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Remans, Tony; Schenk, Peer M.; Manners, John M.; Grof, Christopher P. L.; Elliott, Adrian R.

doi:10.1023/a:1007654318401

Cited by 42 publications

(2 citation statements)

References 9 publications

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“…After incubation at 37°C for 30 min reactions were stopped by the addition of 400 lL of 400 mM Na 2 CO 3 solution. GUS activity was quantified by measuring light absorbance at 415 nm in spectrophotometer (Hitachi U-1900 UV/VIS, Japan) [9]. Absorbance A 415 was also measured from PLBs-containing uninfected microtubes to determine light absorption by plant release compounds, as well as from inoculated microtubes in the absence of PLBs to measure the total enzymatic activity in the inoculum used for infections.…”

Section: Quantification Of Bacterial Attachment To Vkd's Plbs Via Spementioning

confidence: 99%

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem’s Delight Orchid Protocorm-Like Bodies

Gnasekaran

Subramaniam

2015

Indian J Microbiol

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Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem's Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5a was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5a that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD's PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate.

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Section: Quantification Of Bacterial Attachment To Vkd's Plbs Via Spementioning

confidence: 99%

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem’s Delight Orchid Protocorm-Like Bodies

Gnasekaran

Subramaniam

2015

Indian J Microbiol

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“…A further advantage of these proteins is that their fluorescence intensity is directly proportional to protein amount and can be used for quantification [22,23]. Although GFP fluorescence can be quantified by image analysis [24,25], this involves time-consuming steps that can be overcome by spectrofluorometric measurement of intact plant organs or protein extracts from GFP-expressing samples [23,26,27].…”

Section: Introductionmentioning

confidence: 99%

Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies

et al. 2014

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BackgroundFluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons.ResultsWe report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus.ConclusionsLaser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation.

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Engineering green fluorescent protein as a dual functional tag

Paramban

Bugos

2004

Biotech & Bioengineering

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A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.

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Untitled

Cited by 42 publications

References 9 publications

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem’s Delight Orchid Protocorm-Like Bodies

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem’s Delight Orchid Protocorm-Like Bodies

Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies

Engineering green fluorescent protein as a dual functional tag

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