Team:Newcastle/Results/Endophyte1
RESULTS
Visualising Pseudomonas sp.
Re-isolation.
The first result obtained in regards to endophyte colonisation was obtained following engagement with GrowUp Urban Farms. This stakeholder engagement brought two issues to our attention, firstly root wounding methods (as we initially intended) are not accessible to farmers and suggested a seed coating may be more appropriate. Secondly that from a commercial point of view, there is a concern that the genetically modified Pseudomonas sp. would be present in the leaves which may be harvested and eaten.
To address these concerns the team ensured to investigate the presence of the Pseudomonas sp. in both the root and stem/leaves. Furthermore, the team shifted from using a wounding inoculation method to a seed-coating method.
The first set of results came from seed-coating experiments, where 96 Arabdisopsis thaliana seedlings were surface sterilised and coated in a liquid culture of Pseudomonas sp. wild type. Once germinated, 8 seedlings were selected and surface sterilised before being cut at the root and leaves for plating on TSA plates. Wild type Pseudomonas sp. was re-isolated in pure culture from all of these samples and no Pseudomonas sp. was re-isolated from a set of 8 control Arabidopsis thaliana seedlings. These results showed that seed-coating was an appropriate inoculation method which makes our project more accessible for commercial use. Figure 1. Pseudomonas sp. on a TSA plate that has been re-isolated from Arabidopsis thaliana seedlings which have been inoculated by the seed-coating method suggested by GrowUp Urban Farms. The team decided that the most valuable way to assess the endophytic relationship between Pseudomonas sp. and its host plant was to use microscopy. The team were instructed by Dr Vasilios Andriotis to use bright field microscopy techniques. 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. A Pseudomonas sp. inoculated Arabidopsis thaliana root without DAPI filter. 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. A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed and DAPI stained.Endophytic bacteria 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. Figure 21. Bright field microscopy of an Arabidopsis thaliana seedling root at x100 magnification,showing transformed Pseudomonas sp. living as an endophyte in the intercellular spaces. A selection of seedlings were again selected for microscopy, this time negative control E. coli DH5α inoculated seedlings were examined. Microscopy revealed that though E. coli was present on the root surface, there was no sign of endophytic relationship like that of Pseudomonas sp.. Further images were taken of transformant Pseudomonas sp. inoculated seedlings to aid visual demonstration of our transformed endophyte working.
Figure 23.Bright field microscopy showing the root of our negative control Arabidopsis thaliana at100x magnification, where E. coli DH5α was visible on the surface of the root in small numbers but not inside the root as an endophyte.
REFERENCES & Attributions
Attributions: Frank Eardley and Lewis Tomlinson