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Frontiers in Plant Science 2014Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the...
Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant-Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.
PubMed: 25520733
DOI: 10.3389/fpls.2014.00681 -
Biochimica Et Biophysica Acta Aug 2014The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on... (Review)
Review
The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the 'archetypal' A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Adenosine Triphosphatases; Agrobacterium tumefaciens; Bacterial Secretion Systems; DNA; Fimbriae, Bacterial; Gram-Negative Bacteria; Periplasmic Proteins; Protein Binding; Protein Folding; Protein Transport
PubMed: 24389247
DOI: 10.1016/j.bbamcr.2013.12.019 -
The FEBS Journal Dec 2015Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in... (Comparative Study)
Comparative Study Review
Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2 His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships.
Topics: Agrobacterium tumefaciens; Eukaryotic Cells; Humans; Prokaryotic Cells; Zinc Fingers
PubMed: 26365095
DOI: 10.1111/febs.13503 -
Genes Feb 2023In the post-genomics era, -mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in...
In the post-genomics era, -mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber ( L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the () gene or RNAi of the gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The -mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.
Topics: Cucumis sativus; Agrobacterium tumefaciens; Transformation, Genetic; Plants, Genetically Modified; Mutagenesis
PubMed: 36980873
DOI: 10.3390/genes14030601 -
Applied and Environmental Microbiology Feb 2021GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic...
GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels. Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.
Topics: Agrobacterium tumefaciens; Arsenates; Lipid Metabolism; Phosphates
PubMed: 33361371
DOI: 10.1128/AEM.02261-20 -
MBio May 2021The growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains...
The growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in to WT GPR and provided only partial complementation to cells lacking GPR. -specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. Bacterial growth and division are extensively studied in model systems (, , and ) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some and species. The latter phylum includes the family , in which many species, including , exhibit polar growth. Current research aims to identify growth pole (GP) factors. The growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.
Topics: Agrobacterium tumefaciens; Apolipoproteins; Cell Cycle Proteins; Cell Division; Cell Polarity; Cell Wall; Green Fluorescent Proteins
PubMed: 34006657
DOI: 10.1128/mBio.00764-21 -
International Journal of Molecular... Jun 2021() is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the...
() is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of mediated by (). was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. -mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene () (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of with the gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when LBA4404 (A = 1.0) and (A = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer for producing high-value products and fundamental studies.
Topics: Agrobacterium tumefaciens; Anti-Bacterial Agents; Biotechnology; Cinnamates; Clone Cells; DNA, Bacterial; Euglena gracilis; Gene Expression; Genes, Reporter; Hygromycin B; Microalgae; Mutagenesis, Insertional; Nuclear Transfer Techniques; Transformation, Genetic; Transgenes
PubMed: 34208268
DOI: 10.3390/ijms22126299 -
Microbial Genomics Nov 2020is an efficient tool for creating transgenic host plants. The first step in the genetic transformation process involves chemotaxis, which is crucial to the survival of...
is an efficient tool for creating transgenic host plants. The first step in the genetic transformation process involves chemotaxis, which is crucial to the survival of in changeable, harsh and even contaminated soil environments. However, a systematic study of its chemotactic signalling pathway is still lacking. In this study, the distribution and classification of chemotactic genes in the model C58 and 21 other strains were annotated. Local blast was used for comparative genomics, and hmmer was used for predicting protein domains. Chemotactic phenotypes for knockout mutants of ternary signalling complexes in C58 were evaluated using a swim agar plate. A major cluster, in which chemotaxis genes were consistently organized as MCP (methyl-accepting chemotaxis protein), CheS, CheY1, CheA, CheR, CheB, CheY2 and CheD, was found in , but two coupling CheW proteins were located outside the '' cluster. In the ternary signalling complexes, the absence of MCP atu0514 significantly impaired chemotaxis, and the absence of CheA (atu0517) or the deletion of both CheWs abolished chemotaxis. A total of 465 MCPs were found in the 22 strains, and the cytoplasmic domains of these MCPs were composed of 38 heptad repeats. A high homology was observed between the chemotactic systems of the 22 strains with individual differences in the gene and receptor protein distributions, possibly related to their ecological niches. This preliminary study demonstrates the chemotactic system of , and provides some reference for sensing and chemotaxis to exogenous signals.
Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Chemotaxis; Computer Simulation; Genome, Bacterial; Methyl-Accepting Chemotaxis Proteins; Phylogeny; Plants; Sequence Alignment; Signal Transduction
PubMed: 33118922
DOI: 10.1099/mgen.0.000460 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
Fungal Biology Sep 2019The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon...
The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102-148 transformants per 10 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.
Topics: Agrobacterium tumefaciens; Ascomycota; China; Cucumis melo; DNA, Bacterial; Fungal Proteins; Mutagenesis, Insertional; Plant Diseases; Transformation, Genetic; Virulence
PubMed: 31416586
DOI: 10.1016/j.funbio.2019.05.011