Showing papers on "Transformation (genetics) published in 2014"
TL;DR: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings and offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.
Abstract: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium- mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.
135 citations
TL;DR: It was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens, and the major cereals are now transformed quite efficiently.
Abstract: Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites, which are the basis of tissue culture and transformation in dicotyledons, in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was determined that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.
121 citations
TL;DR: A highly efficient in planta transient transformation method in onion epidermis is developed by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression.
Abstract: Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.
109 citations
TL;DR: This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum, and it is revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404.
Abstract: Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.
108 citations
TL;DR: This study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C. zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway.
Abstract: The unicellular green alga Chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid But the genetic toolbox for this alga is not available In the present study, an efficient transformation system was established for C zofingiensis The transformation system utilized a modified norflurazon-resistant phytoene desaturase (PDS-L516F, with an leucine-phenylalanine change at position 516) as the selectable marker Three promoters from endogenous PDS, nitrate reductase (NIT), and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) genes were tested, with the RBCS promoter demonstrating the highest transformation efficiency Inclusion of the first intron of the PDS gene further enhanced the efficiency by 91 % Both particle bombardment and electroporation methods were examined, and the latter gave a fourfold higher transformation efficiency The introduction of PDS-L516F, which exhibited a 33 % higher desaturation activity than the unaltered enzyme, enabled C zofingiensis to produce 321 % more total carotenoids (TCs) and 541 % more astaxanthin The enhanced accumulation of astaxanthin in transformants was revealed to be related to the increase in the transcripts of PDS, β-carotenoid ketolase (BKT), and hydroxylase (CHYb) genes Our study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway In addition, the engineered C zofingiensis might serve as an improved source of natural astaxanthin
106 citations
TL;DR: A high-efficiency nuclear transformation method for the diatom Phaeodactylum tricornutum using an electroporation system that offers a number of advantages, including simplicity, general utility, low-cost and high efficiency.
102 citations
TL;DR: These novel protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplast as a starting material for the development of genetically-engineered oil palm plants.
Abstract: Background
Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.
Methodology/Principal Findings
We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.
Conclusions/Significance
We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.
96 citations
TL;DR: Overproduction of proline might play an important role against salt shock and cellular integrity in pigeonpea plants.
Abstract: Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ∆1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.
92 citations
TL;DR: The results suggest that bacterial lignin transformation is an adaptive trait that can be exploited to engineer combinatorial arrays with defined product profiles, a prerequisite for scale-up production using different plant sources.
Abstract: Engineering the microbial transformation of lignocellulosic biomass is essential to developing modern biorefining processes that alleviate reliance on petroleum-derived energy and chemicals. Many current bioprocess streams depend on the genetic tractability of Escherichia coli with a primary emphasis on engineering cellulose/hemicellulose catabolism, small molecule production, and resistance to product inhibition. Conversely, bioprocess streams for lignin transformation remain embryonic, with relatively few environmental strains or enzymes implicated. Here we develop a biosensor responsive to monoaromatic lignin transformation products compatible with functional screening in E. coli. We use this biosensor to retrieve metagenomic scaffolds sourced from coal bed bacterial communities conferring an array of lignin transformation phenotypes that synergize in combination. Transposon mutagenesis and comparative sequence analysis of active clones identified genes encoding six functional classes mediating lignin transformation phenotypes that appear to be rearrayed in nature via horizontal gene transfer. Lignin transformation activity was then demonstrated for one of the predicted gene products encoding a multicopper oxidase to validate the screen. These results illuminate cellular and community-wide networks acting on aromatic polymers and expand the toolkit for engineering recombinant lignin transformation based on ecological design principles.
82 citations
TL;DR: A chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum‐based diesel.
Abstract: In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.
74 citations
TL;DR: This study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.
Abstract: Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.
TL;DR: An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii that provides a quick, efficient, economical and high-frequency transformation method for microalgae.
Abstract: Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 μM acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.
TL;DR: The β-tubulin promoter regions of all recognized species of genus Nannochloropsis were obtained, and all five marine species by electroporation were transformed, and the PCR amplified double stranded DNA fragments (PCR fragments) based transformation system was established in these Nann Cochloropsis species, which showed much higher transformation efficiency.
Abstract: Nannochloropsis are model species for investigating biofuel production by algae. To develop them into an integrated photons-to-fuel production platform, high efficiency transformation methods are necessary. Here, we obtained the beta-tubulin promoter regions of all recognized species of genus Nannochloropsis, and successfully transformed all five marine species by electroporation. In addition, the PCR amplified double stranded DNA fragments (PCR fragments) based transformation system was established in these Nannochloropsis species, which showed much higher transformation efficiency (10.7-61.2 x 10 (6), 1.5-13-fold) than that of linearized plasmid based transformation. The cotransformation of N. salina using a circular plasmid containing a non-selectable GUS gene and a PCR fragment containing only a selection marker cassette was also achieved and found to be very efficient (over 50%). This simple and highly efficient transformation protocol reported in our study provided a useful tool for gene functional analysis and genetic engineering of the oleaginous Nannochloropsis species.
TL;DR: In this article, several polycyclic aromatic hydrocarbons (PAHs) were selected as model molecules to investigate their potential transformation on Fe(III)-saturated clay minerals under various mineralogical and environmental conditions.
Abstract: Clay-driven transformation of polycyclic aromatic hydrocarbons (PAHs) is critically influenced by their molecular structure and clay surface properties. In the present study, several PAHs were selected as model molecules to investigate their potential transformation on Fe(III)-saturated clay minerals under various mineralogical and environmental conditions. Results suggest that the reactivity of PAHs is highly correlated with their ionization potential (IP) values. PAHs with IP lower than a threshold between 7.5 and 7.6 prefer undergoing a one-electron transfer reaction. Otherwise, Fe(III)-smectite is unable to degrade PAHs with IP above it. The electron-transfer process leads to the reduction of Fe(III) to Fe(II) and formation of organic radical cations, which are more stable in clay interlayers than at other clay sites. Subsequent reactions of radical cations with oxygenic species (such as H2O) result in formation of oxygenated products. The surface chemical properties, i.e., the hydration of cations, fraction of Fe(III), layer charge location, and type of ligands, strongly affect the interaction between PAHs and Fe(III), thus modulate the reactivity of surface Fe(III)-species on the clay minerals. This study provides the first direct evidence for clay-catalyzed transformation of PAHs supporting the plausibility of their in situ degradation in soils, and demonstrates that abiotic reactions with surface-bound Fe(III) may affect or even dominate the long-term behavior of PAHs in soils, particularly in the presence of swelling clay minerals.
TL;DR: Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules, here it is found that the rj4/rj4 genotype soybeans were fully complemented by theexpression of the Rj 4 gene.
Abstract: Soybeans exhibit a nitrogen-fixing symbiosis with soil bacteria of the genera Bradyrhizobium and Ensifer/Sinorhizobium in a unique organ, the root nodule. It is well known that nodulation of soybean is controlled by several host genes referred to as Rj (rj) genes. Among these genes, a dominant allele, Rj4, restricts nodulation with specific bacterial strains such as B. elkanii USDA61 and B. japonicum Is-34. These incompatible strains fail to invade the host epidermal cells as revealed by observations using DsRed-labeled bacteria. Here, we describe the molecular identification of the Rj4 gene by using map-based cloning with several mapping populations. The Rj4 gene encoded a thaumatin-like protein (TLP) that belongs to pathogenesis-related (PR) protein family 5. In rj4/rj4 genotype soybeans and wild soybeans, we found six missense mutations and two consecutive amino acid deletions in the rj4 gene as compared with the Rj4 allele. We also found, using hairy root transformation, that the rj4/rj4 genotype soybeans were fully complemented by the expression of the Rj4 gene. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules.
TL;DR: The objective of this article is to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basic physics and molecular biology behind them.
TL;DR: In this paper, an improved Agrobacterium-mediated transformation protocol was described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1, which significantly improved transformation frequency and event quality.
Abstract: An improved
Agrobacterium
-mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.
This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.
TL;DR: The susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains is examined and it is concluded that VIP1 is not important for Agrobacteria-mediated transformation or VirE2 subcellular localization.
Abstract: SUMMARYAgrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into thehost genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1(VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction withand targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 over-expressing plants to transformation by numerous Agrobacterium strains. In no instance could we detectaltered transformation susceptibility. We also used confocal microscopy to examine the subcellular localiza-tion of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein,in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleusof Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts andtobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to thecytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpres-sion. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionallydetect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. Thevast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacteri-um-mediated transformation or VirE2 subcellular localization.Keywords: Agrobacterium, plant transformation, VirE2 interacting protein 1, VirE2, Arabidopsis thaliana,Nicotiana benthamiana, protein subcellular localization.INTRODUCTIONAgrobacterium tumefaciens, a soil-borne pathogen,induces neoplastic growths on plants by transportingtransferred DNA (T-DNA), a region of its Ti (tumor-induc-ing) plasmid, to the host cell and integrating it intothe host genome. During the process of transformation,Ti-plasmid-encoded virulence (vir) genes are induced byphenolic molecules secreted from wounded plants. SomeVir proteins function in the bacterium, whereas others [vir-ulence effector proteins VirD2, VirD5, VirE2, VirE3, VirF andGALLS (from Agrobacterium rhizogenes)], are secreted bythe bacterium into the plant via a type IV secretion system(T4SS) made up of 11 VirB proteins and VirD4 (for reviews,see Gelvin, 2003, 2009, 2010a,b; McCullen and Binns, 2006;Pitzschke and Hirt, 2010; Lacroix and Citovsky, 2013b).The VirD2 endonuclease, in conjunction with VirD1,nicks the Ti-plasmid at T-DNA border sequences and cova-lently attaches to the 5
TL;DR: This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension‐cultured cells and by polyethylene glycol (PEG)‐mediated DNA transformation intoprotoplasts derived from rice leaf sheaths.
Abstract: Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension-cultured cells and by polyethylene glycol (PEG)-mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG-mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided.
TL;DR: The Agrobacterium-mediated transformation of barley is a valuable tool for determining gene function by over-expression of a gene of interest or by RNAi-based gene silencing and uses a hygromycin resistance gene to allow selection of transgenic plants.
Abstract: Agrobacterium-mediated transformation of barley is a valuable tool for determining gene function by over-expression of a gene of interest or by RNAi-based gene silencing. The method is based on the inoculation of immature embryos with Agrobacterium and uses a hygromycin resistance gene to allow selection of transgenic plants. The protocol described leads to average transformation efficiencies of 25 % meaning that large numbers of fertile transgenic plants can be produced.
TL;DR: A compatible IL-cellulase system based on the cellulase was developed, and its use significantly improved the saccharification rate of rice straw from 53% to 88% versus the control, demonstrating its potential for efficient transformation of lignocellulose to glucose in a single-step process.
TL;DR: An efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants is developed, which provides a useful platform for future genetic engineering studies in this economically important crop.
Abstract: Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by PCR, Southern blot analysis and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by RT-PCR analysis. Transformation efficiency varied from 9.4% to 18.2% depending on the cultivars, selectable marker genes and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop.
TL;DR: A simple and efficient transient transformation system is developed, which can be used in homologous expression or reverse genetic study of the plants and ThbZIP1 is found to mediate stress tolerance by regulating the expression of SOD and POD genes.
Abstract: In this study, we developed a simple and efficient transient transformation system, which can be used in homologous expression or reverse genetic study of the plants. A system for characterizing gene function in response to stress tolerance was also developed based on this transformation method. The overexpression and RNAi-silencing of a bZIP gene from Tamarix hispida, ThbZIP1, were performed in T. hispida using this transformation method. Real-time PCR showed that the expression of ThbZIP1 was highly up- and down-regulated in the plants with overexpression and RNAi-silenced expression of ThbZIP1, respectively, when compared with control plants (transiently transformed with empty pROK2). A physiological study showed that ThbZIP1 can enhance the activities of both peroxidase (POD) and superoxide dismutase (SOD), and decrease electrolyte leakage rate and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) under salt stress conditions. Furthermore, ThbZIP1 is found to mediate stress tolerance by regulating the expression of SOD and POD genes. These results suggested that this transient transformation system is an effective method for determining the function of a gene in response to abiotic stress in plants.
TL;DR: The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae.
Abstract: Summary
Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.
TL;DR: This is the first report of an Agrobacterium-based transformation system for L. bicolor, which will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.
Abstract: Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD600) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.
TL;DR: Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.
TL;DR: A simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap), which will promote future molecular biological study in M. polymorpha.
Abstract: The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.
TL;DR: It is shown that RNAi can be expressed in phytopathogenic fungi by direct transfection with a plant virus-based vector and that the approach also can be used to obtain foreign protein expression in fungi, anticipating this approach will find wider application as a tool in functional genomics of filamentous fungi.
Abstract: RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.
TL;DR: Optimal parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates are presented and the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii is confirmed through molecular methods.
Abstract: Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH2)3]8Si8Mg6O12(OH)4), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×102 transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.
TL;DR: In this article, the authors used transcriptome sequencing to determine whether the full set of genes known to be required for competence in other naturally transformable bacteria is part of the SigH regulon.
Abstract: Staphylococcus aureus is a major human pathogen. Hospital infections caused by methicillin-resistant strains (MRSA), which have acquired resistance to a broad spectrum of antibiotics through horizontal gene transfer (HGT), are of particular concern. In S. aureus, virulence and antibiotic resistance genes are often encoded on mobile genetic elements that are disseminated by HGT. Conjugation and phage transduction have long been known to mediate HGT in this species, but it is unclear whether natural genetic transformation contributes significantly to the process. Recently, it was reported that expression of the alternative sigma factor SigH induces the competent state in S. aureus. The transformation efficiency obtained, however, was extremely low, indicating that the optimal conditions for competence development had not been found. We therefore used transcriptome sequencing to determine whether the full set of genes known to be required for competence in other naturally transformable bacteria is part of the SigH regulon. Our results show that several essential competence genes are not controlled by SigH. This presumably explains the low transformation efficiency previously reported, and demonstrates that additional regulating mechanisms must be involved. We found that one such mechanism involves ComK1, a transcriptional activator that acts synergistically with SigH.