Progress 02/01/17 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major problem encountered was the unreliability of in-house generated antibodies to PIF1, PIF4 and PIF5. I was overly optimistic about the prospects of these antibodies detecting their targets in planta. As a result, both of my goals were hindered substantially. Additionally, a group in Sweden published their results on the discovery of two E3 ligases involved in the degradation of PIF4. As a result, I thought that it would be wise (and in line with my proposed project goals, specifically goal number 2) to test whether these E3 ligases were involved in the degradation of other PIFs. What opportunities for training and professional development has the project provided?In the course of conducting this project, I had the opportunity to meet regularlywith the supervising PI (Peter Quail), present my work in lab meetings and in PGEC seminars, I have had multiple opportunites to describe my work to colleagues and get their feedback. I have also developed proficiency in new scientific tools, especially those involving the analysis of sequencing data and protein biochemistry. In addition, my collaboration with a group in Sweden has contributed towards me starting a new position at that research institute. I am hopeful that members of the Quail lab will be able to pick up where I left off on this project and that the goals will ultimately be addressed, resulting in a peer-reviewed publication on which I would be a co-author. This would also help in my professional development. How have the results been disseminated to communities of interest?Since the results were inconclusive, they remain unpublished. I did present the research during semi-annual PGEC seminars. I also discussed the results with collaborators and colleagues. As I mentioned aboved, I am optimistic that someone will pick up where I left off and these results will ultimately be published in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Unfortunately, neither goal was accomplished. Issues with antibodies prevented me from achieving the first goal and by the time the antibodies became useable, insufficient time remained to address the questions posed in this goal. The second goal was partially addressed by a group in Umea, Sweden. They found two E3 ligases involved in the degradation of PIF4. Given that this research was published early in the course of my work, I attempted to adjust my research goals accordingly. I initiated a collaboration with this group and obtained seeds from them with the goal of testing whether these recently identified ligases were involved in the degradation of other PIFs (PIF1, PIF3 and PIF5). Unfortunately, the unreliability of antibodies mentioned above hindered the progress on these experiments and the results were inconclusive.
Publications
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Progress 02/01/18 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major problem encountered thus far is the unreliability of in-house generated antibodies to PIF1, PIF4 and PIF5. As I described, I sought out alternatives but was left with no viable options until the antibodies to PIF1 and PIF4 became available/useable. Unfortunately, insufficient time remains for me to continue using these antibodies to further the goals of the project. I am hopeful that other members of the Quail lab will be able to pick up where I left off to address these goals. What opportunities for training and professional development has the project provided?As a result of regular meetings with the supervising PI (Peter Quail), lab meetings and PGEC seminars, I have had multiple opportunites to describe my work to colleagues and get their feedback. I have also developed proficiency in new scientific tools, especially those involving the analysis of sequencing data and protein biochemistry. Initiating a collaboration with other scientists although helped me with my professional development, especially since I will be startinga second postdoctoral project at the home institute of my collaborators. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Progress towards completing the first goal, to determine whether or not PIF1, PIF4 and PIF5 participate in the MAD mechanism of signal feedback attenuation in Arabidopsis thaliana remains stalled as a result of the unreliability of antibodies generated in the lab against these proteins. Preliminary tests on recently obtained PIF1 and PIF4 antibodies have shown reliable detection of target proteins, but insufficient time remains for me to test the accumulation of these protein under the targetted conditions. The second goal, to uncover additional E3 ligase(s) functioning in the degradation of PIF3 has also stalled. I performed work in collaboration with a group at the Umeå Plant Science Centre in Sweden led by Dr. Ove Nilsson. They found that a pair of E3 ligases (called BOP1 and BOP2) function in the degradation of PIF4 and I attempted to both replicate their results and to testwhether these ligases function in the degradation of PIF1, PIF3 and PIF5. These experiments were unsuccessful due to aforementioned issues with antibody reliability.
Publications
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major problem encountered thus far is the unreliability of in-house generated antibodies to PIF1, PIF4 and PIF5. As I described, I have sought out alternatives from both companies and other labs and am optimistic that these antibodies will enable me to complete my goals. What opportunities for training and professional development has the project provided?As a result of regular meetings with the supervising PI (Peter Quail), lab meetings and PGEC seminars, I have had multiple opportunites to describe my work to colleagues and get their feedback. I have also developed proficiency in new scientific tools, especially those involving the analysis of sequencing data and protein biochemistry. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?As described above, I have initiated a collaboration with Dr. Ove Nilsson in Umeå and aim to test whether the E3ligases recently characterized in his lab also function in the degradation of PIF1, PIF3 and PIF5. I also will test the newly obtained antibodies against PIF4 and PIF5 to determine whether they will enable me to study PIF4 and PIF5 abundance in the lrb triple mutant.
Impacts
What was accomplished under these goals?
Progress towards completing the first goal, to determine whether or not PIF1, PIF4 and PIF5 participate in the MAD mechanism of signal feedback attenuation in Arabidopsis thaliana has been stalled as a result of the unreliability of antibodies generated in the lab against these proteins. The antibodies recognize in vitro produced protein, but efforts to detect proteins from plant tissue extract have thus far been unsuccessful. I have recently ordered antibodies against PIF4 and PIF5 from a company and hope that they will enable me to complete the first goal. The second goal, to uncover additional E3 ligase(s) functioning in the degradation of PIF3 is still in progress. A group at the Umeå Plant Science Centre in Sweden led by Dr. Ove Nilsson recently published results describing the discovery of a pair of E3 ligases called BOP1 and BOP2 that function in the degradation of PIF4. I have initiated a collaboration with Dr. Nilsson and aim to test whether these ligases function in the degradation of PIF1, PIF3 and PIF5. Furthermore, they have successfully used an antibody against PIF4 which I intend to use in addressing the first goal.
Publications
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