Direct DNA Uptake Research Articles

CeO2 Nanoparticles-Regulated Plasmid Uptake and Bioavailability for Reducing Transformation of Extracellular Antibiotic Resistance Genes

The direct uptake of extracellular DNA (eDNA) via transformation facilitates the dissemination of antibiotic resistance genes (ARGs) in the environment. CeO2 nanoparticles (NPs) have potential in the regulation of conjugation-dominated ARGs propagation, whereas their effects on ARGs transformation remain largely unknown. Here, CeO2 NPs at concentrations lower than 50 mg L-1 have been applied to regulate the transformation of plasmid-borne ARGs to competent Escherichia coli (E. coli) cells. Three types of exposure systems were established to optimize the regulation efficiency. Pre-incubation of competent E. coli cells with CeO2 NPs at 0.5 mg L-1 inhibited the transformation (35.4%) by reducing the ROS content (0.9-fold) and cell membrane permeability (0.9-fold), thereby down-regulating the expression of genes related to DNA uptake and processing (bhsA, ybaV, and nfsB, 0.7-0.8 folds). Importantly, CeO2 NPs exhibited an excellent binding capacity with the plasmids, decreasing the amounts of plasmids available for cellular uptake and down-regulating the gene expression of DNA uptake (bhsA, ybaV, and recJ, 0.6-0.7 folds). Altogether, pre-exposure of plasmids with CeO2 NPs (10 and 25 mg L-1) suppressed the transformation with an efficiency of 44.5-51.6%. This study provides a nano-strategy for controlling the transformation of ARGs, improving our understanding on the mechanisms of nanomaterial-mediated ARGs propagation.

Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea.

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 103 transformants μg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 103 transformants μg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms.IMPORTANCE Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way that this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here, we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.

Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Gene editing in tomatoes.

Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes

ABSTRACTLysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

Protoplast Transformation as a Plant-Transferable Transient Expression System.

The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

Fate of a Larch Unedited tRNA Precursor Expressed in Potato Mitochondria

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNA(His). We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNA(His) precursor was processed in the isolated organelles. By contrast, no mature tRNA(His) was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these "foreign" editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3'-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

Plastid transformants of tomato selected using mutations affecting ribosome structure

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.

Evidence that direct DNA uptake through cut shoots leads to genetic transformation of Solanum aviculare Forst.

The reporter genes GUS, NPTII and BAR, either separately or in combination, have been exploited to determine if DNA which can directly enter plants, circulate within the plant and enter nuclei, can also integrate into the genome in a manner which will permit gene expression. Feeding of either seed-derived or adventitious cut shoots of Solanum aviculare with the GUS gene followed by rooting of the shoots and growing on, resulted in all tissues of the plant showing GUS activity as detected cytochemically. Southern blot analysis of plants derived from the adventitious shoots confirmed the presence of the reporter gene in roots. Reporter gene expression was observed also in the F1 generation. If GUS and NPTII or GUS, NPTII and BAR were fed together, then in each case it was possible to have both expression and Southern blot confirmation of each of the genes. There was a relatively high rate of transformation of approximately 5% of the fed stems across all experiments conducted during the present study.

Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts.

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.

Exogenous DNA Uptake via Cellular Permeabilization and Expression of Foreign Gene in Wheat Zygotic Embryos.

Exogenous DNA uptake and transient expression of the GUS reporter gene was studied in wheat (Triticum aestivum L.) zygotic embryos employing a simple procedure of cellular permeabilization by membrane interactive agents like saponin and toluene. Uptake of expression vectors with different promoters was detected in mature and immature embryos. The frequency of GUS expression was detected to be higher in mature embryos than immature embryos probably due to a certain degree of disorganization of the plasmalemma during the dehydrated state of the embryos. Of the various permeabilizing agents employed, saponin and toluene were detected as potential membrane permeabilizers without adversely affecting embryo viability. Variations in GUS gene expression were not significant among different genotypes of T. aestivum CPAN1676, PBW343 and HD2329, thus indicative of the relative genotype independence of this process. The results support the proposed idea of direct DNA uptake as an alternate and simple method for inserting foreign DNA into plant cells and its use for transformation of higher plants.

An Arabidopsis cyclin promoter region is active in transgenic maize plants

We have produced transgenic maize carrying Arabidopsis cyclin promoter and beta-glucuronidase chimeric fusion (cycB1;1At::GUS) by direct DNA uptake into embryogenic cell suspension protoplasts to study the activity of this cyclin promoter in a heterologous cell system. The transgenic maize lines exhibiting different levels of reporter gene expression show that regulatory elements from cycB1;1At gene as a dicot promoter can function in maize. Expression patterns revealed by fluorimetric, as well as, histochemical GUS reporter enzymatic assays indicated cell division-dependent expression of the GUS gene driven by this cycB1;At;1 promoter with elevated activity in proliferating cells and meristems. Treatment of tissues with inhibitors of mitosis increased the activity of cycB1;1At promoter, that suggests a link between mitotic phase of cell division and activity of cycB1;1At regulatory elements in transgenic maize.

Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants

Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and -pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods.

Meristem, cell division and S phase-dependent activity of wheat histone H4 promoter in transgenic maize plants

We have studied the activity of a wheat histone H4 promoter in transgenic maize plants produced by direct DNA uptake into embryogenic cell suspension protoplasts. Expression patterns revealed by fluorimetric as well as histochemical GUS reporter enzyme assays and Northern hybridisation indicated the cell division-dependent expression of the GUS gene driven by the histone H4 promoter in proliferating cells and meristems. Changes in gene expression associated with the progression through the cell cycle in maize cell-suspension cultures were also investigated after partial synchronisation of cell division by aphidicolin or hydroxyurea. The transgene expression regulated by the wheat histone H4 promoter coincided with elevated levels of mRNAs from other S phase-associated genes such as maize histone H4, histone H2B and proliferating cell nuclear antigen ( PCNA) . The reporter gene expression was low during mitosis when accumulation of the transcripts of a B-type mitotic cyclin CycB1;zm;1 could be detected. The presented data indicate that the used 720-bp-long promoter region can provide replication-dependent expression for the GUS reporter gene in transgenic maize. In these maize plants, external hormone stimuli generated by a synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D) or abscisic acid (ABA) have modified the histone H4 promoter-driven GUS gene transcription. These findings support the usefulness of the H4::GUS transgenic plants for further studies on cell cycle activation/inactivation by mitogenic or stress related stimuli in maize.

STUDIES TO IMPROVE TRANSIENT GUSA EXPRESSION IN A. TUMEFACIENS-MEDIATED TRANSFORMATION OF RICE

The direct-DNA uptake methods has been routinely used in transforming rice. The recent development of rice genetic transformation using A. tumefaciens as a natural vector necessitate the investigation of factors that will promote optimum gene expression and T-DNA transfer in the recalcitrant and economically important indica rice varieties. Activities of three different promoters were investigated for transient expression using gusA after particle bombardment of suspension cells. Actin and ubiquitin promoters were found to induce higher GUS activity compared to the chimeric promoter of mannopine synthethase and a truncated cauliflower mosaic virus 35S promoter. A binary vector pCGN1558 was modified to contain these promoters in front of the gusA-intron. A. tumefaciens strain EHA101 and A136 were transformed with the derivatives of the binary vector and transformed colonies were tested for infectivity on coleoptile bases of IR54 and IR72 seedlings. GUS activity of the infected coleoptile bases showed that the A. tumefaciens colonies were comparable to but not better than the positive control At656. Possible reasons for the obtained results were explained with emphasis on the importance of the compatibility of the A. tumefaciens strains with the binary vector, the border sequences, and the Ti-plasmid present.

Transformation and Regeneration of Creeping Bentgrass (Agrostis palustris Huds.) Protoplasts

An efficient plant regeneration system from protoplasts of creeping bentgrass (Agrostis palustris Huds.) cultures is essential for gene transfer to these grasses through direct DNA uptake into protoplasts. A simple and efficient plant regeneration system for protoplasts isolated from embryogenic suspension cultures of seven creeping bentgrass cultivars was established. Suspension cultures were derived from embryogenic callus cultures established from surface‐sterilized mature seeds. Four creeping bentgrass cultivars were tested to determine their effect as feeders on plating efficiencies, callus development, and regeneration of protoplasts. Embryogenic suspension cultures from all four cultivars worked as feeders with plating efficiencies ranging from 0.05 to 0.32%. Protoplast derived calli formed within 3 wk after isolation and were regenerated with, or without, the addition of cytokinin to the regeneration medium. Some cultivars required the use of a particular feeder to regenerate plants. Plants were regenerated from all the cultivars tested. The bar gene, which confers resistance to the herbicide bialaphos [2‐amino‐4‐(hydroxymethylphosphinyl)butanoic acid], was transformed into protoplasts by means of either PEG or electroporation. Bialaphos‐resistant colonies were obtained from five creeping bentgrass cultivars. Resistant colonies of the cultivar Cobra were regenerated into plants. One hundred fifty‐three Cobra transformants analyzed were resistant to five times the field rate of commercial formulation of the herbicide. Molecular characterization of the transformants revealed the stable integration of the bar gene into the genome and expression of transcripts corresponding to the bar gene.

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv 'Igri') were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.

Stable transformation of barley via direct DNA uptake. Electroporation- and PEG-mediated protoplast transformation.

Transformation technology has great potential for application in many areas of fundamental and applied plant science; the introduction and expression of transgenes will facilitate new approaches in genetics, biochemistry, and physiology and stimulate progress in these fields. In addition, gene transfer techniques will allow the genetic manipulation of crop plants toward improved agronomic characteristics. Indeed, in those plant species that are amenable to Agrobacterium—mediated transformation the use of transgenic plants has already had a significant impact in a number of areas of research, and genetically engineered plants with various different modified traits are undergoing field trials or are actually coming to market.