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Applied and Environmental Microbiology Mar 2022The discovery of new and efficient genetic engineering technologies for will broaden the capacity for fundamental research on this genus and its utilization as a...
The discovery of new and efficient genetic engineering technologies for will broaden the capacity for fundamental research on this genus and its utilization as a transgenic vehicle. In this study, we aim to develop an efficient recombineering system for species. We examined isolates of and the closely related genus to identify pairs of ET-like recombinases that would aid in the recombineering of species. Four pairs of ET-like recombinases, named RecETh1h2h3h4, RecETh1h2P3, RecET, and RecETh, were identified in Agrobacterium tumefaciens strain B6, Rhizobium leguminosarum bv. trifolii WSM597, sp. strain LC145, and sp. strain Root483D2, respectively. Eight more candidate recombineering systems were generated by combining the new ET-like recombinases with Redγ or Pluγ. The PluγET system, the RecETh1h2h3h4 system, and the PluγETh system were determined to be the most efficient recombineering systems for the type strains A. tumefaciens C58, A. tumefaciens EHA105, and Rhizobium rhizogenes NBRC 13257, respectively. The utility of these systems was demonstrated by knocking out the - fusion gene in C58, the gene in EHA105, and the 3'-to-5' exonuclease gene and endoglucanase gene in NBRC 13257. Our work provides an effective genetic manipulation strategy for species. is a powerful transgenic vehicle for the genetic manipulation of numerous plant and fungal species and even animal cells. In addition to improving the utility of as a transgenic vehicle, genetic engineering tools are important for revealing crucial components that are functionally involved in transfer DNA (T-DNA) translocation events. This work developed an efficient and versatile recombineering system for . The successful genome modification of strains revealed that this new recombineering system could be used for the genetic engineering of .
Topics: Agrobacterium tumefaciens; Genetic Engineering; Recombinases; Rhizobium; Rhizobium leguminosarum
PubMed: 35044833
DOI: 10.1128/aem.02499-21 -
Frontiers in Plant Science 2022Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue...
Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with . Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as cell density, infection time and co-culture duration were studied to optimize transformation efficiency. strains EHA105 and pBI121 binary vector harboring the (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L NAA, 0.5 mg·L 2,4-D and 0.5 mg·L TDZ was optimal for callus induction and MS medium containing 0.5 mg·L NAA, 1.0 mg·L 2,4-D and 0.5 mg·L TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L and 200 mg·L, respectively. Among various combinations analyzed, the best transformation result was obtained the 25 min of infection of at 0.6 OD and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in .
PubMed: 36589115
DOI: 10.3389/fpls.2022.996690 -
Plants (Basel, Switzerland) Nov 2022Overgrowth of has frequently been found in -mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death....
Overgrowth of has frequently been found in -mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28-30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of subsequent to transformation.
PubMed: 36432853
DOI: 10.3390/plants11223124 -
Plants (Basel, Switzerland) May 2022Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments...
Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock 'Carrizo' citrange [ (L.) Osb. × (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured cells in a 1/10 MS, 0.5 g/L 2-(-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.
PubMed: 35684233
DOI: 10.3390/plants11111457 -
The Plant Journal : For Cell and... Oct 2021Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well...
Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.
Topics: Agrobacterium tumefaciens; Fungal Proteins; Green Fluorescent Proteins; Light; Luminescent Measurements; Microorganisms, Genetically-Modified; Molecular Farming; Operon; Plant Immunity; Plant Leaves; Recombinant Proteins; Nicotiana
PubMed: 34369027
DOI: 10.1111/tpj.15454 -
Frontiers in Plant Science 2020Hairy roots derived from the infection of a plant by ) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules.... (Review)
Review
Hairy roots derived from the infection of a plant by ) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules. Hairy roots are able to produce and secrete complex active glycoproteins from a large spectrum of organisms. They are also adequate to express plant natural biosynthesis pathways required to produce specialized metabolites and can benefit from the new genetic tools available to facilitate an optimized production of tailor-made molecules. This adaptability has positioned hairy root platforms as major biotechnological tools. Researchers and industries have contributed to their advancement, which represents new alternatives from classical systems to produce complex molecules. Now these expression systems are ready to be used by different industries like pharmaceutical, cosmetics, and food sectors due to the development of fully controlled large-scale bioreactors. This review aims to describe the evolution of hairy root generation and culture methods and to highlight the possibilities offered by hairy roots in terms of feasibility and perspectives.
PubMed: 32194578
DOI: 10.3389/fpls.2020.00033 -
Antimicrobial Resistance and Infection... Mar 2022Agrobacterium spp. are infrequent agents of bloodstream infections linked to healthcare-associated outbreaks. However, it is unclear if outbreaks also occur across...
BACKGROUND
Agrobacterium spp. are infrequent agents of bloodstream infections linked to healthcare-associated outbreaks. However, it is unclear if outbreaks also occur across larger geographic areas. Triggered by two local clusters from putative point sources, our aim was to detect potential additional clusters in Switzerland.
METHODS
We performed a nationwide descriptive study of cases in Switzerland based on a prospective surveillance system (Swiss Centre for Antibiotic Resistance, anresis.ch), from 2008 to 2019. We identified patients with Agrobacterium spp. isolated from blood cultures and used a survey to collect clinical-epidemiological information and susceptibility testing results. We performed whole genome sequencing (WGS) of available clinical isolates and determined their relatedness by single nucleotide polymorphism (SNP) variant calling analysis.
RESULTS
We identified a total of 36 cases of Agrobacterium spp. from blood samples over 10 years. Beyond previously known local clusters, no new ones were identified. WGS-based typing was performed on 22 available isolates and showed no clonal relationships between newly identified isolates or to those from the known clusters, with all isolates outside these clusters being at least 50 SNPs apart.
CONCLUSION AND RELEVANCE
Agrobacterium spp. bacteraemia is infrequently detected and, given that it may be healthcare-associated and stem from a point source, occurrence of multiple episodes should entail an outbreak investigation. With the help of the national antimicrobial resistance surveillance system we identified multiple clinical cases of this rare pathogen but found no evidence by WGS that suggested a nation-wide outbreak.
Topics: Agrobacterium; Bacteremia; Humans; Prospective Studies; Retrospective Studies; Switzerland
PubMed: 35264215
DOI: 10.1186/s13756-022-01086-y -
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 -
Frontiers in Plant Science 2023(L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly...
(L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly sought-after as a functional food. Its self-fertilisation capability and adaptability to challenging environments further contribute to its potential as a sustainable agricultural option. To harness its unique traits, genetic transformation in is crucial. In this study, we optimised the Agrobacterium-mediated transformation protocol for callus, resulting in a transformation rate of regenerated plants of approximately 20%. The protocol's effectiveness was confirmed through successful GUS staining, GFP expression, and the generation of albino plants via gene inactivation. These results validate the feasibility of genetic manipulation and highlight the potential for trait enhancement in .
PubMed: 37746024
DOI: 10.3389/fpls.2023.1270150 -
Plants (Basel, Switzerland) Jul 2021Duckweed () is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives...
Duckweed () is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives for livestock. It is also an ideal system for basic biological research and various practical applications. In this study, we attempt to establish a micropropagation technique and -mediated transformation in . The plant-growth regulator type and concentration and -mediated transformation were evaluated for their effects on duckweed callus induction, proliferation, regeneration, and gene transformation efficiency. Calli were successfully induced from 100% of explants on Murashige and Skoog (MS) medium containing 25.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ). MS medium containing 4.5 μM 2,4-D and 2.0 μM TDZ supported the long-lasting growth of calli. Fronds regenerated from 100% of calli on Schenk and Hildebrandt (SH) medium containing 1.0 μM 6-benzyladenine (6-BA). We also determined that 200 μM acetosyringone in the cocultivation medium for 1 day in the dark was crucial for transformation efficiency (up to 3 ± 1%). Additionally, we propose that both techniques will facilitate efficient high-throughput genetic manipulation in Lemnaceae.
PubMed: 34451621
DOI: 10.3390/plants10081576