Conversion of a putative Agrobacterium sugar-binding protein into a FRET sensor with high selectivity for sucrose.
ABSTRACT Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.
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ABSTRACT: Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2-800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI: http://dx.doi.org/10.7554/eLife.01741.001.eLife Sciences 04/2014; 3:e01741. DOI:10.7554/eLife.01741 · 8.52 Impact Factor
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ABSTRACT: Intracellular metabolites play a crucial role in characterizing and regulating corresponding cellular activities. Tracking intracellular metabolites in real time by traditional means was difficult until the powerful toolkit of genetically encoded biosensors was developed. Over the past few decades, iterative improvements of these biosensors have been made, resulting in the effective monitoring of metabolites. In this review, we introduce and discuss the recent advances in the use of genetically encoded biosensors for tracking some key metabolites, such as ATP, cAMP, cGMP, NADH, reactive oxygen species, sugar, carbon monoxide, and nitric oxide. A brief phylogeny of fluorescent proteins and several typical construction modes for genetically encoded biosensors are also described. We also discuss the development of novel RNA-based sensors, which are genetically encoded biosensors active at the transcriptional level.Biotechnology Journal 11/2013; 8(11):1280-91. DOI:10.1002/biot.201300001 · 3.71 Impact Factor
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ABSTRACT: The gene-for-gene concept has historically been applied to describe a specific resistance interaction wherein single genes from the host and the pathogen dictate the outcome. These interactions have been observed across the plant kingdom and all known plant microbial pathogens. In recent years, this concept has been extended to susceptibility phenotypes in the context of transcription activator like (TAL) effectors that target SWEET sugar transporters. However, as this interaction has only been observed in rice, it was not clear whether the gene-for-gene susceptibility was unique to that system. Here we show, through a combined systematic analysis of the TAL effector-complement of Xanthomonas axonopodis pv. manihotis (Xam), and RNA-Sequencing to identify targets in cassava, that TAL20Xam668 specifically induces the sugar transporter MeSWEET10a to promote virulence. Designer TAL effectors (dTALEs) complement TAL20Xam668 mutant phenotypes, demonstrating that MeSWEET10a is a susceptibility gene in cassava. Sucrose uptake-deficient Xam do not lose virulence, indicating that sucrose may be cleaved extracellularly and taken up as hexoses into Xam. Together, our data suggest that pathogen hijacking of plant nutrients is not unique to rice blight but also plays a role in bacterial blight of the dicot cassava.Molecular Plant-Microbe Interactions 08/2014; 27(11). DOI:10.1094/MPMI-06-14-0161-R · 4.46 Impact Factor