Transient Genetic Transformation Research Articles

Generation of CRISPR-edited birch plants without DNA integration using Agrobacterium-mediated transformation technology

CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Deep learning and targeted metabolomics-based monitoring of chewing insects in tea plants and screening defense compounds.

Tea is an important cash crop that is often consumed by chewing pests, resulting in reduced yields and economic losses. It is important to establish a method to quickly identify the degree of damage to tea plants caused by leaf-eating insects and screen green control compounds. This study was performed through the combination of deep learning and targeted metabolomics, in vitro feeding experiment, enzymic analysis and transient genetic transformation. A small target damage detection model based on YOLOv5 with Transformer Prediction Head (TPH-YOLOv5) algorithm for the tea canopy level was established. Orthogonal partial least squares (OPLS) was used to analyze the correlation between the degree of damage and the phenolic metabolites. A potential defensive compound, (-)-epicatechin-3-O-caffeoate (EC-CA), was screened. In vitro feeding experiments showed that compared with EC and epicatechin gallate, Ectropis grisescens exhibited more significant antifeeding against EC-CA. In vitro enzymatic experiments showed that the hydroxycinnamoyl transferase (CsHCTs) recombinant protein has substrate promiscuity and can catalyze the synthesis of EC-CA. Transient overexpression of CsHCTs in tea leaves effectively reduced the degree of damage to tea leaves. This study provides important reference values and application prospects for the effective monitoring of pests in tea gardens and screening of green chemical control substances.

A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.

DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.

Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods.
 Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression.

An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

Assessing Carnivorous Plants for the Production of Recombinant Proteins.

The recovery of recombinant proteins from plant tissues is an expensive and time-consuming process involving plant harvesting, tissue extraction, and subsequent protein purification. The downstream process costs can represent up to 80% of the total cost of production. Secretion-based systems of carnivorous plants might help circumvent this problem. Drosera and Nepenthes can produce and excrete out of their tissues a digestive fluid containing up to 200 mg. L-1 of natural proteins. Based on the properties of these natural bioreactors, we have evaluated the possibility to use carnivorous plants for the production of recombinant proteins. In this context, we have set up original protocols of stable and transient genetic transformation for both Drosera and Nepenthes sp. The two major drawbacks concerning the proteases naturally present in the secretions and a polysaccharidic network composing the Drosera glue were overcome by modulating the pH of the plant secretions. At alkaline pH, digestive enzymes are inactive and the interactions between the polysaccharidic network and proteins in the case of Drosera are subdued allowing the release of the recombinant proteins. For D. capensis, a concentration of 25 μg of GFP/ml of secretion (2% of the total soluble proteins from the glue) was obtained for stable transformants. For N. alata, a concentration of 0.5 ng of GFP/ml secretions (0.5% of total soluble proteins from secretions) was reached, corresponding to 12 ng in one pitcher after 14 days for transiently transformed plants. This plant-based expression system shows the potentiality of biomimetic approaches leading to an original production of recombinant proteins, although the yields obtained here were low and did not allow to qualify these plants for an industrial platform project.

Lectins modulate the microbiota of social amoebae

The social amoeba Dictyostelium discoideum maintains a microbiome during multicellular development; bacteria are carried in migrating slugs and as endosymbionts within amoebae and spores. Bacterial carriage and endosymbiosis are induced by the secreted lectin discoidin I that binds bacteria, protects them from extracellular killing, and alters their retention within amoebae. This altered handling of bacteria also occurs with bacteria coated by plant lectins and leads to DNA transfer from bacteria to amoebae. Thus, lectins alter the cellular response of D. discoideum to bacteria to establish the amoebae's microbiome. Mammalian cells can also maintain intracellular bacteria when presented with bacteria coated with lectins, so heterologous lectins may induce endosymbiosis in animals. Our results suggest that endogenous or environmental lectins may influence microbiome homeostasis across eukaryotic phylogeny.

Development of Efficient Protocols for Stable and Transient Gene Transformation for Wolffia Globosa Using Agrobacterium.

Members of the Wolffia genus are fascinating plants for many biologists as they are the smallest flowering plants on Earth and exhibit a reduced body plan that is of great interest to developmental biologists. There has also been recent interest in the use of these species for bioenergy or biorefining. Molecular and developmental studies have been limited in Wolffia species due to the high genome complexity and uncertainties regarding the stable genetic transformation. In this manuscript we present new protocols for both stable and transient genetic transformation for Wolffia globosa using Agrobacterium tumefaciens. For the transient transformation, we used Wolffia fronds whereas we used clusters for the stable transformation. As proof of concept we transformed two synthetic promoter constructs driving expression of the GUS marker gene, that have previously been used to monitor auxin and cytokinin output in a variety of species. Using these approaches we obtained a Transformation Efficiency (TE) of 0.14% for the stable transformation and 21.8% for the transient transformation. The efficiency of these two methods of transformation are sufficient to allow future studies to investigate gene function. This is the first report for successful stable transformation of W. globosa.

Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

BackgroundTheobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.ResultsAgrobacteriumtumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis.ConclusionsLeaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5–7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0119-5) contains supplementary material, which is available to authorized users.

Grapevine (Vitis vinifera L.) promoter analysis by biolistic-mediated transient transformation of cell suspensions

Expression of alcohol dehydrogenase promoters from Vitis vinifera L. and Arabidopsis thaliana was investigated in homologous and heterologous cell systems after bombardment of chimeric genes. The effect of several parameters including wounding, DNA delivery and construct type on luciferase/beta-glucuronidase activities was evaluated. In parallel, alcohol dehydrogenase activity was assayed in normal and gaseous nitrogen-treated cells. Compared to A. thaliana , results showed large differences in reporter gene activity according to the Adh promoter leader sequence and the cell suspension system. The V. vinifera cell system was found to be appropriate to investigate Adh promoter functioning. A novel protocol, based on transient genetic transformation of grape-One cells by biolistic is proposed to study grapevine promoter expression, either in air or in response to anaerobiosis.

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens.

Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated transient genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists.

Gene overexpression and gene silencing in Birch using an Agrobacterium-mediated transient expression system

As transient expression systems are effective methods for the functional characterization of genes, a transient gene expression and silencing system was developed for Betula platyphylla Suk (Chinese Birch). Firstly, the cinnamoyl-CoA reductase (CCR) gene and its promoter were isolated from Chinese Birch. The vectors for overexpression of CCR and RNAi-based silence of CCR were constructed and transformed into Agrobacterium, respectively. Overexpression and silence of the CCR gene were respectively, performed on Birch seedlings using an Agrobacterium-mediated transient expression system. The expression levels of CCR were determined using real-time PCR. The results showed that the transcripts of CCR notably increased in the Birch plants transformed with the CCR overexpression construct, and notably decreased in plants transformed with the silencing construct when compared with nontransgenic plants. These studies confirmed that this transient genetic transformation system works well on Birch plants, and can be used for the functional characterization of genes and protein production in Birch.

Transient genetic transformation of embryogenic callus of Cocos nucifera

Coconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods, and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical applications, such as the improvement of coconut micropropagation via somatic embryogenesis.

Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment

Transient gene expression is an indispensable tool for studying functions of gene products. In the case of plants, transient introduction of genes by Agrobacterium infiltration is a method of choice for many species. However, this technique does not work efficiently in Arabidopsis leaf tissue, the most widely used model system for basic plant biology research. Here we present an optimized protocol for biolistic delivery of plasmid DNA into the epidermis of Arabidopsis leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol yields efficient and reproducible transient expression of diverse genes and is exemplified here for use in a functional assay of a transcription repressor and for the subcellular localization and cell-to-cell movement of plant viral movement protein. This protocol is suitable for studies of biological function and subcellular localization of the gene product of interest directly in planta by utilizing different types of activity-based assays. Using this procedure, the data are obtained after 2-4 d of work.

TRANSIENT TRANSFORMATION OF A CHLORARACHNIOPHYTE ALGA, LOTHARELLA AMOEBIFORMIS (CHLORARACHNIOPHYCEAE), WITH uidA AND egfp REPORTER GENES(1).

A transient genetic transformation system was established for a chlorarachniophyte alga, Lotharella amoebiformis K. Ishida et Y. Hara. We first isolated sequences that contain a putative promoter for a RUBISCO SSU (rbcS) gene and a terminator for another copy of rbcS gene from L.amoebiformis. With those promoter and terminator sequences, we developed two expression vectors, pLaRGus and pLaRGfp, which code uidA and egfp genes, respectively. The cells were then transformed with each vector using a microparticle bombardment system. When the cells were transformed with the pLaRGus, β-glucuronidase (GUS) staining dyed several cells blue. Green fluorescent protein (GFP) fluorescence was observed in the cells transformed with pLaRGfp. The highest transient transformation efficiency, 35 per 2 × 10(7) cells, was detected from the GUS staining. This study demonstrates that two reporter genes are expressed in L.amoebiformis cells when rbcS promoter and terminator are used. The conditions of transformation were also optimized. This is the first report of successful genetic transformation in chlorarachniophyte algae.

Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium.

Agrobacterium-mediated genetic transformation of plants, a unique example of transkingdom DNA transfer, requires the presence of several proteins encoded by the host cell. One such cellular factor is VIP1, an Arabidopsis protein proposed to interact with and facilitate import of the bacterial DNA-protein transport (T) complexes into the plant cell nucleus. Thus, VIP1 is required for transient expression of the bacterial DNA, an early step in the transformation process. However, the role of VIP1 in subsequent transformation events leading to the stable expression of bacterial DNA was unexplored. Here, we used reverse genetics to dissect VIP1 functionally and demonstrate its involvement in the stable genetic transformation of Arabidopsis plants by Agrobacterium. Our data indicate that the ability of VIP1 to interact with the VirE2 protein component of the T-complex and localize to the cell nucleus is sufficient for transient genetic transformation, whereas its ability to form homomultimers and interact with the host cell H2A histone in planta is required for tumorigenesis and, by implication, stable genetic transformation.