Rational Design of Novel Fluorescent Enzyme Biosensors for Direct Detection of Strigolactones

Chesterfield, Rebecca J.; Whitfield, Jason; Pouvreau, Benjamin; Cao, Da; Alexandrov, Kirill; Beveridge, Christine A.; Vickers, Claudia E.

doi:10.1021/acssynbio.0c00192

Cited by 20 publications

(4 citation statements)

References 60 publications

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“…A recent study has provided an exciting new approach to measure SL binding in vivo as well as in vitro. These SL biosensors incorporate a circularly permuted GFP (cpGFP) protein into an external loop joining alpha‐helices 6 and 7 of DAD2 or ShHTL7 (Chesterfield et al, 2020 ). SL induces conformational shifts in DAD2 or ShHTL7 that also affect the conformation of cpGFP, reducing its fluorescence.…”

Section: Discussionmentioning

confidence: 99%

“…A second fluorescent protein fused to the biosensor enables ratiometric measurements of fluorescence that bypass problems with varying biosensor abundance. In tobacco protoplasts, the DAD2‐based biosensor can detect rac ‐GR24 concentrations as low as 100 nM (Chesterfield et al, 2020 ). Because this system is sensitive to even single amino acid shifts in the placement of the cpGFP and must be fine‐tuned for each SL receptor, it may be better suited for screening for agonist or antagonist molecules of the receptor than evaluating the effects of receptor mutations.…”

Section: Discussionmentioning

confidence: 99%

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Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

et al. 2022

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DWARF14 (D14) is an ɑ/β‐hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)‐type proteins in the SUPPRESSOR OF MAX2 1‐LIKE (SMXL) family. We used CRISPR‐Cas9 to generate knockout alleles of the two homoeologous D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b double mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 from Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24ent‐5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by rac‐GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to rac‐GR24, but required an active catalytic triad. We used this platform to evaluate the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

et al. 2022

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“…Adequate crop nutrition, particularly phosphate, have been found to inhibit strigolactone exudation, with a minimal impact on the germination and parasitism of Striga spp. (Chesterfield et al, 2020). Additionally, AMF enhances crop phosphorus absorption with favorable response on plant growth and health, thereby partially offsetting the parasitic impacts of Striga spp.…”

Section: Current Trends In Striga Managementmentioning

confidence: 99%

Trenchant microbiological-based approach for the control of Striga: Current practices and future prospects

Olowe

Akanmu

Fadiji

et al. 2023

Front. Sustain. Food Syst.

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Striga species are obligate parasitic weeds most of which are members of the Orobanchaceae family. They are commonly associated with staple crops and constitute threats to food security, especially in Sub-Saharan Africa. They pose deleterious impacts on staple cereal crops like maize and pearl millet, resulting in 7–10 billion dollars yield losses or, in extreme infestations, entire crop losses. Farmers' limited knowledge about the weed (genetics, ecology, nature of the damage caused, complex life cycle, interactions with its host and associated microbes) and their attitude toward its control have negatively affected its management and sustainability. With the present Striga management such as mechanical, chemicals, cultural and biological measures, it is extremely difficult to achieve its active management due to nature of the association between host plants and parasites, which requires highly selective herbicides. The use of soil microbes has not been well explored in the management of Striga infection in African countries. However, many soil microorganisms have been considered viable biological control techniques for fighting parasitic weeds, due to their vast action and roles they play in the early stage of host-Striga interaction. Their application for pest control is well perceived to be cost-effective and eco-friendly. In this review, we gave a comprehensive overview of major knowledge gaps and challenges of smallholders in Striga management and highlighted major potentials of microbial-based approach with respect to the mechanisms of host-Striga-microbe interactions, and the metagenomics roles on Striga management that include understanding the microbe and microbial systems of Striga-infested soil.

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“…( g ) Schematic of the ratiometric DAD2 cpGFP (circularly permuted green fluorescent protein) biosensor design. Strigolactone (ligand) induced conformational change is propagated into cpGFP, modulating its fluorescence [ 12 ]. The C-terminal LSSmOrange fusion provides an internal fluorescent control; RFU—relative fluorescence units.…”

Section: Figurementioning

confidence: 99%

Genetically Encoded Fluorescent Biosensors for Biomedical Applications

et al. 2021

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One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Genetically encoded fluorescent biosensors constitute a class of imaging agents that enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically encoded fluorescent biosensors in drug screening. This review summarizes results of the studies that have been conducted in the last years toward the fabrication of genetically encoded fluorescent biosensors for biomedical applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.

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Rational Design of Novel Fluorescent Enzyme Biosensors for Direct Detection of Strigolactones

Cited by 20 publications

References 60 publications

Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

Trenchant microbiological-based approach for the control of Striga: Current practices and future prospects

Genetically Encoded Fluorescent Biosensors for Biomedical Applications

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