Hello everybody,
I'm working on an arabidopsis MAPKKK gene against different biotic and abiotic stresses. The T-DNA knock out mutant gives an early flowering phenotype that actually makes difficult for me to get phenotype against pathogens. The plant starts flowering after three weeks of its germination. So far i've been able to determine its role against a bacterial pathogen and drought and ABA, which is enough to assume its role against different stimuli. I'm running out of time for my PhD but my PI is pressing me to do more disease assays despite the fact that he doesn't pays much attention about my research and the plant growth conditions are miserable.

Anyway i want to know what else downstream work i can do for this gene for example any protein assay to prove its role against pathogens without using plants. I've to focus on pathogen interaction as this is the policy of the department. I talked about doing yeast two hybrid analysis but again the PI insists on doing phenotypic study first. Also I suspect this gene could also be a receptor as MAPKKKs function on the top of signaling network. What assay I can do to determine whether its a receptor or not? Perhaps this is the most important question that i want to ask.

My problem seems to be more related with my PI's attitude but i want to talk with him in detail and before discussion i need to gather some ideas.
I've tried my best to focus on the question but it seems frustration is becoming a part of my PhD and i need some counseling too.....

cheers

hi,

i can't really answer your technical question and I don't have the slightest idea about plant work but i may offer a suggestion for your PI thing.

my suggestion is: try being as diplomatic as you can and don't go head first into something you are not 100% sure of.
actually: don't go head first even if you KNOW you are right - this won't help you in the long run.
the PI is the one who calls the shots, that's how the system is set up. If you can't convince him/her or steer him/her in the direction you think would be good - don't push it.
you still need letters of recommendation, authorships on papers you participated on, contacts etc. from that person.. so unless he/she is a total a** go easy on this and try to manipulate gently rather than push vigorously.

well,.. my few cents anyway from a "been there in MY PhD thesis" point of view

Thanks Warsel, your suggestions are valuable. This P.I. thing is universal and all that I/we need is to be patient...

coming back to the point, any suggestions on my question????

cheers


P.S. I've deleted my last post, I think I was too manic and cynical of my fellows.

Hi!

I can feel your pain and frustation, IMO the best way to go is to do a litle bit of what your PI wants and at the same time do what interests you on the background. Once you have results on your interest branch you can show them to your PI, after that he will probably be fine with your approach.

If the protein is a receptor on the top of a signalling pathway it will probably phosphorilate. Phosphorilated proteins have different electrophoretic mobilities from the same proteins non-phosphorilated.
So your protein will appear as 2 distinctive bands. If you have an antibody against the protein you just have to perform a western blot in which you load extract of the nock-out (negative control), your plant after strong exposition to the pathogen. drought or whatever stimuli and your plant without the stimuli. If you see variation in the band densities, thats it.
If you don't have an antibody but you know the protein sequence you can predict the protein weight, Isoelectric point and gravy index(hydropobicity). With this info you can perform 2D electrophoresis and locate your protein by it's caracteristics. (Applies the same for phosphorilation).
If it doesn't phosphorilate then it is probable that it interacts with other proteins or DNA, to probe this you should perform native electrophoresis. If it is interacting the mobility will change. To help keep the interacting molecule bound you can use paraformaldehyde, sodium bisulfite...
Still, I don't work with plants but I find quite surprising that been the first receptor it responds to such different stimuli.