Using seedlings inoculated with wild type Pseudomonas sp. as a positive control,initial microscopy used only water to mount the samples onto the microscope slide. This method was unsuitable for visualising bacteria in the root as bacteria were indistinguishable from the plant. Our Pseudomonas sp. strain produces a fluorescent siderophore causing green fluorescence so we attempted to view the sample through a GFP filter, this was unsuccessful as the auto-fluorecence of plant tissue was too great to see the bacteria. To overcome this issue, DAPI stain was used to stain nucleic acids of the bacteria. DAPI was added to distilled water at a concentration of 1 microlitre per millilitre and this was used to mount the sample and a DAPI filter was used to visualise the sample. At 40x magnification bacteria were visible and this revealed a biofilm was present on the surface of the root, however it was unclear if the bacteria were inside the root so seedlings were washed in distilled water. Examination of the cleaned seedlings was challenged by the fact that root hair cells had wrapped around the main root during the washing process meaning that it was unclear if bacteria were inside the main root or merely on the surface.

To overcome the issue of root hair cells wrapping, the team used 15% glycerol in place of water for mounting samples. This was highly successful as the viscosity of the glycerol caused root hair cells to spread and revealed clear signs of colonisation. Pseudomonas sp. was present in the intercellular spaces along both the root and hypocotyl. These bacteria were still motile and we could see them moving in real time.

Figure 2. Bright field microscopy of a DAPI stained Arabidopsis thaliana. Wild type Pseudomonas sp. is visible in the intercellular spaces.

Figure 3. Bright field microscopy through a DAPI filter, Pseudomonas sp. is visible on the root surface.

We were able to take images of the bright field microscopy with and without a DAPI filter before merging the images to provide a clear visual representation of how the bacteria live within the plant.

Figure 4. A Pseudomonas sp. inoculated Arabidopsis thaliana root without DAPI filter.

Figure 5. A Pseudomonas sp. inoculated Arabidopsis thaliana root with DAPI filter.

Figure 5. An overlay of Figure 4 and Figure 5, clearly showing Pseudomonas sp in the intercellular spaces of plant cells.

Visualising E. coli

As we had a positive control in the form of wildtype Pseudomonas sp. the team decided a to use E. coli DH5α as a negative control since it is not known to be a plant coloniser. A selection of seedlings inoculated by the seed-coating method were again selected for microscopy. Microscopy revealed that though E. coli was present on the root surface in small numbers, there was no sign of an endophytic relationship like that of Pseudomonas sp., showing us what our transformant should and should not look like when examined.

Figure 7. Bright field microscopy showing the root of our negative control Arabidopsis thaliana where E. coli DH5α was visible on the surface of the root but not inside the root as an endophyte.

Figure 8.Bright field microscopy without DAPI filter showing the root surface of our negative control Arabidopsis thaliana.

Figure 9.Bright field microscopy with DAPI filter showing the root surface of our negative control Arabidopsis thaliana.

Figure 10. an overlay of Figure 8 and Figure 9, showing the root surface of our negative control Arabidopsis thaliana.

Visualising the transformant

A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed with distilled water and mounted on a microscope slide with the same solution of DAPI and glycerol. Endophytic Pseudomonas sp. were visible inside the root and hypocotyl of both Arabidopsis thaliana and Eruca sativa seedlings showing that transformation had not altered the bacteria’s ability to colonise. This showed that our endophytic chassis was a plant coloniser and works as expected.

Figure 11. Bright field microscopy of an Arabidopsis thaliana seedling root showing transformed Pseudomonas sp. living as an endophyte in the intercellular spaces.

Figure 12. Bright field microscopy of an Arabidopsis thaliana seedling root inoculated with Pseudomonas sp.without DAPI filter.

Figure 13. Bright field microscopy of an Arabidopsis thaliana seedling root inoculated with Pseudomonas sp.with DAPI filter.

Figure 14. An overlay of Figure 12 and Figure 13, showing transformed Pseudomonas sp. in the intercellular spaces of an Arabidopsis thaliana seedling.