A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae Nora R Zöllner, Juying Long, Congfeng Song, Jacob Sharkey, Michael M Wudick, Eliza P I Loo, Mayuri Sadoine, Violetta Applegate, Astrid Höppner, Sander H J Smits, Bing Yang, Wolf B Frommer Pnas Nexus, 2026 The virulence of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) of rice, critically depends on the activation of SWEET sucrose uniporters of the host. To date, the role of SWEET-released sucrose for virulence remains unclear. We here identified the sux locus of Xoo consisting of a LacI-type repressor (SuxR), an outer membrane TonB-like porin (SuxA), an inner membrane MFS H+-symporter (SuxC), and a cytosolic sucrose hydrolase (SuxB). Structural and functional analyses demonstrate that SuxB has exclusive sucrose hydrolase activity. Mutant analyses show that the transporter SuxC and the sucrose hydrolase are necessary for growth of bacteria on sucrose, while SuxA is not essential, likely due to the ability of other porins to transport sucrose across the outer membrane. Consistent with a role of SuxR as a sucrose repressor, transcriptome studies show sucrose-dependent regulation of the suxA/suxB genes. Besides a role of sucrose for reproduction, we found that sucrose promotes motility, extracellular polysaccharides production, biofilm formation, and virulence. Notably, the SuxC sucrose H+-symporter and the sucrose hydrolase SuxB were required for full virulence of Xoo on indica and japonica rice varieties. Our findings indicate that pathogen-induced sucrose efflux via SWEETs provides sucrose to Xoo, that Xoo uses the sux gene cluster to acquire and utilize sucrose, and that sucrose promotes bacterial fitness and xylem colonization.
Fluorophore-based Genetically Encoded Biosensors for Ratiometric Fluorescence Imaging in Microbes Erdem Sunal, Vanessa Castro-Rodriguez, Mayuri Sadoine Journal of Visualized Experiments, 2025 Investigating small-molecule dynamics within microbes is essential for comprehensive studies of microbial function. Both intra-organism and inter-organism small molecule dynamics play critical roles in microbial physiology, symbiosis, and disease. However, monitoring these dynamics remains highly challenging using most existing techniques. Fluorophore-based genetically encoded biosensors are powerful tools for tracking small-molecule dynamics in vivo and hold high potential for driving new discoveries. These biosensors are most commonly used in fluorescence imaging, often in combination with perfusion devices that allow precise control over environmental conditions. When integrated with advanced imaging techniques, this approach provides high-resolution, spatially and temporally resolved data, enabling insights into single-cell microbial responses. Despite their promise, implementing such biosensors remains technically challenging. Understanding the key steps is crucial for broader adoption. Here, we present a protocol designed to support the effective deployment of newly engineered biosensors into microbes for quantitative ratiometric fluorescence imaging under controlled conditions.
A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein J. Obinna Ejike, Mayuri Sadoine, Yi Shen, Yuuma Ishikawa, Erdem Sunal, Sebastian Hänsch, Anna B. Hamacher, Wolf B. Frommer, Michael M. Wudick, Robert E. Campbell, Thomas J. Kleist Biochemistry, 2024 Genetically encoded sensors enable quantitative imaging of analytes in live cells. State-of-the-art sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP are susceptible to artifacts caused by differing expression levels or sensor distribution in vivo. Hence, our lab developed dual-FP Matryoshka technology introduced by a single cassette that contains a stable large Stokes shift (LSS) reference FP nested within a reporter FP (cpEGFP), allowing simple construction of intensiometric sensors with the capacity for ratiometric quantification. The first-generation Green-Orange (GO) Matryoshka cassette established proof of concept but required custom optical setups to maximize achievable dynamic range. Here, we present a genetically encoded calcium sensor that employs optimized second-generation Green-Apple (GA) Matryoshka technology that incorporates a newly designed red LSSmApple fluorophore. LSSmApple provides improved excitation spectrum overlap with cpEGFP, allowing for monochromatic co-excitation with blue light. The exceptionally large Stokes shift of LSSmApple results in improved emission spectrum separation from cpEGFP, which minimizes fluorophore bleed-through and facilitates imaging using standard dichroics and red fluorescent protein (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new fluorescent protein, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.
Monitoring nutrients in plants with genetically encoded sensors: achievements and perspectives Mayuri Sadoine, Roberto De Michele, Milan Župunski, Guido Grossmann, Vanessa Castro-Rodríguez Plant Physiology, 2023 Understanding mechanisms of nutrient allocation in organisms requires precise knowledge of the spatiotemporal dynamics of small molecules in vivo. Genetically encoded sensors are powerful tools for studying nutrient distribution and dynamics, as they enable minimally invasive monitoring of nutrient steady-state levels in situ. Numerous types of genetically encoded sensors for nutrients have been designed and applied in mammalian cells and fungi. However, to date, their application for visualizing changing nutrient levels in planta remains limited. Systematic sensor-based approaches could provide the quantitative, kinetic information on tissue-specific, cellular, and subcellular distributions and dynamics of nutrients in situ that is needed for the development of theoretical nutrient flux models that form the basis for future crop engineering. Here, we review various approaches that can be used to measure nutrients in planta with an overview over conventional techniques, as well as genetically encoded sensors currently available for nutrient monitoring, and discuss their strengths and limitations. We provide a list of currently available sensors and summarize approaches for their application at the level of cellular compartments and organelles. When used in combination with bioassays on intact organisms and precise, yet destructive analytical methods, the spatiotemporal resolution of sensors offers the prospect of a holistic understanding of nutrient flux in plants.
OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels Thomas J. Kleist, I Winnie Lin, Sophia Xu, Grigory Maksaev, Mayuri Sadoine, Elizabeth S. Haswell, Wolf B. Frommer, Michael M. Wudick Biomolecules, 2022 Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive 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 fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, the OzTrac sensors reported osmolality changes as a proportional change in the emission ratio of the two fluorescent protein domains. Live-cell imaging revealed an 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.
Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology Mayuri Sadoine, Yuuma Ishikawa, Thomas J. Kleist, Michael M. Wudick, Masayoshi Nakamura, Guido Grossmann, Wolf B. Frommer, Cheng-Hsun Ho Plant Physiology, 2021 The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.
Affinity Series of Genetically Encoded Förster Resonance Energy-Transfer Sensors for Sucrose Mayuri Sadoine, Mira Reger, Ka Man Wong, Wolf B. Frommer ACS Sensors, 2021 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 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.
Sensors for the quantification, localization and analysis of the dynamics of plant hormones Reika Isoda, Akira Yoshinari, Yuuma Ishikawa, Mayuri Sadoine, Rüdiger Simon, Wolf B. Frommer, Masayoshi Nakamura Plant Journal, 2021 Summary Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non‐invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.
Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses Mayuri Sadoine, Vanessa Castro-Rodríguez, Tobias Poloczek, Helene Javot, Erdem Sunal, Michael Wudick, Wolf Frommer Bio Protocol, 2020 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.
Cotranslational Incorporation into Proteins of a Fluorophore Suitable for smFRET Studies Mayuri Sadoine, Michele Cerminara, Michael Gerrits, Jörg Fitter, Alexandros Katranidis ACS Synthetic Biology, 2018 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.
Integrating nano-biosensors into plants: A smart approach for plant disease management to enhance agriculture resilience VC Rodriguez, M Sadoine Nano-Delivering Plant Biostimulants and Anti-Pathogenic Agents, 533-560 , 2026 2026
A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae NR Zöllner, J Long, C Song, J Sharkey, MM Wudick, EPI Loo, M Sadoine, ... PNAS nexus 5 (1), pgaf412 , 2026 2026 Citations: 3
Fluorophore-based Genetically Encoded Biosensors for Ratiometric Fluorescence Imaging in Microbes E Sunal, V Castro-Rodriguez, M Sadoine Journal of visualized experiments: JoVE , 2025 2025
A Monochromatically Excitable Green–Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein JO Ejike, M Sadoine, Y Shen, Y Ishikawa, E Sunal, S Hänsch, ... Biochemistry 63 (1), 171-180 , 2024 2024 Citations: 8
Monitoring nutrients in plants with genetically encoded sensors: achievements and perspectives M Sadoine, R De Michele, M Župunski, G Grossmann, ... Plant Physiology 193 (1), 195-216 , 2023 2023 Citations: 21
OzTracs: optical osmolality reporters engineered from mechanosensitive ion channels TJ Kleist, IW Lin, S Xu, G Maksaev, M Sadoine, ES Haswell, WB Frommer, ... Biomolecules 12 (6), 787 , 2022 2022 Citations: 2
Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology M Sadoine, Y Ishikawa, TJ Kleist, MM Wudick, M Nakamura, ... Plant Physiology 187 (2), 485-503 , 2021 2021 Citations: 65
Sucrose-dependence of sugar uptake, quorum sensing and virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae M Sadoine, J Long, C Song, Y Arra, WB Frommer, B Yang bioRxiv, 2021.08. 22.457195 , 2021 2021 Citations: 6
Affinity series of genetically encoded high sensitivity Förster Resonance Energy Transfer sensors for sucrose M Sadoine, M Reger, KM Wong, WB Frommer ACS sensors 6 (5), 1779–1784 , 2021 2021 Citations: 20
Sensors for quantification, localization and analysis of dynamics of plant hormones R Isoda, A Yoshinari, Y Ishikawa, M Sadoine, R Simon, WB Frommer, ... 2020 Citations: 102
Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses M Sadoine, V Castro-Rodríguez, T Poloczek, H Javot, E Sunal, ... Bio-protocol 10 (19), e3773-e3773 , 2020 2020 Citations: 1
Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies M Sadoine, M Cerminara, J Fitter, A Katranidis Bio-protocol 8 (12), e2881-e2881 , 2018 2018 Citations: 1
Cotranslational incorporation into proteins of a fluorophore suitable for smFRET studies M Sadoine, M Cerminara, M Gerrits, J Fitter, A Katranidis ACS synthetic biology 7 (2), 405-411 , 2018 2018 Citations: 13
Selective dual-labeling of cell-free synthesized proteins for single-molecule FRET studies M Sadoine RWTH Aachen University , 2018 2018
Selective double-labeling of cell-free synthesized proteins for more accurate smFRET studies M Sadoine, M Cerminara, N Kempf, M Gerrits, J Fitter, A Katranidis Analytical chemistry 89 (21), 11278-11285 , 2017 2017 Citations: 20
Cell-Free Synthesis of Site-Specifically Double-Labeled Proteins for More Accurate Single-Molecule FRET Studies M Sadoine, M Cerminara, N Kempf, A Katranidis, J Fitter Biophysical Journal 112 (3), 31a , 2017 2017
Differential regulation of glucose transport activity in yeast by specific cAMP signatures C Bermejo, F Haerizadeh, MSC Sadoine, D Chermak, WB Frommer Biochemical Journal 452 (3), 489-497 , 2013 2013 Citations: 24
MOST CITED SCHOLAR PUBLICATIONS
Sensors for quantification, localization and analysis of dynamics of plant hormones R Isoda, A Yoshinari, Y Ishikawa, M Sadoine, R Simon, WB Frommer, ... 2020 Citations: 102
Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology M Sadoine, Y Ishikawa, TJ Kleist, MM Wudick, M Nakamura, ... Plant Physiology 187 (2), 485-503 , 2021 2021 Citations: 65
Differential regulation of glucose transport activity in yeast by specific cAMP signatures C Bermejo, F Haerizadeh, MSC Sadoine, D Chermak, WB Frommer Biochemical Journal 452 (3), 489-497 , 2013 2013 Citations: 24
Monitoring nutrients in plants with genetically encoded sensors: achievements and perspectives M Sadoine, R De Michele, M Župunski, G Grossmann, ... Plant Physiology 193 (1), 195-216 , 2023 2023 Citations: 21
Affinity series of genetically encoded high sensitivity Förster Resonance Energy Transfer sensors for sucrose M Sadoine, M Reger, KM Wong, WB Frommer ACS sensors 6 (5), 1779–1784 , 2021 2021 Citations: 20
Selective double-labeling of cell-free synthesized proteins for more accurate smFRET studies M Sadoine, M Cerminara, N Kempf, M Gerrits, J Fitter, A Katranidis Analytical chemistry 89 (21), 11278-11285 , 2017 2017 Citations: 20
Cotranslational incorporation into proteins of a fluorophore suitable for smFRET studies M Sadoine, M Cerminara, M Gerrits, J Fitter, A Katranidis ACS synthetic biology 7 (2), 405-411 , 2018 2018 Citations: 13
A Monochromatically Excitable Green–Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein JO Ejike, M Sadoine, Y Shen, Y Ishikawa, E Sunal, S Hänsch, ... Biochemistry 63 (1), 171-180 , 2024 2024 Citations: 8
Sucrose-dependence of sugar uptake, quorum sensing and virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae M Sadoine, J Long, C Song, Y Arra, WB Frommer, B Yang bioRxiv, 2021.08. 22.457195 , 2021 2021 Citations: 6
A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae NR Zöllner, J Long, C Song, J Sharkey, MM Wudick, EPI Loo, M Sadoine, ... PNAS nexus 5 (1), pgaf412 , 2026 2026 Citations: 3
OzTracs: optical osmolality reporters engineered from mechanosensitive ion channels TJ Kleist, IW Lin, S Xu, G Maksaev, M Sadoine, ES Haswell, WB Frommer, ... Biomolecules 12 (6), 787 , 2022 2022 Citations: 2
Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses M Sadoine, V Castro-Rodríguez, T Poloczek, H Javot, E Sunal, ... Bio-protocol 10 (19), e3773-e3773 , 2020 2020 Citations: 1
Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies M Sadoine, M Cerminara, J Fitter, A Katranidis Bio-protocol 8 (12), e2881-e2881 , 2018 2018 Citations: 1
Integrating nano-biosensors into plants: A smart approach for plant disease management to enhance agriculture resilience VC Rodriguez, M Sadoine Nano-Delivering Plant Biostimulants and Anti-Pathogenic Agents, 533-560 , 2026 2026
Fluorophore-based Genetically Encoded Biosensors for Ratiometric Fluorescence Imaging in Microbes E Sunal, V Castro-Rodriguez, M Sadoine Journal of visualized experiments: JoVE , 2025 2025
Selective dual-labeling of cell-free synthesized proteins for single-molecule FRET studies M Sadoine RWTH Aachen University , 2018 2018
Cell-Free Synthesis of Site-Specifically Double-Labeled Proteins for More Accurate Single-Molecule FRET Studies M Sadoine, M Cerminara, N Kempf, A Katranidis, J Fitter Biophysical Journal 112 (3), 31a , 2017 2017
