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Biomedicines Jul 2022underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important...
underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important plant species. The bacterium initiates infection by colonizing and forming biofilms on plant surfaces, and thus, novel compounds are required to prevent its growth and biofilm formation. In this study, we investigated the ability of tannic acid, which is ubiquitously present in woody plants, to specifically inhibit the growth and biofilm formation of . Tannic acid showed antibacterial activity and significantly reduced the biofilm formation on polystyrene and on the roots of as determined by 3D bright-field and scanning electron microscopy (SEM) images. Furthermore, tannic acid dose-dependently reduced the virulence features of , which are swimming motility, exopolysaccharide production, protease production, and cell surface hydrophobicity. Transcriptional analysis of cells (Abs600 nm = 1.0) incubated with tannic acid for 24 h at 30 °C showed tannic acid most significantly downregulated the gene, which is required for adhesion to surfaces. Tannic acid at 100 or 200 µg/mL limited the iron supply to and similarly reduced the biofilm formation to that performed by 0.1 mM EDTA. Notably, tannic acid did not significantly affect germination even at 400 µg/mL. The findings of this study suggest that tannic acid has the potential to prevent growth and biofilm formation by and thus infections resulting from colonization.
PubMed: 35884920
DOI: 10.3390/biomedicines10071619 -
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 -
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 -
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 -
Journal of Applied Microbiology Nov 2022In the current study the anti-virulence and anti-biofilm activities of the cinnamic acid derivative, 3-methoxycinnamic acid, was investigated against Agrobacterium...
AIMS
In the current study the anti-virulence and anti-biofilm activities of the cinnamic acid derivative, 3-methoxycinnamic acid, was investigated against Agrobacterium tumefaciens.
METHODS AND RESULTS
Based on the disc diffusion test and β-galactosidase activity assay, 3-methoxycinnamic acid was shown to interfere with the quorum sensing (QS) system of A. tumefaciens. Crystal violet staining assay, phenol-sulfuric acid method, Bradford protein assay and confocal laser scanning microscopy (CLSM) revealed that the biofilm formation of A. tumefaciens was inhibited after the treatment of 3-methoxycinnamic acid. Employing high-performance liquid chromatography (HPLC) analysis of culture supernatant revealed that the production of 3-oxo-octanoylhomoserine lactone (3-oxo-C8-HSL) decreased concentration-dependently after treatment with 3-methoxycinnamic acid. Swimming and chemotaxis assays also indicated that 3-methoxycinnamic acid had a good effect on reducing the motility and chemotaxis of A. tumefaciens. In addition, the RT-qPCR, molecular docking and simulations further demonstrated that 3-methoxycinnamic acid could competitively inhibit the binding of 3-oxo-C8-HSL to TraR and down-regulate virulence-related genes.
CONCLUSIONS
3-Methoxycinnamic acid is proved to have good anti-virulence and anti-biofilm activities against A. tumefaciens.
SIGNIFICANCE AND IMPACT OF THE STUDY
This is the first study that investigates the anti-virulence and anti-biofilm activities of 3-methoxycinnamic acid against A. tumefaciens. With its potential QS-related virulence and biofilm inhibitory activities, 3-methoxycinnamic acid is expected to be developed as a potent pesticide or adjuvant for the prevention and treatment of crown gall caused by A. tumefaciens.
Topics: Agrobacterium tumefaciens; Molecular Docking Simulation; Gentian Violet; Quorum Sensing; Biofilms; 4-Butyrolactone; Phenols; Pesticides; beta-Galactosidase
PubMed: 35951737
DOI: 10.1111/jam.15774 -
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,... 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 -
Proceedings of the National Academy of... Feb 2022Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly...
Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: " clustering" in which the replication origins of all four replicons interact, and "Ch1-Ch2 alignment" in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepB for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Cell Cycle; Chromosomes, Bacterial; Genome, Bacterial; Replicon
PubMed: 35101983
DOI: 10.1073/pnas.2115854119 -
The New Phytologist Jan 2022Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover...
Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen.
Topics: Agrobacterium tumefaciens; Carbon; Solanum lycopersicum; Plant Roots; Plant Tumors; Virulence
PubMed: 34655498
DOI: 10.1111/nph.17810 -
Biotechnology and Bioengineering Oct 2021Agrobacterium tumefaciens is a soil-borne bacterium that is known for its DNA delivery ability and widely exploited for plant transformation. Despite continued interest...
Agrobacterium tumefaciens is a soil-borne bacterium that is known for its DNA delivery ability and widely exploited for plant transformation. Despite continued interest in improving the utility of the organism, the lack of well-characterized engineering tools limits the realization of its full potential. Here, we present a synthetic biology toolkit that enables precise and effective control of gene expression in A. tumefaciens. We constructed and characterized six inducible expression systems. Then, we optimized the one regulated by cumic acid through amplifier introduction and promoter engineering and evaluated its 15 cognate promoters. To establish fine-tunability, we constructed a series of spacers and a promoter library to systematically modulate both translational and transcriptional rates. We finally demonstrated the application of the tools by co-expressing genes with altered expression levels using a single signal. This study provides precise expression tools for A. tumefaciens, facilitating rational engineering of the bacterium for advanced plant biotechnological applications.
Topics: Agrobacterium tumefaciens; Gene Expression Regulation, Bacterial; Genetic Engineering; Promoter Regions, Genetic
PubMed: 34180537
DOI: 10.1002/bit.27872