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Biotechnology Advances Dec 2021Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice... (Review)
Review
Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice for gene delivery into plants. Ever since, optimized Agrobacterium strains have been developed with additional (genetic) modifications that were mostly aimed at enhancing the transformation efficiency, although an optimized strain also exists that reduces unwanted plasmid recombination. As a result, a collection of very useful strains has been created to transform a wide variety of plant species, but has also led to a confusing Agrobacterium strain nomenclature. The latter is often misleading for choosing the best-suited strain for one's transformation purposes. To overcome this issue, we provide a complete overview of the strain classification. We also indicate different strain modifications and their purposes, as well as the obtained results with regard to the transformation process sensu largo. Furthermore, we propose additional improvements of the Agrobacterium-mediated transformation process and consider several worthwhile modifications, for instance, by circumventing a defense response in planta. In this regard, we will discuss pattern-triggered immunity, pathogen-associated molecular pattern detection, hormone homeostasis and signaling, and reactive oxygen species in relationship to Agrobacterium transformation. We will also explore alterations that increase agrobacterial transformation efficiency, reduce plasmid recombination, and improve biocontainment. Finally, we recommend the use of a modular system to best utilize the available knowledge for successful plant transformation.
Topics: Agrobacterium tumefaciens; Gene Transfer Techniques; Plants, Genetically Modified; Recombination, Genetic; Transformation, Genetic
PubMed: 33290822
DOI: 10.1016/j.biotechadv.2020.107677 -
Phytopathology Apr 2023The phytopathogenic bacterium causes crown gall disease in plants, characterized by the formation of tumor-like galls where wounds were present. Nowadays, however, the... (Review)
Review
The phytopathogenic bacterium causes crown gall disease in plants, characterized by the formation of tumor-like galls where wounds were present. Nowadays, however, the bacterium and its Ti (tumor-inducing) plasmid is better known as an effective vector for the genetic manipulation of plants and fungi. In this review, I will briefly summarize some of the major discoveries that have led to this bacterium now playing such a prominent role worldwide in plant and fungal research at universities and research institutes and in agricultural biotechnology for the production of genetically modified crops. I will then delve a little deeper into some aspects of biology and discuss the diversity among agrobacteria and the taxonomic position of these bacteria, the diversity in Ti plasmids, the molecular mechanism used by the bacteria to transform plants, and the discovery of protein translocation from the bacteria to host cells as an essential feature of -mediated transformation.
Topics: Plant Tumor-Inducing Plasmids; Crops, Agricultural; Plant Diseases; Plants, Genetically Modified; Agrobacterium tumefaciens; Plant Tumors; Plasmids
PubMed: 37098885
DOI: 10.1094/PHYTO-11-22-0432-IA -
Transgenic Research Apr 2023Agrobacterium tumefaciens-mediated plant transformation has become routine work across the world to study gene function and the production of genetically modified... (Review)
Review
Agrobacterium tumefaciens-mediated plant transformation has become routine work across the world to study gene function and the production of genetically modified plants. However, several issues hamper the transformation process in a profound way, both directly and indirectly. One of the major concerns is the overgrowth of Agrobacterium, which occasionally appears after the co-cultivation phase of the explant. This phenomenon is reported in several species and seems to spoil the whole transformation process. There are multiple approaches being employed to counter this unwanted growth of bacteria in a few plant species. In reality, once the overgrowth appears, it becomes nearly impossible to cure it. Hence, for the prevention of this phenomenon, numerous factors are regulated. These factors are: explant nature, A. tumefaciens strain, T-DNA vector, co-cultivation (time and condition), acetosyringone, washing medium, antibiotics (type, concentration, combination, incubation period), etc. In this article, we discuss these factors based on available reports. It can be of immense help in formulating viable strategies to control A. tumefaciens overgrowth.
Topics: Agrobacterium tumefaciens; Plants; Transformation, Genetic; Plants, Genetically Modified
PubMed: 36806963
DOI: 10.1007/s11248-023-00338-w -
Methods in Molecular Biology (Clifton,... 2015Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated procedure on leaves from in vitro proliferated shoots....
Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated procedure on leaves from in vitro proliferated shoots. Regardless of the sufficient regeneration levels achieved from leaf explants of some commercial strawberry genotypes, the regeneration of transformed strawberry plants remains difficult and seems to be strongly genotype dependent. In fact, the main factors that play an important role in the success of strawberry genetic transformation are the availability of an efficient regeneration protocol and of an appropriate selection procedure of the putative transgenic shoots. The strawberry genetic transformation protocol herein described relates to three genotypes resulted from our experience with the highest regeneration and transformation efficiency. The study includes two octoploid Fragaria × ananassa cultivars, Sveva and Calypso, and a diploid F. vesca cultivar (Alpina W.O.). All the different steps related to the leaf tissue Agrobacterium infection, coculture, and selection of regenerating adventitious shoots, as well as the following identification of selected lines able to proliferate and root on the selective agent (kanamycin), will be described.
Topics: Agrobacterium tumefaciens; Fragaria; Genetic Engineering; Plant Leaves; Plant Roots; Soil; Transformation, Genetic
PubMed: 25416261
DOI: 10.1007/978-1-4939-1658-0_18 -
ACS Synthetic Biology Aug 2022is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including...
is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between and host cells promotes infection efficacy. Thus, improving the strength and specificity of to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here, we demonstrate a methodology for engineering genetically encoded exogeneous adhesins at the surface of . We identified an autotransporter gene we named Aat that is predicted to show canonical β-barrel and passenger domains. We engineered the β-barrel scaffold and linker (Aat) to display synthetic adhesins susceptible to rewire to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.
Topics: Adhesins, Bacterial; Agrobacterium tumefaciens; HeLa Cells; Humans; Protein Transport
PubMed: 35881049
DOI: 10.1021/acssynbio.2c00069 -
Methods in Molecular Biology (Clifton,... 2015Populus trichocarpa Nisqually-1 is a clone of black cottonwood that is widely used as a model woody plant. It was the first woody plant to have a full genome sequence...
Populus trichocarpa Nisqually-1 is a clone of black cottonwood that is widely used as a model woody plant. It was the first woody plant to have a full genome sequence and remains today as the model for growth, metabolism, development, and adaptation for all woody dicotyledonous plants. It is one of the best-annotated plant genomes available. It is also currently studied to improve bioenergy feedstocks and to learn about responses to environmental variation that may result from climate change. It is the best characterized woody plant for lignin biosynthesis. In spite of its role as a model woody plant, many important genetic applications have been limited because it was particularly difficult for DNA transformation. The ability to transform P. trichocarpa is a central component of a systems biology approach to the study of metabolic and developmental processes, where in combination with genome and transcriptome sequencing, all the expressed genes for specific pathways can be defined, cloned, and characterized for biological function. We previously reported on a method for Agrobacterium-mediated genetic transformation in P. trichocarpa(Song et al. Plant Cell Physiol 47: 1582-1589, 2006). Since then, we have optimized the protocol based on many experiments that varied in tissue manipulation, media, DNA constructs and Agrobacterium strains. A modified step-by-step protocol for Agrobacterium-mediated transformation of stem explants is described here. The health of the tissue explants and the time of cocultivation are among the critical steps in the protocol for successful transformation. This updated protocol should be helpful to many laboratories that are currently carrying out P. trichocarpa transformation. It should also encourage many labs that have not yet had success with P. trichocarpa to try again.
Topics: Agrobacterium tumefaciens; Coculture Techniques; Genetic Engineering; Plant Roots; Plant Shoots; Populus; Regeneration; Transformation, Genetic
PubMed: 25416271
DOI: 10.1007/978-1-4939-1658-0_28 -
Methods in Molecular Biology (Clifton,... 2022Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. An efficient...
Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. An efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, F. oxysporum, the causal agent of Apple replant disease (ARD) in China. Four parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidial concentration of F. oxysporum, and co-culture temperature and time have a significant influence on all parameters. Transformants emit green fluorescence under fluorescence microscopy. The integration of a mitotically stable hygromycin resistance gene (hph) in the genome is confirmed by PCR. The transformation efficiency can reach up to 300 transformants per 10 conidia under optimal conditions. This ATMT method is an efficient tool for insertional mutagenesis of F. oxysporum.
Topics: Agrobacterium tumefaciens; DNA, Bacterial; Fusarium; Mutagenesis, Insertional; Spores, Fungal; Transformation, Genetic
PubMed: 34686977
DOI: 10.1007/978-1-0716-1795-3_6 -
Current Topics in Microbiology and... 2018Agrobacterium tumefaciens attaches stably to plant host tissues and abiotic surfaces. During pathogenesis, physical attachment to the site of infection is a prerequisite... (Review)
Review
Agrobacterium tumefaciens attaches stably to plant host tissues and abiotic surfaces. During pathogenesis, physical attachment to the site of infection is a prerequisite to infection and horizontal gene transfer to the plant. Virulent and avirulent strains may also attach to plant tissue in more benign plant associations, and as with other soil microbes, to soil surfaces in the terrestrial environment. Although most A. tumefaciens virulence functions are encoded on the tumor-inducing plasmid, genes that direct general surface attachment are chromosomally encoded, and thus this process is not obligatorily tied to virulence, but is a more fundamental capacity. Several different cellular structures are known or suspected to contribute to the attachment process. The flagella influence surface attachment primarily via their propulsive activity, but control of their rotation during the transition to the attached state may be quite complex. A. tumefaciens produces several pili, including the Tad-type Ctp pili, and several plasmid-borne conjugal pili encoded by the Ti and At plasmids, as well as the so-called T-pilus, involved in interkingdom horizontal gene transfer. The Ctp pili promote reversible interactions with surfaces, whereas the conjugal and T-pili drive horizontal gene transfer (HGT) interactions with other cells and tissues. The T-pilus is likely to contribute to physical association with plant tissues during DNA transfer to plants. A. tumefaciens can synthesize a variety of polysaccharides including cellulose, curdlan (β-1,3 glucan), β-1,2 glucan (cyclic and linear), succinoglycan, and a localized polysaccharide(s) that is confined to a single cellular pole and is called the unipolar polysaccharide (UPP). Lipopolysaccharides are also in the outer leaflet of the outer membrane. Cellulose and curdlan production can influence attachment under certain conditions. The UPP is required for stable attachment under a range of conditions and on abiotic and biotic surfaces. Other factors that have been reported to play a role in attachment include the elusive protein called rhicadhesin. The process of surface attachment is under extensive regulatory control and can be modulated by environmental conditions, as well as by direct responses to surface contact. Complex transcriptional and post-transcriptional control circuitry underlies much of the production and deployment of these attachment functions.
Topics: Agrobacterium tumefaciens; Bacterial Adhesion; Bacterial Proteins; Fimbriae, Bacterial; Flagella; Virulence
PubMed: 29998422
DOI: 10.1007/82_2018_96 -
International Journal of Molecular... May 2023The transformation efficiency (TE) was improved by a series of special chemical and physical methods using immature embryos from the cultivar Fielder, with the PureWheat...
The transformation efficiency (TE) was improved by a series of special chemical and physical methods using immature embryos from the cultivar Fielder, with the PureWheat technique. To analyze the reaction of immature embryos infected, which seemed to provide the necessary by in PureWheat, a combination of scanning electron microscopy (SEM), complete transcriptome analysis, and metabolome analysis was conducted to understand the progress. The results of the SEM analysis revealed that were deposited under the damaged cortex of immature embryos as a result of pretreatment and contacted the receptor cells to improve the TE. Transcriptome analysis indicated that the differentially expressed genes were mainly enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, plant-pathogen interaction, plant hormone signal transduction, and the MAPK (Mitogen-activated protein kinase) signaling pathway. By analyzing the correlation between differentially expressed genes and metabolites, the expression of many genes and the accumulation of metabolites were changed in glucose metabolism and the TCA cycle (Citrate cycle), as well as the amino acid metabolism; this suggests that the infection of wheat embryos with is an energy-demanding process. The shikimate pathway may act as a hub between glucose metabolism and phenylpropanoid metabolism during infection. The downregulation of the gene and upregulation of the gene led to the accumulation of lignin precursors through phenylpropanoid metabolism. In addition, several metabolic pathways and oxidases were found to be involved in the infection treatment, including melatonin biosynthesis, benzoxazinoid biosynthesis, betaine biosynthesis, superoxide dismutase, and peroxidase, suggesting that wheat embryos may be under the stress of and, thus, undergo an oxidative stress response. These findings explore the physiological and molecular changes of immature embryos during the co-culture stage of the PureWheat technique and provide insights for -mediated transgenic wheat experiments.
Topics: Agrobacterium tumefaciens; Triticum; Transcriptome; Plants, Genetically Modified; Gene Expression Profiling; Glucose
PubMed: 37176157
DOI: 10.3390/ijms24098449 -
Plant & Cell Physiology Dec 2021Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of...
Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.
Topics: Agrobacterium tumefaciens; Marchantia; Plants, Genetically Modified; Transduction, Genetic; Transformation, Genetic
PubMed: 34383076
DOI: 10.1093/pcp/pcab126