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

ACS Synth Biol. 2020 Aug 21;9(8):2107-2118. doi: 10.1021/acssynbio.0c00192. Epub 2020 Aug 11.

Abstract

Strigolactones are plant hormones and rhizosphere signaling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica. The biosensors were constructed via domain insertion of circularly permuted GFP. The biosensors exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue rac-GR24, and the ShHTL7 biosensor also responded to a specific antagonist. To overcome biosensor sensitivity to changes in expression level and protein degradation, an additional strigolactone-insensitive fluorophore, LSSmOrange, was included as an internal normalization control. Other plant hormones and karrikins resulted in no fluorescence change, demonstrating that the biosensors report on compounds that specifically bind the SL receptors. The DAD2 biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. These biosensors have applications in high-throughput screening for agrochemical compounds, and may also have utility in understanding strigolactone mediated signaling in plants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis
  • Biosensing Techniques / methods*
  • Gene Expression / drug effects
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Heterocyclic Compounds, 3-Ring / analysis*
  • Heterocyclic Compounds, 3-Ring / metabolism
  • Heterocyclic Compounds, 3-Ring / pharmacology
  • Lactones / analysis*
  • Lactones / metabolism
  • Lactones / pharmacology
  • Petunia / metabolism
  • Plant Proteins / antagonists & inhibitors
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Protein Domains
  • Proteolysis / drug effects
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Striga / metabolism

Substances

  • GR24 strigolactone
  • Heterocyclic Compounds, 3-Ring
  • Lactones
  • Plant Proteins
  • Recombinant Fusion Proteins
  • orobanchol
  • Green Fluorescent Proteins