Plant Biotechnology Journal (Plant Biotechnol)

Publications in this journal

• Article: Marker-free genetic engineering of the chloroplast in the green microalga Chlamydomonas reinhardtii.

  Hsu-Ching Chen, Anastasios Melis
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  ABSTRACT: The work applied a transgene expression method based on the replacement of an inactive rbcL gene as the selection marker in Chlamydomonas reinhardtii chloroplasts. The native rbcL gene in strain CC2653 has a point mutation that causes early translation termination, thus resulting in a photosynthesis mutant. Recovery of rbcL function for selection is offered along with the heterologous expression of the alcohol dehydrogenase ADH1 gene from Saccharomyces cerevisiae in the Chlamydomonas chloroplast. The CrCpADH1 gene was inserted via double homologous recombination in the psaB-rbcL chloroplast intergenic region of recipient strain CC2653, using the psaB and rbcL gene sequences for the double homologous recombination. This transformation conferred a functional rbcL gene and expression of the CrCpADH1 transgene in the recipient strain. This method alleviated the need to use antibiotics for selection, resulting in a negligible number of false positives during screening, and attaining a transformation efficiency greater than 90%. The approach also ensured segregation of chloroplast DNA copies, so as to achieve homoplasmy of the transformant chloroplast DNA, with a concomitant elimination of recipient strain Cp DNA. High levels of steady-state CrCpADH1 transcripts were detected in the homoplasmic transformants. However, CrCpADH1 protein levels were attenuated under continuous illumination growth conditions due to oxygen accumulation in the cells. Under conditions of low oxygen partial pressure, or anoxia, accumulation of CrCpADH1 protein in the cells and ethanol in the growth medium was observed. A metabolic pathway for ethanol production is proposed in Chlamydomonas, mediated by the chloroplast-localized CrCpADH1 transgenic enzyme.
  Plant Biotechnology Journal 05/2013;
• Article: A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

  Eva C Thuenemann, Ann E Meyers, Jeanette Verwey, Edward P Rybicki, George P Lomonossoff
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  ABSTRACT: Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.
  Plant Biotechnology Journal 05/2013;
• Article: Accelerating adoption of genetically modified crops in Africa through a trade liability regime.

  Stuart J Smyth, William A Kerr, Peter W B Phillips
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  ABSTRACT: Given the apparently unbridgeable divide that has developed between the 25 odd countries that grow and trade GM crops and the evolving EU regulatory hurdles, it may be time to consider alternative strategies for realizing a global market for agricultural products. Africa is one area of the world where the battle over GM agriculture is being played out, yet it is the continent where GM could have the greatest positive impact. Numerous African nations, given their long-standing trade connections to European nations, fear that allowing the commercialization of GM crops could lead to comingling of GM and conventional products and, hence, the loss of export opportunities to the EU. These are legitimate concerns. One potential solution that warrants serious consideration would be to establish a pool of funds that could be accessed by African agricultural commodity exporters in instances where exports to Europe are rejected. A production levy could be imposed in leading industrial adopting nations (i.e., Australia, Canada and the United States). The revenue raised would provide an endowment fund that could be used to offset the costs arising from import refusals. African-sourced shipments rejected by the EU will most certainly have alternate markets, but could receive a reduced price or incur higher costs associated with serving alternate markets. The intent of the fund would be to compensate for the real difference between the net returns contracted with European importers and the final market price received. This article examines the feasibility of establishing such a fund and discusses the funding options.
  Plant Biotechnology Journal 04/2013;
• Article: Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification.

  Yoshikazu Yuki, Mio Mejima, Shiho Kurokawa, Tomoko Hiroiwa, Yuko Takahashi, Daisuke Tokuhara, Tomonori Nochi, Yuko Katakai, Masaharu Kuroda, Natsumi Takeyama, [......], Michiyo Abe, Yingju Chen, Ushio Nakanishi, Takehiro Masumura, Yoji Takeuchi, Hiroko Kozuka-Hata, Hiroaki Shibata, Masaaki Oyama, Kunisuke Tanaka, Hiroshi Kiyono
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  ABSTRACT: Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.
  Plant Biotechnology Journal 04/2013;
• Article: Early anther ablation triggers parthenocarpic fruit development in tomato

  Medina M, Roque E, Pineda B, Cañas L, Rodriguez-Concepción M, Beltrán JP, Gómez-Mena C
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  ABSTRACT: Fruit set and fruit development in tomato is largely affected by changes in environmental conditions, therefore autonomous fruit set independent of fertilization is a highly desirable trait in tomato. Here, we report the production and characterization of male-sterile transgenic plants that produce parthenocarpic fruits in two tomato cultivars (Micro-Tom and Moneymaker). We generated male-sterility using the cytotoxic gene barnase targeted to the anthers with the PsEND1 anther-specific promoter. The ovaries of these plants grew in the absence of fertilization producing seedless, parthenocarpic fruits. Early anther ablation is essential to trigger the developing of the transgenic ovaries into fruits, in the absence of the signals usually generated during pollination and fertilization. Ovaries are fully functional and can be manually pollinated to obtain seeds. The transgenic plants obtained in the commercial cultivar Moneymaker show that the parthenocarpic development of the fruit does not have negative consequences in fruit quality. Throughout metabolomic analyses of the tomato fruits, we have identified two elite lines which showed increased levels of several health promoting metabolites and volatile compounds. Thus, early anther ablation can be considered a useful tool to promote fruit set and to obtain seedless and good quality fruits in tomato plants. These plants are also useful parental lines to be used in hybrid breeding approaches.
  Plant Biotechnology Journal 04/2013;
• Article: Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

  Yajun Li, Jiachang Zhang, Juan Zhang, Ling Hao, Jinping Hua, Liusheng Duan, Mingcai Zhang, Zhaohu Li
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  ABSTRACT: LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses.
  Plant Biotechnology Journal 04/2013;
• Article: Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits.

  Yuko Ogo, Kenjiro Ozawa, Tsutomu Ishimaru, Tsugiya Murayama, Fumio Takaiwa
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  ABSTRACT: Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.
  Plant Biotechnology Journal 04/2013;
• Article: Camelina seed transcriptome: a tool for meal and oil improvement and translational research.

  Huu T Nguyen, Jillian E Silva, Ram Podicheti, Jason Macrander, Wenyu Yang, Tara J Nazarenus, Jeong-Won Nam, Jan G Jaworski, Chaofu Lu, Brian E Scheffler, Keithanne Mockaitis, Edgar B Cahoon
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  ABSTRACT: Camelina (Camelina sativa), a Brassicaceae oilseed, has received recent interest as a biofuel crop and production platform for industrial oils. Limiting wider production of camelina for these uses is the need to improve the quality and content of the seed protein-rich meal and oil, which is enriched in oxidatively unstable polyunsaturated fatty acids that are deleterious for biodiesel. To identify candidate genes for meal and oil quality improvement, a transcriptome reference was built from 2047 Sanger ESTs and more than 2 million 454-derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of approximately 60K transcripts from 22 597 putative genes includes camelina homologues of nearly all known seed-expressed genes, suggesting a high level of completeness and usefulness of the reference. These sequences included candidates for 12S (cruciferins) and 2S (napins) seed storage proteins (SSPs) and nearly all known lipid genes, which have been compiled into an accessible database. To demonstrate the utility of the transcriptome for seed quality modification, seed-specific RNAi lines deficient in napins were generated by targeting 2S SSP genes, and high oleic acid oil lines were obtained by targeting FATTY ACID DESATURASE 2 (FAD2) and FATTY ACID ELONGASE 1 (FAE1). The high sequence identity between Arabidopsis thaliana and camelina genes was also exploited to engineer high oleic lines by RNAi with Arabidopsis FAD2 and FAE1 sequences. It is expected that these transcriptomic data will be useful for breeding and engineering of additional camelina seed traits and for translating findings from the model Arabidopsis to an oilseed crop.
  Plant Biotechnology Journal 04/2013;
• Article: RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field-grown sugarcane.

  Je Hyeong Jung, Wilfred Vermerris, Maria Gallo, Jeffrey R Fedenko, John E Erickson, Fredy Altpeter
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  ABSTRACT: The agronomic performance, cell wall characteristics and enzymatic saccharification efficiency of transgenic sugarcane plants with modified lignin were evaluated under replicated field conditions. Caffeic acid O-methyltransferase (COMT) was stably suppressed by RNAi in the field, resulting in transcript reduction of 80%-91%. Along with COMT suppression, total lignin content was reduced by 6%-12% in different transgenic lines. Suppression of COMT also altered lignin composition by reducing syringyl units and p-coumarate incorporation into lignin. Reduction in total lignin by 6% improved saccharification efficiency by 19%-23% with no significant difference in biomass yield, plant height, stalk diameter, tiller number, total structural carbohydrates or brix value when compared with nontransgenic tissue culture-derived or transgenic control plants. Lignin reduction of 8%-12% compromised biomass yield, but increased saccharification efficiency by 28%-32% compared with control plants. Biomass from transgenic sugarcane lines that have 6%-12% less lignin requires approximately one-third of the hydrolysis time or 3- to 4-fold less enzyme to release an equal or greater amount of fermentable sugar than nontransgenic plants. Reducing the recalcitrance of lignocellulosic biomass to saccharification by modifying lignin biosynthesis is expected to greatly benefit the economic competitiveness of sugarcane as a biofuel feedstock.
  Plant Biotechnology Journal 04/2013;
• Article: Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3(ixr1-2) gene in transgenic tobacco.

  Dipak K Sahoo, Jozsef Stork, Seth Debolt, Indu B Maiti
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  ABSTRACT: Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis-sense mutation in the primary cell wall-specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis-sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3(ixr1-2) derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3(ixr1-2) , codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24-CESA3(ixr1-2) displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3(ixr1-2) in tobacco. Secondly, during enzymatic saccharification, transgenic leaf- and stem-derived cellulose is 54%-66% and 40%-51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24-AtCESA3(ixr1-2) caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells.
  Plant Biotechnology Journal 04/2013; 11(3):362-72.
• Article: A global view of transcriptome dynamics during flower development in chickpea by deep sequencing.

  Vikash K Singh, Rohini Garg, Mukesh Jain
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  ABSTRACT: Measurement of gene expression can provide important clues about gene function and molecular basis of developmental processes. Here, we have analysed the chickpea transcriptome in vegetative and flower tissues by exploiting the potential of high-throughput sequencing to measure gene expression. We mapped more than 295 million reads to quantify the transcript abundance during flower development. We detected the expression of more than 90% genes in at least one tissue analysed. We found quite a large number of genes were differentially expressed during flower development as compared to vegetative tissues. Further, we identified several genes expressed in a stage-specific manner. Various transcription factor families and metabolic pathways involved in flower development were elucidated. The members of MADS-box family were most represented among the transcription factor genes up-regulated during various stages of flower development. The abundant expression of several well-known genes implicated in flower development in chickpea flower development stages confirmed our results. In addition, we detected the expression specificities of lineage-specific genes during flower development. The expression data presented in this study is the most comprehensive dataset available for chickpea as of now and will serve as resource for unraveling the functions of many specific genes involved in flower development in chickpea and other legumes.
  Plant Biotechnology Journal 04/2013;
• Article: A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism.

  Damar L López-Arredondo, Luis Herrera-Estrella
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  ABSTRACT: Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large-scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.
  Plant Biotechnology Journal 03/2013;
• Article: Activation domains for controlling plant gene expression using designed transcription factors.

  Jianquan Li, Ryan Blue, Bryan Zeitler, Tonya L Strange, Jocelynn R Pearl, David H Huizinga, Steve Evans, Philip D Gregory, Fyodor D Urnov, Joseph F Petolino
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  ABSTRACT: Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.
  Plant Biotechnology Journal 03/2013;
• Article: Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing.

  Guo-Qing Song, Kenneth C Sink, Aaron E Walworth, Meridith A Cook, Richard F Allison, Gregory A Lang
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  ABSTRACT: Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes.
  Plant Biotechnology Journal 03/2013;
• Article: Biological and biochemical characterization of HIV-1 Gag/dgp41 virus-like particles expressed in Nicotiana benthamiana.

  Sarah A Kessans, Mark D Linhart, Nobuyuki Matoba, Tsafrir Mor
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  ABSTRACT: The transmembrane HIV-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus-based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.
  Plant Biotechnology Journal 03/2013;
• Article: Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.

  Nataliya Kovalchuk, Wei Jia, Omid Eini, Sarah Morran, Tatiana Pyvovarenko, Stephen Fletcher, Natalia Bazanova, John Harris, Kontanze Beck-Oldach, Yuri Shavrukov, Peter Langridge, Sergiy Lopato
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  ABSTRACT: Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter-driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.
  Plant Biotechnology Journal 03/2013;
• Article: Orthology Guided Assembly in highly heterozygous crops: creating a reference transcriptome to uncover genetic diversity in Lolium perenne.

  Tom Ruttink, Lieven Sterck, Antje Rohde, Christian Bendixen, Pierre Rouzé, Torben Asp, Yves Van de Peer, Isabel Roldan-Ruiz
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  ABSTRACT: Despite current advances in next-generation sequencing data analysis procedures, de novo assembly of a reference sequence required for SNP discovery and expression analysis is still a major challenge in genetically uncharacterized, highly heterozygous species. High levels of polymorphism inherent to outbreeding crop species hamper De Bruijn Graph-based de novo assembly algorithms, causing transcript fragmentation and the redundant assembly of allelic contigs. If multiple genotypes are sequenced to study genetic diversity, primary de novo assembly is best performed per genotype to limit the level of polymorphism and avoid transcript fragmentation. Here, we propose an Orthology Guided Assembly procedure that first uses sequence similarity (tBLASTn) to proteins of a model species to select allelic and fragmented contigs from all genotypes and then performs CAP3 clustering on a gene-by-gene basis. Thus, we simultaneously annotate putative orthologues for each protein of the model species, resolve allelic redundancy and fragmentation and create a de novo transcript sequence representing the consensus of all alleles present in the sequenced genotypes. We demonstrate the procedure using RNA-seq data from 14 genotypes of Lolium perenne to generate a reference transcriptome for gene discovery and translational research, to reveal the transcriptome-wide distribution and density of SNPs in an outbreeding crop and to illustrate the effect of polymorphisms on the assembly procedure. The results presented here illustrate that constructing a non-redundant reference sequence is essential for comparative genomics, orthology-based annotation and candidate gene selection but also for read mapping and subsequent polymorphism discovery and/or read count-based gene expression analysis.
  Plant Biotechnology Journal 02/2013;