Agrobacterium Tumefaciens-mediated Gene Transfer Research Articles

Establishment of a gene recombination method for a major human skin commensal fungus, Malassezia restricta, using Agrobacterium tumefaciens-mediated gene transfer system.

Malassezia restricta is the most predominant fungus in the microbiome of human skin. This microorganism can cause or exacerbate Malassezia-associated skin dermatitis, seborrheic dermatitis, atopic dermatitis, and pityriasis versicolor. The virulence factors of M. restricta have not been analyzed because a gene recombination system has not been developed. In this study, we established an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system, optimized for generating gene-deficient mutants of M. restricta. A mutant of FKB1 gene, which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus, was generated using the ATMT system. Subsequently, the FKB1 gene was reintroduced into the FKB1 gene-deficient mutant for obtaining a gene-complemented strain. The wild-type strain of M. restricta was sensitive to tacrolimus, whereas the FKB1 gene-deficient mutant was resistant to tacrolimus; the phenotypic drug susceptibility in the mutant was restored by reintroducing the FKB1 gene. Contrastingly, the FKB1 gene-deficient mutant was not resistant to cyclosporine A, which also inhibits calcineurin by binding to cyclophilin A. The gene recombination system for M. restricta will facilitate in elucidating the molecular mechanisms causing Malassezia-associated dermatitis.

Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review.

Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise from the unintended genetic modifications (GM) that have been reported as side-effects of such techniques. Here, we systematically review the scientific literature for studies that have investigated unintended genomic alterations in plants modified by the following GM techniques: Agrobacterium tumefaciens-mediated gene transfer, biolistic bombardment, and CRISPR-Cas9 delivered via Agrobacterium-mediated gene transfer (DNA-based), biolistic bombardment (DNA-based) and as ribonucleoprotein complexes (RNPs). The results of our literature review show that the impact of such techniques in host genomes varies from small nucleotide polymorphisms to large genomic variation, such as segmental duplication, chromosome truncation, trisomy, chromothripsis, breakage fusion bridge, including large rearrangements of DNA vector-backbone sequences. We have also reviewed the type of analytical method applied to investigate the genomic alterations and found that only five articles used whole genome sequencing in their analysis methods. In addition, larger structural variations detected in some studies would not be possible without long-read sequencing strategies, which shows a potential underestimation of such effects in the literature. As new technologies are constantly evolving, a more thorough examination of prospective analytical methods should be conducted in the future. This will provide regulators working in the field of genetically modified and gene-edited organisms with valuable information on the ability to detect and identify genomic interventions.

Agrobacterium tumefaciens-Mediated Gene Transfer in a Major Human Skin Commensal Fungus, Malassezia globosa

Although the fungal microbiome in human skin mainly comprises lipophilic yeasts, including Malassezia species, these microorganisms can cause various dermatitis conditions, including pityriasis versicolor, seborrheic dermatitis, folliculitis, and atopic dermatitis, depending on the host condition. Both Malassezia globosa and Malassezia restricta are major species implicated in Malassezia-related dermatitis. However, the pathogenicity of these microorganisms has not been revealed at the genetic level owing to the lack of a genetic recombination system. Therefore, we developed a gene recombination system for M. globosa using Agrobacterium tumefaciens-mediated gene transfer of the target gene FKB1, which encodes the FKBP12 protein that binds the calcineurin inhibitor tacrolimus. The wild-type strain of M. globosa was sensitive to tacrolimus, whereas the FKB1 deletion mutant was resistant to tacrolimus. Reintroduction of FKB1 into the FKB1 deletion mutant restored wild-type levels of susceptibility to tacrolimus. Moreover, an FKB1-eGFP fusion gene was generated and expression of this fusion protein was observed in the cytoplasm. This newly developed gene recombination system for M. globosa will help further our understanding of the pathogenesis of M. globosa-related dermatitis at the genetic level.

P351 Genetic recombination between the main human skin commensal fungus, Malassezia restricta, and M. globosa using an Agrobacterium tumefaciens-mediated gene transfer system

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjects: Malassezia restricta and M. globosa cause or exacerbate Malassezia -associated skin, seborrheic, and atopic dermatitis, as well as pityriasis versicolor, but the virulence factors remain unclear because between-species genetic recombination has not yet been achieved. We built an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system that generated gene-knockout mutants of both Malassezia species.Materials and Methods: The binary vector pAg1-Δfkb1::NAT1 was introduced into M. restricta CBS 7877 and M. globosa CBS 7966 via ATMT; FKB1 was replaced. Then, FKB1 was re-introduced into the mutants to counteract the deficiencies.Results and Discussion: The medium acetosyringone concentration and temperature, as well as the co-cultivation ratios of A. tumefaciens and the Malassezia strains, affected recombination efficacy. We generated a mutant of the FKB1 gene (which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus). Wild-type M. restricta and M. globosa were tacrolimus-sensitive, while the FKB1 mutants were tacrolimus-resistant; drug susceptibility was restored by reintroducing FKB1.ConclusionStudies on recombination between M. restricta and M. globosa will aid elucidation of the molecular mechanisms underlying Malassezia -associated dermatitis.

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

Agrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Improving T-DNA Transfer to Tamarix hispida by Adding Chemical Compounds During Agrobacterium tumefaciens Culture.

Agrobacterium tumefaciens-mediated gene transfer is the most commonly used method for plant genetic engineering. However, during the period of A. tumefaciens culture, the effects of Agrobacterium culture before inoculation on genetic transformation are poorly understood. In the present study, we investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes, including virA, virB1, virC1, virD2, virD4 virE2, and virG. Spe treatment significantly induced the expression of all the studied genes, including virA, virB1, virC1, virD1, virD2, virD4, virE2, and virG. DTT treatment decreased reactive oxygen species accumulation. AS treatment activated the expression of the genes virA, virB1, virC1, virD1, virD2, virD4 and virG. All these effects resulted in increased T-DNA transfer. Additionally, combined Spe, 5-AzaC, DTT, and AS treatment improve Agrobacterium infection to a greater extent compared with their use alone, increasing T-DNA transfer by more than 8-fold relative to no treatment. Therefore, to improve genetic transformation, pretreatment of Agrobacterium during the culture period is important for improving genetic transformation efficiency.

Investigation of <i>Agrobacterium</i>-mediated Transient dsRNA Expression in Tobacco

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

The effect of magnetic field strength on shoot regeneration and Agrobacterium tumefaciens-mediated gene transfer in flax (Linum usitatissimum L.)

This study was conducted to determine the effects of magnetic field (MF) strength on shoot regeneration and Agrobacterium tumefaciens-mediated gene transfer in flax (Linum usitatissimum L.). Seeds of flax cv. Madaras were exposed to different MF strengths (0 – control, 75, 150, and 300 millitesla (mT)) for 24 h by using an electromagnetic generator system fabricated in laboratory conditions. After sterilization, seeds were germinated on MS (Murashige and Skoog) medium in Magenta vessels. Hypocotyl explants excised from 7-days-old seedlings were used for regeneration. GV2260 strain of Agrobacterium tumefaciens was used in transformation studies. Inoculated hypocotyls were cultured on MS medium containing 1 mg/l BAP (6-benzylaminopurine) and 0.02 mg/l NAA (naphthaleneacetic acid) for 2 days by co-cultivation. Then, they were transferred to MS medium containing the same growth regulators, 100 mg/l kanamycin and 500 mg/l Duocid for selection. The presence of the nptII gene was verified by PCR (polymerase chain reaction) analysis in putative transgenic plants. The highest results with respect to shoot regeneration and transformation frequency were obtained from treatments of 75 mT MF strength.

Chimaeric Rift Valley Fever Virus-Like Particle Vaccine Candidate Production in Nicotiana benthamiana.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg-1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

Generation of transgenic cell suspension cultures of the model legume Medicago truncatula: a rapid method for Agrobacterium mediated gene transfer

Plant cell suspension cultures are used in basic research and applied biotechnology. In both cases, the transfer and stable integration of heterologous genes is a required technique. This report describes a rapid method for transformation of cell cultures of Medicago truncatula, the model species for the legume family. Accession A17 from the cultivar Jemalong is the reference genotype selected for the sequencing of the genome and therefore most studies on Medicago are carried out on this accession line. However, this line has a low embryogenic capacity and is poorly responsive to transformation protocols that rely on somatic embryogenesis. An alternative method for transformation of suspension cultures of this line, which does not depend on leaf transformation or somatic embryogenesis, was therefore needed. The method described herein uses Agrobacterium tumefaciens mediated gene transfer, allowing the transformation of Medicago callus tissue and the following establishment of liquid suspension cell cultures approximately 2 months after transformation. Kanamycin resistance was used to select for positive transformation events and the screening was facilitated by visualization of a fluorescent marker, which was fused to the gene of interest. This new protocol reduces the time between transformation and cell culture establishment, and allows the generation of transgenic suspension cultures of Medicago reference accession A17.

Efficient genetic transformation of sour orange, Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus

Transgenic Citrus aurantium plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and Basta herbicide as the selective agent. In the present research, hypocotyl, and epicotyls parts of C. aurantium plant were inoculated with A. tumefaciens strain EHA-105. Then, explants were transferred into the regeneration medium and shoot growth containing various concentrations of kinetin and IAA growth regulators. In order to detect transgenic shoots, herbicide Basta (ammonium glufosinate) was added at a rate of 1 mg/L to the regeneration medium. Production buds happened in explants which Marker BAR gene resistant to Basta herbicides was received. After PCR, to approve the final transgenic plants, transformation efficiency was evaluated by 9.16%. Regeneration of explants treated with hormones showed a significant difference at 1% compared with control explants. The Maximum regeneration was showed in 0.5 mg/L kinetin treatment. These results indicated that the combination between kinetin and IAA could be effective in increasing regeneration.

Heterologous expression of Infectious bursal disease virus VP2 gene in Chlorella pyrenoidosa as a model system for molecular farming

Infectious bursal disease is a highly contagious and immunosuppressive disease of chickens. Available vaccines induce some sort of stress during vaccination program in chickens. In the present study, an attempt was made to overexpress IBDV VP2 in edible algae. These algae expressing IBDV VP2 could be used to vaccinate animals in stress free environment. IBDV VP2 over expression was carried under CaMV35S promoter and the gene expression cassette was cloned into pART27 binary vector. IBDV expressing construct was transformed into Chlorella pyrenoidosa through Agrobacterium tumefaciens mediated gene transfer. Transformed C. pyrenoidosa cells were selected through PCR screening test. Further, expression of IBDV VP2 protein in transformed C. pyrenoidosa was confirmed by western blot analysis to ensure the production of IBDV VP2 protein. This study will provide a new platform to generate oral vaccine against IBDV by using C. pyrenoidosa as model platform.

Concerning the role of cinnamoyl CoA reductase gene in phenolic acids biosynthesis in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge produces salvianolic acid, a water-soluble phenolic acid that is widely used in treating cardiovascular disease. The objective of this study is to employ AtPAP1 (Production of Anthocyanin Pigmentation) and SmCCR1 (cinnamoyl CoA reductase) for improving the phenolic acids of S. miltiorrhiza. First, results showed that AtPAP1 significantly increased the promoter activities of SmCCR1 by transient transformation of tobacco. To enrich the precursors available for phenolic acid biosynthesis, we present a strategy of combinational genetic manipulation by Agrobacterium tumefaciens-mediated gene transfer that simultaneously over-expressed AtPAP1 and down-regulated SmCCR1. Compared with the control, the amount of lignin was significantly reduced and its composition was changed in transgenic plants. While, the main phenolic acids, including salvianolic acid B and rosmarinic acid, were induced 2.0- and 1.8-fold compared with control lines in two-month-old plants. Meanwhile, the contents of total phenolics and total flavonoids were significantly improved in transformed plants. Expression of related genes throughout the phenylpropanoid pathways were altered, following the same trends as shown for their products, i.e., phenolic acids accumulation and lignin reduction. These results demonstrate that such methods for genetic modification, which increase the expression of transcription factors while suppressing that of branch genes, are of great value when genetically engineering plants for other secondary products.

Function Analysis of Caffeoyl-CoA O-Methyltransferase for Biosynthesis of Lignin and Phenolic Acid in Salvia miltiorrhiza

In this study, we cloned a full-length cDNA and the genomic DNA sequence of SmCCoAOMT (GenBank ID JQ007585) from Salvia miltiorrhiza. The 744-bp open-reading frame encodes a protein of 247 amino acids that shares 95% similarity with one in Vitis vinifera. Real-time quantitative PCR analysis revealed that SmCCoAOMT is most highly expressed in the stems and can be induced by methyl jasmonate (MeJA) and XC-1 treatment. To evaluate its function in vivo, we generated RNA interference transgenic plants through Agrobacterium tumefaciens-mediated gene transfer. Compared with untransformed control plants, the transgenics had significantly less lignin and the expression of lignin-biosynthetic genes SmCCR and SmCOMT was depressed. In 90-day-old roots from plants of transgenic line M5, accumulations of rosmarinic acid and salvianolic acid B (Sal B) were greatly reduced by 0.89- and 0.69-fold, respectively. This low-Sal B phenotype was stable in the roots, with the level of accumulation being approximately 43.58mgg-1 dry weight, which was 52% of the amount measured in the untransformed control. Our results suggest that SmCCoAOMT is involved in lignin biosynthesis and affects the accumulation of phenolic acids. This study also provides potential guidance for using lignin-related genes to genetically engineer Salvia miltiorrhiza.

The effect of magnetic field strength on Agrobacterium tumefaciens-mediated gene transfer in Rapeseed (Brassica napus L.)

The effect of magnetic field strength on Agrobacterium tumefaciens-mediated gene transfer in Rapeseed (Brassica napus L.)

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under well-watered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to non-transgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17-24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes.

Transfering Shrunken 2 (Sh2) gene coding for adp-glucose pyrophosphorylase enzyme into some inbred maize lines by Agrobacterium tumefaciens mediated gene transfer

The Shrunken 2 (Sh2) gene was proved to be involved in starch biosynthesis as it codes for two large subunits of ADP-glucose pyrophosphorylase - an enzyme that plays an important role in regulating starch biosynthesis in cereal crops. The aim of this study was to transfer Sh2 gene into some inbred maize lines and regenerate transgenic maize plants possessing transgene for further study of the role of Sh2 gene in the enhancement of starch biosynthesis in maize. The Agrobacterium tumefaciens strains C58 and DHA105 harboring transformation vector pCambia1301/Ubi/Sh2 containing Sh2 gene and monocot-specific Ubiquitin promoter were used to transfer Sh2 gene into immature embryos of the maize lines. PCR amplification of Ubi and Sh2 genes was performed to confirm the presence of the transgene in T0 transgenic plants. While, Southern and Northern hybridizations were carried out to confirm stable integration and expression of Sh2 gene in T0 and T1 transgenic plants, respectively. Transgenic plants were regenerated from transformed calli of six maize lines H95, H240, H26, H14, H4 and CML161 with the transformation frequencies from 1.08 to 1.77%. A. tumefaciens strain DHA105 gave higher transformation frequencies than that of C58 strain and the values ranged from 1.60 to 4.31%. The transgenic maize plants possessed 1 to 3 copies of transgene. Many transgenic plants grew normally and set seeds. The study of the effect of transgene in starch biosynthesis in transgenic maize lines will be performed.

Transfering Shrunken 2 (Sh2) gene coding for adp-glucose pyrophosphorylase enzyme into some inbred maize lines by Agrobacterium tumefaciens mediated gene transfer

Transfering Shrunken 2 (Sh2) gene coding for adp-glucose pyrophosphorylase enzyme into some inbred maize lines by Agrobacterium tumefaciens mediated gene transfer