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Plant Communications Apr 2024Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated...
Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocols and low efficiency of current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of the regenerative activity-dependent in planta injection delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues. Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues. The method was successfully used for transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (Ipomoea pes-caprae). Compared with traditional transformation methods, RAPID has a much higher transformation efficiency and shorter duration, and it does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.
Topics: Plants, Genetically Modified; Agrobacterium tumefaciens; Ipomoea batatas
PubMed: 38243598
DOI: 10.1016/j.xplc.2024.100822 -
Plant Biotechnology Journal Jul 2024Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be...
Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.
Topics: Gene Editing; Transformation, Genetic; Agrobacterium; Plants, Genetically Modified; CRISPR-Cas Systems; Plant Leaves; Kalanchoe; Gene Transfer Techniques
PubMed: 38425137
DOI: 10.1111/pbi.14318 -
The New Phytologist Jun 2024
PubMed: 38837421
DOI: 10.1111/nph.19881 -
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 -
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 -
Nature Plants Sep 2023Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes. Transfer DNA (T-DNA) originating from...
Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.
Topics: Gene Editing; Genome, Plant; Retroelements; Genes, Plant; Arabidopsis
PubMed: 37653336
DOI: 10.1038/s41477-023-01495-w -
Annual Review of Phytopathology Sep 2023Among plant-associated bacteria, agrobacteria occupy a special place. These bacteria are feared in the field as agricultural pathogens. They cause abnormal growth... (Review)
Review
Among plant-associated bacteria, agrobacteria occupy a special place. These bacteria are feared in the field as agricultural pathogens. They cause abnormal growth deformations and significant economic damage to a broad range of plant species. However, these bacteria are revered in the laboratory as models and tools. They are studied to discover and understand basic biological phenomena and used in fundamental plant research and biotechnology. Agrobacterial pathogenicity and capability for transformation are one and the same and rely on functions encoded largely on their oncogenic plasmids. Here, we synthesize a substantial body of elegant work that elucidated agrobacterial virulence mechanisms and described their ecology. We review findings in the context of the natural diversity that has been recently unveiled for agrobacteria and emphasize their genomics and plasmids. We also identify areas of research that can capitalize on recent findings to further transform our understanding of agrobacterial virulence and ecology.
Topics: Agrobacterium; Virulence; Biological Evolution; Ecology; Genomics
PubMed: 37164023
DOI: 10.1146/annurev-phyto-021622-125009 -
Molecular Biotechnology Aug 2023Plant transformation based on Agrobacterium-mediated transformation is a technique that mimics the natural agrobacterium system for gene(s) introduction into crops.... (Review)
Review
Plant transformation based on Agrobacterium-mediated transformation is a technique that mimics the natural agrobacterium system for gene(s) introduction into crops. Through this technique, various crop species have been improved/modified for different trait/s, showing a successful genetic transformation so far. This technique has many advantages over other transformation methods such as stable integration of transgene, cost effective. However, there are many limitations of this technology such as mostly the crops are recalcitrant to agrobacterium, low transformation efficiency, transgene integration as well as off targets. So, it's very important to explore the major limitations and possible solutions for Agrobacterium-mediated transformation in order to increase its genetic transformation efficiency. Therefore, the present review article gives a comprehensive study how the transgenic crops are developed using Agrobacterium-mediated transformation, crops that have already been modified through this method, and risks associated with transgenic plants based on Agrobacterium-mediated transformation. Moreover, the challenges and problems associated with Agrobacterium-mediated transformation and how those problems can be solved in future for a successful genetic transformation of crops using modern biotechnology techniques such as CRISPR/Cas9 systems. The present review article will be really helpful for the audience those working on Genome editing of crops using Agrobacterium-mediated transformation and will opens many ways for future plant genetic transformation.
PubMed: 37573566
DOI: 10.1007/s12033-023-00826-8 -
Methods in Molecular Biology (Clifton,... 2024The yeast two-hybrid system is a powerful and commonly used genetic tool to investigate the interaction between artificial fusion proteins inside the nucleus of yeast....
The yeast two-hybrid system is a powerful and commonly used genetic tool to investigate the interaction between artificial fusion proteins inside the nucleus of yeast. Here, we describe how to use the Matchmaker GAL4-based yeast two-hybrid system to detect the interaction of the Agrobacterium type VI secretion system (T6SS) sheath components TssB and TssC. The bait and prey gene are expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD) and GAL4 activation domain (AD, prey/library fusion protein), respectively. When bait and prey fusion proteins interact in yeast nucleus, the DNA-BD and AD are brought into proximity, thus activating transcription of reporter genes. This technology can be widely used to identify interacting partners, confirm suspected interactions, and define interacting domains.
Topics: Humans; Agrobacterium; DNA; Gene Library; Saccharomyces cerevisiae
PubMed: 37930532
DOI: 10.1007/978-1-0716-3445-5_15 -
Chemosphere Aug 2023Extracellular polymeric substances (EPS) are highly hydrated matrices produced by bacteria, containing various polymers such as polysaccharides, proteins, lipids, and... (Review)
Review
Extracellular polymeric substances (EPS) are highly hydrated matrices produced by bacteria, containing various polymers such as polysaccharides, proteins, lipids, and DNA. Extracellular polymer concentrations, ions, and functional groups provide physical stability to the EPS. Constituents of EPS form the three-dimensional architecture and help acquire nutrition for the bacteria. Structural and functional diversity of the extracellular polymer depends on the specific glycosyltransferases, polymerase and transporter proteins. These enzymes are encoded by specific genes present in operons such as crd, alg, wca, and gum reported in Agrobacterium, Pseudomonas, Enterobacteriaceae, and Xanthomonas. The operons regulate the biosynthesis of extracellular polymers such as curdlan, alginate, colonic acid, and xanthan, respectively. Various functional groups in the EPS, such as carbonyl, hydroxyl, phosphoryl, and amide, provide the sorption site for interaction with environmental pollutants. Hydrophobic interactions and coordinate bonds mainly dominate the binding of EPS with environmental pollutants. EPS binds, emulsifies, and solubilizes the organic compounds, enhancing the degradation process. EPS binds with heavy metals through complexation, surface adsorption, precipitation, and ion exchange mechanisms. The biodegradability efficiency and nontoxicity properties of EPS make it an excellent biopolymer for decontaminating environmental pollutants. This review summarizes an overview of the biosynthetic mechanisms and interaction of the bacterial extracellular polymer with environmental pollutants. Interaction mechanisms of pollutants with EPS and EPS-mediated bioremediation will help develop removal applications. Moreover, understanding the genes responsible for EPS production, and implementation of new genetic methodology can be helpful for the enhanced biosynthesis of EPS to control pollution by sequestrating more environmental pollutants.
Topics: Extracellular Polymeric Substance Matrix; Environmental Pollutants; Metals, Heavy; Bacteria; Polymers; Adsorption
PubMed: 37164199
DOI: 10.1016/j.chemosphere.2023.138876