hCT production when two different promoters and two different cultivars were used. These results showed that organ specific expression in potato led to nearly 3 fold higher hCT accumulation than constitutive expression and Kardal cultivar expressed hCT about 2 times higher than Marfona cultivar.
01 Jan 2016
TL;DR: The results suggested that the GhEF1A8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.
Abstract: The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene expression and/or tissue-specific expression patterns in plants.To obtain promoter sequences of plants with new properties,we analyzed the expression traits of the cotton(Gossypium hirsutum)translation elongation factor 1A gene family.The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants,and showed much higher transcript levels in stems and leaves.Its promoter(GhEF1A1.7)and the 5’untranslated region(5’UTR),comprising a regulatory region named PGhEF1A8,were isolated from cotton and studied in stably transformed tobacco plants.The regulatory region sequences were fused to theβ-glucuronidase(GUS)reporter gene to characterize its expression pattern in tobacco.Histochemical and fluorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco,including leaves,stems,flowers,and roots.The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus(CaMV)35S promoter::GUS plants,but as same as CaMV 35S promoter::GUS plants in flower and root tissues.GUS expression levels decreased 2-10-fold when the 5’UTR was absent from PGhEF1A8.Deletion analysis of the PGhEF1A8 sequence showed that the region-647to-323 might possess negative elements that repress transgene expression in tobacco plants.The results suggested that the GhEF1A8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.
Journal Article•
TL;DR: Transgenic N. benthamiana engineered with dehD from Rhizobium sp.
Abstract: A dehalogenase D gene (dehD) capable of degrading monochloroacetic acid (MCA) has been previously isolated from Rhizobium sp. RC1 and characterized. The 804-bp dehD gene was inserted into pCAMBIA under the control of the cauliflower mosaic virus 35S promoter and designated pCAMdehD, with a total size of 10,592 bp. The pCAMdehD was introduced into tobacco (Nicotiana benthamiana) via Agrobacterium-mediated transformation. The integration and expression of dehD in N. benthamiana was confirmed by PCR and reverse transcription PCR, respectively. MCA-resistant transformants were selected in tissue cultures containing 60 µg/L MCA. Analysis of plants using a leaf-painting assay revealed that transgenic N. benthamiana was resistant to 4.0 g/L MCA compared with 2.0 g/L for non-transformed controls. The use of dehD could thus be advantageous for herbicide-resistant plant breeding systems, and it is also a suitable marker gene for plant-transformation studies. To the best of our knowledge, this is the first report detailing transgenic N. benthamiana engineered with dehD from Rhizobium sp. RC1.