Unravelling DELLA’s protein-protein interactions via co-Immunoprecipitation in wheat protoplasts Hi everyone, i’m yoni Bultinck Intern at Bayer and helping Karel with the research about DELLA. Della is a protein that has been very important in the Green Revolution. I would like to take you with me into the history of the Green Revolution. This revolution had an important social role. Because of the population growth in the world they had to find a solution by wich the yield increased. Norman Ernest Borlaug found out that when he put an mutation in the DELLA protein, it resolted in an smaller crop, that had an dwarfing propertie. When the plant is less high it brings a lot of positive properties with it. The plant will be less sensitive to fall down. Bayer wants to know more about the DELLA protein in wheat, because wheat is an imortant and frequently used crop. When we know more about DELLA we can do research about its interacting partners. When the interacting partners are found, Bayer can do further research to increase the yield even more than in the Green Revolution. Yoni Bultinck

How DELLA controls GA responses Workflow and main methods used Results Conclusion

The discovery of Rht had a major impact on wheat breeding Green Revolution Lodging Norman Borlaug DELLA v v v v

Normal pathway TF TF GA TF TF GID1 TF DELLA TF GA growth response

The Green Revolution TF TF TF DELLA TF TF TF GA growth response

Workflow to identify novel DELLA interaction partners Main Workflow Control Steps

Transfection + + PEG solution Incubate 24h DELLA GFP + After we have the protoplast they will be transformed with our DELLA and his interacting partners, next to the DELLA protein there is an Reporter Gene. Reporter genes are often used as an indication of whether a certain gene has been taken up. So we put the protoplasts and the DNA into the same tube and we add an PEG solution (polyethylene glycol) this PEG solution ensures that the DNA comes in the nucleus of the protoplasts. After an incubation of 24h we can see the results. I will shown you some results later. If there are a lot of protoplasts transformed the goal is to isolate the DELLA from other unnecessary elements. So first of all, because DELLA is situated in the nucleus of the protoplasts, we have to break the nucleus open. This happens with an extraction buffer. Incubate 24h

Workflow to identify novel DELLA interaction partners Main Workflow Control Steps

Detect the nucleus with different buffers Protein extraction Detect the nucleus with different buffers Shown by anti-H3 signal Histone Before we do an protein extraction we first test which extraction buffer breaks the nucleus open. We test the BASIS buffer it’s ?? And the RIPA this is a stronger buffer because it have more detergents in it. So we thought the BASIS buffer don’t break the nucleus open. As we know for sure is that the HISTON protein is setteled in the nucleus, they are the chief protein components of chromatin, acting as spools around which DNA winds. This is already the Result on an western blot ( i will explain the technique later how it works)  with as primair antibody an anti-histon. So as you can see on the picture with both extraction buffers the nucleus is breaking open because in both you can see a signal at the height of ?? Opzoeken!!! BASIS buffer: 10 mL DELLA extraction buffer 50 mM TRIS (pH = 7.5): 0.5 ml from 1M 150 mM NaCl: 0.3 mL from 5M Pre-cooled milli-Q: 9.2 mL 1 complete EDTA tablet Roche 0.5% Triton X-100 = 50 µL in 10 mL 1 mM PMSF: 100 µL to 10 mL   RIPA buffer 0.1% SDS: 50 µL from 20% SDS 1% Sodium deoxycholate

Workflow to identify novel DELLA interaction partners Main Workflow Control Steps

Co-Immunoprecipitation Identification of protein-protein interactions with GFP-tagged DELLA protein Co-IP shares the fundamental principle of the specific antigen-antibody reaction: an antibody against a specific target protein (in our case it’s anti-GFP) forms an immune complex with that target in a sample. Because GFP is hanging on DELLA The complex is then precipitated on a beaded support to which an antibody-binding protein is immobilized. If any protein not precipitated on the beads they will washed away. Finally, the antigen is eluted from the support and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), often followed by Western blot detection to verify the identity of the antigen.

Results – Western Blot Standard method Improved method 98 kDa 98 kDa

Conclusion Improved the efficiency of transient protoplast transformation Selected standard protein extraction buffer to extracts nuclear proteins Optimized co-IP by eluting with SDS instead of Glycine A method is developed to discover interaction partners of a protein in wheat protoplasts

Unravelling DELLA’s working mechanism in wheat by studying protein-protein interactions Thank you for your attention, I hope you have a better idea what Karel and I are doing and if there are question pleas ask them. Yoni Bultinck