Agrobacterium-mediated Plant Transformation Research Articles

Agrobacterium-mediated genetic transformation of plants: The role of host

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.

Differential localization of two plant homologues of Rab5 GTPases in the secretory pathway

Rab5 GTPases are key regulators of vesicular transport. Known plant Rab5 homologues ARA6 and RHA1 have been assigned to the endocytic and/or biosynthetic vesicular trafficking pathway, but conflicting reports in the literature justify further work on these two GTPases. In this project, binary vectors for Agrobacterium-mediated plant transformation were constructed to drive expression in tobacco leaf epidermis of GTP-trapped mutants of ARA6-green fluorescent protein (GFP) and Venus-RHA1. Confocal laser scanning microscopy revealed key differences in the subcellular localization of the two fluorescently tagged GTPase mutants. When present in the GTP-locked configuration ARA6-GFP is primarily found associated with the tonoplast, whereas Venus-RHA1 is significantly cytosolic. Co-expression of the two fluorescently tagged mutant GTPases with the Golgi markers ST-YFP and ST-CFP, respectively, suggest that neither ARA6 nor RHA1 mutants are mis-targeted to the Golgi apparatus and moreover, they do not show any significant colocalization with each other. The results are consistent with the notion that they differ in their roles within the endocytic and biosynthetic vesicular pathway. A future focus to continue this project would be to use GFP-tagged ARA7, the third plant Rab5 homologue, to verify if the high sequence homology between RHA1 and ARA7 warrants overlapping redundant functions or not.

Agrobacterium-mediated transformation of plants: Transfer of vector DNA fragments in the plant genome

The review presents experimental data on the integration in the plant nuclear genome of vector sequences during Agrobacterium-mediated transformation. Main attention is given to the frequency and mechanisms of this event among transgenic plants. The factors influencing the frequency of this event, as well as the ways of developing special constructs for elimination of vector backbone sequences as undesirable from the point of view of commercial use of genetically modified plants, are considered.

Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

Advances in Agrobacterium-mediated plant transformation with enphasys on soybean

Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.

The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation.

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.

An Improved pPZP Vector for Agrobacterium-mediated Plant Transformation

We report a new and improved pPZP vector (pPZP3425) for efficient plant transformation. This vector is derived from the widely used pPZP100 series of binary Agrobacterium vectors. One disadvantage of these vectors is the use of chloramphenicol resistance for selection in Escherichia coli and Agrobacteria. We have therefore included a kanamycin resistance gene for selection in Agrobacterium. Furthermore, the strong 35S CaMV promoter driving the plant resistance gene has been replaced by the weaker nos promoter because it has been shown that the 35S promoter driving the plant resistance marker can lead to ectopic expression of the transgene. During replacement of the 35S promoter, the NcoI site within the plant resistance gene has been removed, and NcoI can now be used for cloning purposes within the expression cassette which consists of an intron-containing gus gene driven by a strong constitutive promoter (35S promoter with doubled enhancer plus omega-element as translational enhancer). Thus, a single vector can conveniently be used for two purposes: (1) for overexpression of proteins by replacing the gus gene by the coding sequence of choice and (2) for creation of promoter:gus fusions by substituting the constitutive promoter by any other promoter. We demonstrate the usefulness of this vector for cloning a promoter:gus fusion and in planta transformation of Arabidopsis.

A modified low copy number binary vector pUN for Agrobacterium-mediated plant transformation

A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.

Identification and Characterization of Plant Genes Involved inAgrobacterium-Mediated Plant Transformation by Virus-Induced Gene Silencing

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized.

Agrobacterium‐mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.

Gateway‐compatible vectors for plant functional genomics and proteomics

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

Genetic transformation of Coffea canephora by vacuum infiltration

Agrobacterium-mediated plant transformation protocol was evaluated as a fast method to obtain genetically modified Coffea canephora plantlets. Leaf explants were used as source material for Agrobacterium tumefaciens-mediated transformation involving a vacuum infiltration protocol, followed by a step of somatic embryogenesis induction and a final selection of the transformed plants. A. tumefaciens strain C58CI containing the binary vector pER10W-35SRed was used. PCR amplification of DsRFP gene and visual detection of the red fluorescent protein demonstrated 33% transformed embryos. The protocol presented here produces reliable transgenic coffee embryos in two months.

Agrobacterium-mediated transformation of plantain (Musa spp.) cultivar Agbagba

An Agrobacterium-mediated plant transformation system was developed for the production of transgenic plantain [Musa spp. cultivar Agbagba (AAB)]. Apical shoot tips were transformed using Agrobacterium strain EHA105 with the binary vector pCAMBIA 1201, having the hygromycin resistance gene as a selection marker and GUS-INT as a reporter gene. Transient expression of the bglucuronidase (uid A) gene was achieved in transformed apical shoot tips. The hygromycin resistant shoots were regenerated 4 to 5 weeks after co-cultivation of explants with Agrobacterium. The two step selection procedure allowed the regeneration of shoots which were uniformly transformed. The integration of the uid A gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. In this study, transformation based on regeneration from apical shoot tips has been demonstrated. This process does not incorporate steps using disorganized cell cultures but uses micropropagation, which has the important advantage that it allows regeneration of homogeneous populations of plants in a short period of time. This study shows the enormous potential for genetic manipulation of Musa species for disease and pest resistance, as well as abiotic factors, using a rapid and non-species specific transformation and regeneration system.

EFFECTS OF REDUCING COMPOUNDS ON AGROBACTERIUM-MEDIATED TRANSFORMATION OF SWEET POTATO

Reducing compounds have so far been used for improvement of Agrobacterium-mediated transformation of crop plants with a strong ability to give rise to tissue browning, but not for that of sweet potato. Therefore, we examined the effects of commercially available five reducing compounds (L-ascorbic acid, L-cysteine, dithiothreitol, 2-mercaptoethanol, and reduced glutathione) on parameters associated with Agrobacterium system prior to applying them to sweet potato transformation. The parameters chosen in this study are enzymatic browning caused by sweet potato leaves, callus formation from the leaves and vir gene expression in Agrobacterium. All five reducing compounds restrained acute browning at concentrations of 20-60 mM, of the buffer in which the wound leaves were incubated. However, the reducing compounds except reduced glutathione arrested callus formation from the healthy leaves at 10 mM or less. Thus, we tested whether reducing compounds at these low concentration ranges can suppress tissue browning of sweet potato leaves which is slowly occurred under hygromycin B, and found that all of them failed. Furthermore, the compounds except 2-mercaptoethanol repressed vir gene expression at concentrations of 1-10 mM, which is essential to transfer T-DNA molecules from the bacteria to plant cells. Based on the results, we discuss usage of the reducing compounds on sweet potato transformation.

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.