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Methods in Molecular Biology (Clifton,... 2024Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidences show that protein-protein interactions are crucial...
Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidences show that protein-protein interactions are crucial in many biological processes in living cells. Genetic (such as yeast two hybrid, Y2H) and biochemical (such as co-immunoprecipitation, co-IP) methods are the commonly used methods to identify the interacting proteins. Immunoprecipitation (IP), a method using a target protein-specific antibody in conjunction with Protein A/G affinity beads, is a powerful tool to identify the molecules interacting with specific proteins. Therefore, co-IP is considered to be one of the standard methods to identify and/or confirm the occurrence of the protein-protein interaction events in vivo. The co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use two different co-Ip protocols as an example to describe the principle, procedure, and experimental problems of co-IP. First, we show the interaction of two Agrobacterium type VI secretion system (T6SS) sheath components TssB and TssC, and secondly, we show the protocol we used for determining the interaction of an epitope-tagged T6SS effector, Tde1 expressed in Agrobacterium with endogenously expressing adaptor/chaperone protein Tap1.
Topics: Male; Humans; Antibodies; Agrobacterium; Epitopes; Foreskin; Immunoprecipitation; Saccharomyces cerevisiae
PubMed: 37930535
DOI: 10.1007/978-1-0716-3445-5_18 -
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 -
Plant Cell Reports Jun 2024A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and...
A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.
Topics: Glycine max; Plant Leaves; Plants, Genetically Modified; Agrobacterium; Gene Expression Regulation, Plant; Nicotiana; Genetic Vectors
PubMed: 38837057
DOI: 10.1007/s00299-024-03245-4 -
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 -
Systematic and Applied Microbiology Jan 2020The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus... (Review)
Review
The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future.
Topics: Agrobacterium; DNA, Bacterial; Genes, Bacterial; Genes, Essential; Genome, Bacterial; Humans; Phylogeny; Rhizobiaceae; Rhizobium
PubMed: 31818496
DOI: 10.1016/j.syapm.2019.126046 -
Molecular Biotechnology Feb 2020Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that... (Review)
Review
Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that resembles a disease-causing microorganism and is often made from weak or killed forms of the microbe. Vaccines are agents that stimulate the body's immune system to recognize the antigen. Now, a new form of vaccine was introduced which will have the power to mask the risk side of conventional vaccines. This type of vaccine was produced from plants which are genetically modified. In the production of edible vaccines, the gene-encoding bacterial or viral disease-causing agent can be incorporated in plants without losing its immunogenic property. The main mechanism of action of edible vaccines is to activate the systemic and mucosal immunity responses against a foreign disease-causing organism. Edible vaccines can be produced by incorporating transgene in to the selected plant cell. At present edible vaccine are developed for veterinary and human use. But the main challenge faced by edible vaccine is its acceptance by the population so that it is necessary to make aware the society about its use and benefits. When compared to other traditional vaccines, edible vaccines are cost effective, efficient and safe. It promises a better prevention option from diseases.
Topics: Administration, Oral; Agrobacterium; Animals; Biolistics; Biological Products; Chlorophyta; Gene Transfer Techniques; Humans; Immunity, Mucosal; Insecta; Lactobacillales; Molecular Farming; Organisms, Genetically Modified; Plant Viruses; Plants, Genetically Modified; Vaccines, Edible; Yeasts
PubMed: 31758488
DOI: 10.1007/s12033-019-00222-1 -
Current Protocols Dec 2022The storage root crop cassava (Manihot esculenta Crantz) is predicted to remain central to future food and economic security for smallholder farming households and...
The storage root crop cassava (Manihot esculenta Crantz) is predicted to remain central to future food and economic security for smallholder farming households and agricultural output in the tropics. Genetic improvement of cassava is required to meet changing farmer and consumer needs, evolving pests and diseases, and challenges presented by climate change. Transgenic and genome editing technologies offer significant potential for introducing desired traits into farmer-preferred varieties and breeding lines, and for studying the biology of this under-investigated crop species. A bottleneck in implementing genetic modification in this species has been access to robust methods for transformation of cassava cultivars and landraces. In this article, we provide a detailed protocol for Agrobacterium-mediated transformation of cassava and regeneration of genetically modified plants. Basic Protocol 1 describes how to establish and micropropagate in vitro cassava plantlets, and Alternate Protocol 1 details how to establish in vitro cultures from field or greenhouse cuttings. Basic Protocol 2 describes all steps necessary for genetic transformation in the model variety 60444, and Alternate Protocol 2 provides details for modifying this method for use with other cultivars. Finally, Basic Protocol 3 describes how to establish plants produced via Basic Protocol 2 and Alternate Protocol 2 in soil in a greenhouse. These methods have proven applications across more than a dozen genotypes and are capable of producing transgenic and gene-edited plants for experimental purposes, for testing under greenhouse and field conditions, and for development of plants suitable for subsequent regulatory approval and product deployment. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment and propagation of in vitro cassava plantlets Alternate Protocol 1: Establishment of in vitro plants from field or greenhouse plants Basic Protocol 2: Genetic transformation of cassava variety 60444 Alternate Protocol 2: Genetic transformation of additional cultivars Basic Protocol 3: Establishment and growth of plants in the greenhouse.
Topics: Manihot; Agrobacterium; Transformation, Genetic; Plants, Genetically Modified; Plant Breeding
PubMed: 36507868
DOI: 10.1002/cpz1.620 -
Methods in Molecular Biology (Clifton,... 2022Rice (Oryza sativa) is an important cereal crop and a model monocot plant for biology research. The reliable system of foreign DNA transformation and expression is a...
Rice (Oryza sativa) is an important cereal crop and a model monocot plant for biology research. The reliable system of foreign DNA transformation and expression is a valuable strategy for basic research and molecular breeding application in rice. The Agrobacterium tumefaciens-mediated foreign DNA transformation system was a powerful tool for genetic research. However, it needs a long period to obtain the stable transformants for further analysis and the transformation rate limits in some organism. Protoplasts are plant cells without a cell wall, and it is much easier for foreign DNA transformation and expression. It has been widely applied in transient expression. Here, we describe a simple method for efficient protoplast isolation and transfection in rice.
Topics: Agrobacterium tumefaciens; Oryza; Plants, Genetically Modified; Protoplasts; Transfection; Transformation, Genetic
PubMed: 35258826
DOI: 10.1007/978-1-0716-2164-6_6 -
Proceedings of the National Academy of... Jan 2021spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of...
spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both and As an example, we generated EHA105 strains with loss-of-function mutations in , which were fully functional for maize () transformation and confirmed the importance of RolB and RolC for hairy root development by K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved strains for more efficient plant transformation and gene editing.
Topics: Agrobacterium; Agrobacterium tumefaciens; CRISPR-Associated Proteins; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; DNA, Plant; Gene Editing; Genes, Plant; Genome, Plant; Mutagenesis; Mutation; Zea mays
PubMed: 33443212
DOI: 10.1073/pnas.2013338118