Klebsiella oxytoca SG-11 labeling by green fluorescent protein gene (gfp) and its colonization in rice seedling roots
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Klebsiella oxytoca
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Three endophytic bacterial strains (Burkholderia sp. no. 25, Enterobacter sp. no. 35 and Klebsiella sp. no. 38) isolated from sweet potato and sugarcane were examined for their ability to incorporate the gene encoding green fluorescent protein (GFP) via conjugation and electroporation. Enterobacter sp. no. 35 and Klebsiella sp. no. 38 were successfully tagged with the gfp gene by conjugation using pTn5kmgfpmut1. Fluorescence microscopic observation of Brassica oleracea inoculated with gfp-tagged endophytes revealed that Enterobacter sp. no. 35 and Klebsiella sp. no. 38 colonized the junctions between the lateral roots and the main roots.
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Azospirillum brasilense
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This paper describes inoculation of 18 bacterial strains isolated from maize to maize, isolation, screening and identification of those strains based on morphology and color, using multi carbon medium, and REP PCR. The result indicated that two diazotrophic bacteria of R1 and R6 with high acetylene reduction activity were obtained. The plasmid of pKK223 GFP harboring gfp (reporter gene) was transferred into R1 and R6, respectively, and two transformants were obtained with the overexpression of GFP, designated as R1A and R6A; and pMGFP2.1 harboring nif H gfp was transferred into R1 and R6, and two transformants carrying pMGFP2.1 were designated as R1B and R6B, respectively. Also the research of colonizing R1A and R6A in maize root tissues, and inducing expression of nif H gfp in R1B and R6B was conducted gnotobiotically. The result showed that R1A and R6A were colonized in intercellular spacer, xylem vessels, wall of xylem vessels and parenchyma of stele predominantly. It was difficult for R1B and R6B to express nif H gfp in root tissues because of the limited nutrients and factors related to dinitrogenas gene expression. Only if 1‰ sucrose was supplemented, nif H gfp could be expressed. Fig 2, Tab 2, Ref 15
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Aspergillus nidulans
Appressorium
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Glyceraldehyde 3-phosphate dehydrogenase
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To explore the colonization of endophytic nitrogen-fixing bacterium Klebsiella variicola in Penniseum sinense, pET28a and the enhanced green fluorescent protein (EGFP) were used as basic elements to construct a recombinant expression vector pET28a-Lac-EGFP with the addition of starter Lac in pUC18 vector using enzymatic digestion and splicing methods. The constructed recombinant vector was transformed into K. variicola GN02 cells with electroporation method and the colonization of EGFP-tagged K. variicola GN02 in the roots of Pennisetum sp. was observed using laser confocal scanning microscopy. The results of restriction enzyme digestion and sequencing analysis indicated that pET28a-Lac-EGFP was successfully constructed and the target gene was successfully transferred into K. variicola GN02 cells. Positive colonies with obvious green fluorescence were observed under ultraviolet (UV) light. Sodium dodecyl sulphate-polyacrylamide gel-electrophoresis (SDS-PAGE) showed that expression of the EGFP-labelled target protein was also successfully induced in K. variicola GN02 cells. Root scanner and confocal microscopy showed that inoculation of K. variicola GN02 strain was more conducive to the root growth of P. sinense and K. variicola GN02 mainly colonized the endothelial layer of Pennisetum sp. roots. However, colonization with a small amount of K. variicola GN02 led to its concentration in the epidermis, middle column and ducts. Our findings suggest that the endophytic nitrogen-fixing bacterium K. variicola accumulates in plant roots and is subsequently dispersed, aggregated or even colonized in susceptible plants. Furthermore, we provided a theoretical basis for the practical used of K. variicola GN02-based microbial fertilizers in P. sinense and gramineous plants.
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Journal Article Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication‐assisted Agrobacterium‐mediated transformation‐treated soybean cotyledons Get access K.R. Finer, K.R. Finer Department of Biological Sciences, Kent State University/Stark Campus, Canton and K.R. Finer, Kent State University, 6000 Frank Ave N.W., Canton, OH 44720, USA (e‐mail: kfiner@stark.kent.edu). Search for other works by this author on: Oxford Academic Google Scholar J.J. Finer J.J. Finer Department of Horticulture and Crop Sciences, OARDC, The Ohio State University, Wooster, OH, USA Search for other works by this author on: Oxford Academic Google Scholar Letters in Applied Microbiology, Volume 30, Issue 5, 1 May 2000, Pages 406–410, https://doi.org/10.1046/j.1472-765x.2000.00737.x Published: 01 May 2000 Article history Received: 31 August 1999 Accepted: 07 February 2000 Published: 01 May 2000
Sonication
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Development of a genetic tool for visualization of photosynthetic bacteria (PSB) is essential for understanding microbial function during their interaction with plant and microflora. In this study, Rhodopseudomonas palustris GJ-22-gfp harboring the vector pBBR1-pckAPT-gfp was constructed using an electroporation transformation method and was used for dynamic tracing of bacteria in plants. The results showed that strain GJ-22-gfp was stable and did not affect the biocontrol function, and the Confocal Laser Scanning Microscopy (CLSM) results indicated it could successfully colonised on the surface of leaf and root of tobacco and rice. In tobacco leaves, cells formed aggregates on the mesophyll epidermal cells. While in rice, no aggregate was found. Instead, the fluorescent cells colonise the longitudinal intercellular spaces between epidermal cells. In addition, the results of strain GJ-22 on the growth promotion and disease resistance of tobacco and rice indicated that the different colonization patterns might be related to the bacteria could induce systemic resistance in tobacco.
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Protoplast
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The recombined plasmid pRP22-GFP contained with gfp gene and chloramphenicol resistant was successfully introduced into two biocontrol agents Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116. After seed inoculation, the survival and colonization of the two strains were studied by periodically retrieving the GFP-tagged strains in the cucumber rhizosphere based on the selective markers. The results showed that both the strains could successfully colonize in the rhizosphere during the whole life of cucumber, and a higher colonization level was observed during anthesis and fruition stages. In pot trials, they could migrate to the nearby non-inoculated spontaneous weed plants, and reestablish in the rhizosphere of plants subsequently grown in the same pot.
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【Objective】 The objective of this study is to investigate bacterial colonization in rice seedlings by GFP-labeled Bacillus pumilus DX01 and lay a solid foundation for the application of phytopathogenic biocontrol.【Method】 Genetically stable mutants of B.pumilus DX01 with a strong GFP expression were identified from the GFP-labeled Tn5 insertion library and used for tracing the bacterial colonization in rice seedlings.【Result】 A total of 1 467 mutants were subjected to qualitative and quantitative analyses of GFP expression by using fluorescence microplate reader,fluorescence activating cell sorter and fluorescent microscopy,and eventually 8 mutants with significantly enhanced GFP expression were identified.Meanwhile,the copy numbers of exogenous DNA integration in the genome of these mutants were detected.The GFP-tagged bacterial cells that released into the rhizosphere of rice seedlings could survive for more than 15 days.Furthermore,decrease of the B.pumilus bacterial cells in undisinfected soils showed more slowly compared with corresponding sterile soils.【Conclusion】 B.pumilus mainly locates in root hair region and later root branch.Meanwhile,the bacterial film formed on rice root surface was also confirmed.B.pumilus can invade plant through the wound of roots or by young root hairs,and they distribute mostly in root cortex cells and intercellular spaces of cortical layer.The marked bacteria were often found in the vascular bundle of rice seedlings.In conclusion,B.pumilus displayed a good colonization ability in the rhizosphere soils of rice seedlings.
Bacillus pumilus
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To visualize simultaneously different populations of pseu-domonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Micro-colonies consisting of mixed populations were frequently observed at the base of the root system, whereas micro-colonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.
Pseudomonas fluorescens
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