Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv

Authors


Author for correspondence: Kirankumar S. Mysore Tel: +580 224 6740 Fax: +580 224 6692 Email: ksmysore@noble.org

Summary

  • • Green fluorescent protein (GFP) labeling of bacteria has been used to study their infection of and localization in plants, but strong autofluorescence from leaves and the relatively weak green fluorescence of GFP-labeled bacteria restrict its broader application to investigations of plant–bacterial interactions.
  • • A stable and broad-host-range plasmid vector (pDSK-GFPuv) that strongly expresses GFPuv protein was constructed not only for in vivo monitoring of bacterial infection, localization, activity, and movement at the cellular level under fluorescence microscopy, but also for monitoring bacterial disease development at the whole-plant level under long-wavelength ultraviolet (UV) light.
  • • The presence of pDSK-GFPuv did not have significant impact on the in vitro or in planta growth and virulence of phytobacteria. A good correlation between bacterial cell number and fluorescence intensity was observed, which allowed us to rapidly estimate the bacterial population in plant leaf tissue. We demonstrated that GFPuv-expressing bacteria can be used to screen plants that are compromised for nonhost disease resistance and Agrobacterium attachment.
  • • The use of GFPuv-labeled bacteria has a wide range of applications in host–bacterial interaction studies and bacterial ecology-related research.

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