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Nature Protocols Jan 2023There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant... (Review)
Review
There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.
Topics: Agrobacterium; Nicotiana; Plants, Genetically Modified; Coculture Techniques; Gene Editing; Genome, Plant; Soil; CRISPR-Cas Systems; Transformation, Genetic
PubMed: 36253612
DOI: 10.1038/s41596-022-00749-9 -
Plant Biotechnology Journal Oct 2021Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is...
Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.
Topics: Agrobacterium; CRISPR-Cas Systems; Cannabis; Gene Editing; Mutagenesis; Plants, Genetically Modified; Transformation, Genetic
PubMed: 33960612
DOI: 10.1111/pbi.13611 -
Metal Ions in Life Sciences Mar 2020Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to...
Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ββα architecture consisting in a β-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a βββαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.
Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Humans; Proteins; Zinc; Zinc Fingers
PubMed: 32851833
DOI: 10.1515/9783110589757-018 -
International Journal of Molecular... Aug 2021species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of... (Review)
Review
species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.
Topics: Agrobacterium; Chromosomes, Plant; DNA Repair; DNA, Bacterial; DNA, Plant; Plants; Transformation, Genetic
PubMed: 34445162
DOI: 10.3390/ijms22168458 -
Advances in Genetics 2022Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA... (Review)
Review
Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA (T-DNA, transferred DNA) in their host genome. Whereas in nature this process results in uncontrolled growth of the infected plant cells (tumors), this capability of has been widely used as a crucial tool to generate transgenic plants, for research and biotechnology. The virulence of relies on a series of virulence genes, mostly encoded on a large plasmid (Ti-plasmid, tumor inducing plasmid), involved in the different steps of the DNA transfer to the host cell genome: activation of bacterial virulence, synthesis and export of the T-DNA and its associated proteins, intracellular trafficking of the T-DNA and effector proteins in the host cell, and integration of the T-DNA in the host genomic DNA. Multiple interactions between these bacterial encoded proteins and host factors occur during the infection process, which determine the outcome of the infection. Here, we review our current knowledge of the mechanisms by which bacterial and plant factors control virulence and host plant susceptibility.
Topics: Virulence; Agrobacterium tumefaciens; Plants, Genetically Modified; Plasmids; Bacteria; Bacterial Proteins; Virulence Factors
PubMed: 37283660
DOI: 10.1016/bs.adgen.2022.08.001 -
Biomolecules Apr 2020Ginsenosides are secondary metabolites that belong to the triterpenoid or saponin group. These occupy a unique place in the pharmaceutical sector, associated with the... (Review)
Review
Ginsenosides are secondary metabolites that belong to the triterpenoid or saponin group. These occupy a unique place in the pharmaceutical sector, associated with the manufacturing of medicines and dietary supplements. These valuable secondary metabolites are predominantly used for the treatment of nervous and cardiac ailments. The conventional approaches for ginsenoside extraction are time-consuming and not feasible, and thus it has paved the way for the development of various biotechnological approaches, which would ameliorate the production and extraction process. This review delineates the biotechnological tools, such as conventional tissue culture, cell suspension culture, protoplast culture, polyploidy, in vitro mutagenesis, hairy root culture, that have been largely implemented for the enhanced production of ginsenosides. The use of bioreactors to scale up ginsenoside yield is also presented. The main aim of this review is to address the unexplored aspects and limitations of these biotechnological tools, so that a platform for the utilization of novel approaches can be established to further increase the production of ginsenosides in the near future.
Topics: Agrobacterium; Biotechnology; Ginsenosides; Transformation, Genetic
PubMed: 32252467
DOI: 10.3390/biom10040538 -
International Journal of Molecular... Jun 2023Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting... (Review)
Review
Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. -mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (, and ) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.
Topics: Plants, Genetically Modified; Transformation, Genetic; Plant Breeding; Gene Transfer Techniques; Agrobacterium
PubMed: 37445824
DOI: 10.3390/ijms241310646 -
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 -
Environmental Microbiology Jan 2018Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity... (Review)
Review
Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity in shaping the ecological and evolutionary dynamics of these facultative pathogens are incompletely understood. Agrobacterium tumefaciens, the causative agent for crown gall disease of plants has proven a productive model for many aspects of interactions between pathogens and their hosts and with other microbes. In this review, we highlight how this past work provides valuable context for the use of this system to examine how heterogeneity and transitions between disease and non-disease environments influence the ecology and evolution of facultative pathogens. We focus on several features common among facultative pathogens, such as the physiological remodelling required to colonize hosts from environmental reservoirs and the consequences of competition with host and non-host associated microbiota. In addition, we discuss how the life history of facultative pathogens likely often results in ecological tradeoffs associated with performance in disease and non-disease environments. These pathogens may therefore have different competitive dynamics in disease and non-disease environments and are subject to shifting selective pressures that can result in pathoadaptation or the within-host spread of avirulent phenotypes.
Topics: Agrobacterium tumefaciens; Biofilms; Biological Evolution; Ecology; Plant Tumors; Plants; Plasmids
PubMed: 29105274
DOI: 10.1111/1462-2920.13976 -
Microbial Cell Factories Oct 2017Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of... (Review)
Review
Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.
Topics: Agrobacterium; Biolistics; Electroporation; Fungi; Gene Transfer Techniques; Genetic Techniques; Genome, Fungal; Protoplasts; Transformation, Genetic
PubMed: 28974205
DOI: 10.1186/s12934-017-0785-7