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Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive sudden osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing green fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, OzTrac sensors reported osmolality changes as a proportional change in emission ratio of the two fluorescent protein domains. Live-cell imaging revealed accumulation of fluorescent sensors in internal aggregates presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

Virulence of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight of rice, depends on induction of host SWEET sucrose efflux transporters. It remained unknown whether secreted sucrose serves bacterial nutrition or host defense. Here we identified the sux sucrose uptake/utilization locus of Xoo and demonstrate that it is necessary and sufficient for sucrose acquisition. Induction of sux genes during infection closely tracked induction of rice SWEET11a. sux mutants were defective in swimming, swarming, extracellular polysaccharide (EPS) production and biofilm formation. EPS synthesis in mutants was restored by the quorum-sensing factor DSF. Notably, transcripts for rate limiting steps in DSF production were unaffected by sucrose, transcripts of the DSF receptor were sucrose-inducible and increased during infection, indicating sensitization to DSF in response to sucrose supply. Sucrose induced the sigma factors RpoN1 and RpoN2 that regulate swimming, EPS and virulence. Furthermore, in contrast to Xanthomonas axonopodis pv. manihotis, virulence of Xoo depended critically on sux gene function. Together, pathogen-induced sucrose efflux from host cells likely induces bacterial sigma factors and sensitizes quorum signaling necessary for biofilm formation and colonization of the xylem, serves as energy source for swimming against the xylem stream, and as nutrient for growth.Author summaryIf we want to efficiently protect plants against infections, we need to understand the disease mechanisms. Bacterial blight is a major scourge for rice production in Asia and Africa. We had found that disease-causing bacteria use a set of keys, so-called TAL effectors, to switch on sugar transporter genes in rice leaves causing sucrose release around the bacteria. A key question was whether the sugars act primarily in activation of host defense, or serve as nutrients and signals for bacterial infection. Here we provide evidence that both the ability to attack as well as the growth of bacteria depend on sucrose uptake. We unravel a regulatory network including transcriptional regulators, quorum sensing, swimming, biofilm production and virulence that all depend on sucrose uptake. These discoveries may prove to be crucial for the development of strategies for protecting rice against this disease.

Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Forster resonance energy-transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sucrose sensor FLIPsuc-90μΔ1 with Km for sucrose of ∼90 μM was available. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar-binding protein. Here, we took a two-step approach to first improve the dynamic range of the FLIPsuc sensor and then expand the detection range from micro- to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may enable investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90μ also detects trehalose in animal cells, the new series of sensors will likely be suitable for investigating trehalose transport and monitor trehalose steady-state levels in vivo.

Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Förster Resonance Energy Transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sensor FLIPsuc-90µΔ1 with a Km for sucrose of ∼90 µM is available for sucrose monitoring. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar binding protein. Here, we took a two-step approach to first systematically improve the dynamic range of the FLIPsuc nanosensor and then expand the detection range from micromolar to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may allow systematic investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90µ also detects trehalose in animal cells, the new series of sensors can be used to investigate trehalose transporter candidates and monitor trehalose steady-state levels in vivo as well.

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.

Förster resonance energy transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics, and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) that provides proteins with two different functional groups for post-translational labeling at the specific amino acid positions. Besides the sulfhydryl group of a cysteine, we make use of an azido group of a p-azido-l-phenylalanine to achieve chemical orthogonality. Herein, we achieve not only a site-specific but, most importantly, also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids cotranslationally using cell-free protein synthesis systems. Here, we demonstrate the cotranslational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that cotranslational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies.

Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter‐dye distance determination.

Single molecule FRET (smFRET) is a powerful tool for looking at protein folding and conformational dynamics (1). The application of smFRET to interesting proteins involved in pathologies or drug targeting is limited when the protein of interest is difficult to express in vivo due to toxicity and where cell-free expression is required. Moreover, site-specific labeling is an important issue for smFRET (2), but there is still a lack in simple and robust methods for site-specific labeling of in vitro synthesized proteins for smFRET studies. The conventional method is to double label the protein via cysteine residues (3), but as a consequence of a single chemistry, labeling specificity is limited and would lead to an inaccurate picture of the studied population (4). Incorporation of unnatural amino acids carrying an orthogonal chemistry can increase labeling specificity as it was reported (4). However, described methods were so far mainly considered for smFRET studies performed on in vivo synthesized proteins.By using human calmodulin (hCaM) as a model protein we have developed an alternative method for site-specific labeling of cell-free synthesized proteins. Site-specificity of our system allowed us to obtain sharper FRET histograms compared to the conventional labeling method. In addition, by demonstrating functionality of the labeled hCaM through binding experiments performed in presence of ligands and partners, we have shown the biological relevance of our method.In summary, we have developed a robust and simple method that enables the accurate study of cell-free synthesized proteins in smFRET. This method can be used for studying proteins that cannot be expressed in vivo, as it would be for toxic or membrane proteins.1. Tan, Y. W., J. A. Hanson, J. W. Chu, and H. Yang. 2014. Confocal single-molecule FRET for protein conformational dynamics. Methods Mol. Biol. 1084:51-62.2. Joo, C., and T. Ha. 2012. Labeling proteins for single-molecule FRET. Cold Spring Harb. Protoc. 2012:1009-1012.3. Kim, Y., S. O. Ho, N. R. Gassman, Y. Korlann, E. V. Landorf, F. R. Collart, and S. Weiss. 2008. Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem. 19:786-791.4. Seo, M. H., T. S. Lee, E. Kim, Y. L. Cho, H. S. Park, T. Y. Yoon, and H. S. Kim. 2011. Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid. Anal. Chem. 83:8849-8854.

Successful colonization and survival in variable environments require a competitive advantage during the initial growth phase after experiencing nutrient changes. Starved yeast cells anticipate exposure to glucose by activating the Hxt5p (hexose transporter 5) glucose transporter, which provides an advantage during early phases after glucose resupply. cAMP and glucose FRET (fluorescence resonance energy transfer) sensors were used to identify three signalling pathways that co-operate in the anticipatory Hxt5p activity in glucose-starved cells: as expected the Snf1 (sucrose nonfermenting 1) AMP kinase pathway, but, surprisingly, the sugar-dependent G-protein-coupled Gpr1 (G-protein-coupled receptor 1)/cAMP/PKA (protein kinase A) pathway and the Pho85 (phosphate metabolism 85)/Plc (phospholipase C) 6/7 pathway. Gpr1/cAMP/PKA are key elements of a G-protein-coupled sugar response pathway that produces a transient cAMP peak to induce growth-related genes. A novel function of the Gpr1/cAMP/PKA pathway was identified in glucose-starved cells: during starvation the Gpr1/cAMP/PKA pathway is required to maintain Hxt5p activity in the absence of glucose-induced cAMP spiking. During starvation, cAMP levels remain low triggering expression of HXT5, whereas cAMP spiking leads to a shift to the high capacity Hxt isoforms.