Imaging GFP streptomyces on arabidopsis roots
Imaging GFP streptomyces on Arabidopsis roots
Streptomyces strains that contain pIJ8660/PermE* and are able to produce the eGFP protein encoded by it can be visualised on the roots of Arabidopsis plants. This can be useful to ascertain whether the strain is able to colonise the surface or the interior of the root.
Preparation and reagents needed
- Arabidopsis thaliana Col-0 seeds
- Streptomyces modified with pIJ9660/PermE* - see Conjugation using ET12567/pUZ8002
- 20% glycerol
- 70% ethanol
- 20% bleach (6% sodium hypochlorite solution)
- Murashige and Skoog with 10% surcrose (MS10)
- Murashige and Skoog with no sucrose (MS0)
- 276 SynaptoRed (optional)
- Sterilise Arabidopsis thaliana seeds by washing in 70% ethanol for 2 minutes followed by 20% bleach for another 2 minutes. Wash 5 times in sterile distilled water.
- Germinate Arabidopsis thaliana Col-0 seeds on MS10 for 9 days under a 12 hour photoperiod.
- Transfer seedlings to MS0 agar using sterile tweezers to gently lift from the stem.
- Allow seedlings to recover from the transfer for 24 hours, before inoculating the base of the stem with 1µl of 1*106cfu/ml of GFP labelled Streptomyces. Allow to dry.
- Grow seedlings for a further 3 days at a 12 hour photoperiod.
- Wash seedlings in 1 μg/ml−1 276 SynaptoRed for 10 min. Alternatively, wash seedlings in 20% glycerol for 20 minutes. 276 SynaptoRed is used as a counterstain for the plant tissue, for improved contrast and visualisation using confocal microscopy.
- Mount a 20mm section of root onto a glass slide with 100μl of the SynaptoRed/20% glycerol solution, or 20% glycerol if the counterstain is not used.
- Samples can be imaged using epifluorescence microscopy but for best results, image using confocal laser scanning microscopy (fCLSM)
- Green fluorescent protein is excited at 488nm, emission is collected through 527.5 +/- 22.5nm bandpass filter.
- SynaptoRed is excited at 543nm and emission is collected through a 587.5 +/- 27.5nm bandpass filter.
Sarah F Worsley , Jake Newitt , Johannes Rassbach, Sibyl F D Batey, Neil A Holmes, J Colin Murrell, Barrie Wilkinson, Matthew I Hutchings - Streptomyces Endophytes Promote Host Health and Enhance Growth across Plant Species.
Protocol adapted for ActinoBase by Jake T. Newitt, PhD student at the University of East Anglia.