2020 Fiscal Year Annual Research Report
| Project/Area Number |
19F19782
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
白須 賢 国立研究開発法人理化学研究所, 環境資源科学研究センター, グループディレクター (20425630)
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| Co-Investigator(Kenkyū-buntansha) |
FISHMAN MAX 国立研究開発法人理化学研究所, 環境資源科学研究センター, 外国人特別研究員
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| Project Period (FY) |
2019-11-08 – 2022-03-31
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| Keywords | parasiticplants / calciumsignaling / phosphoproteomics / DMBQ / haustorium |
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| Outline of Annual Research Achievements |
During 2020, I have further investigated the role of TOR during haustorium formation in Phtheirospermum japonicum. I was able to identify the areas where TOR is expressed in the root of P. japonicum using a reporter gene construct. The expression profile was found to be very specific but there were single cells expressing TOR transcript around the area with the haustorium forms. TOR is activated by both glucose and sucrose in other plants but it was unknown whether haustorium formation in P. japonicum was impacted by sucrose or glucose. I found that low concentrations of sucrose and glucose did not impact DMBQ-induced haustorium formation in P. japonicum and that concentrations as low as 1.5 mM glucose or sucrose could induce haustorium-like formations in P. japonicum. This suggests TOR may play a positive role in haustorium formation.
The phosphoproteomics data also identified three phosphopeptides associated with two receptor-like kinases (RLKs) that are homologous to a DMBQ-induced RLK in the model plant Arabidopsis thaliana. I created tested roots expressing RNAi knockdown constructs for these two RLKs and found that knocking down both RLKs at once disrupted haustorium formation in P. japonicum in half of the transformed roots. This suggests that these two RLKs play a role in haustorium formation in P. japonicum.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
I feel that I was able to further my research and test several more RNAi constructs in P. japonicum like I had planned but I have not transformed A. thaliana with phosphomimic and phosphodead versions of the targeted proteins like I had planned. I have recently made the constructs for transforming A. thaliana with phosphodead and phosphomimic versions of the RLKs of interest and plan to do that experiment soon. I also had difficulty cloning the gene that encodes TOR in P. japonicum and just recently have looked at expression of TOR in N. benthamiana. These experiments were part of my planned research scheme for 2020 and given that I still have to perform these experiments I have chosen “a bit behind schedule.”
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| Strategy for Future Research Activity |
or the remainder of the fellowship I plan to perform several more experiments. I have enough time left to make A. thaliana card1-2 aequorin expressing phosphomimic and phosphodead versions of the RLKs I identified as interesting. I will be able to determine whether the phosphodead and phosphomimic versions of these RLKs can compliment DMBQ-induced calcium signaling in A. thaliana card1-2 plants. I will also verify the phosphosites of these RLKs in P. japonicum following DMBQ-treatment using anti-phospho antibodies that I designed.
I also plan to heterologously express TOR kinase in Nicotiana benthamiana and identify localization of the kinase and whether phosphomimic or phosphodead versions of TOR kinase localize differently than the wild-type version. This heterologous system will also be used to see whether activity of TOR kinase changes in the phosphomimic or phosphodead versions by looking at phosphorylation of a conserved TOR kinase target.
TOR kinase affects the cell cycle in plants and there are reports that it can affect calcium signaling. DMBQ affects the cell cycle at the root tip of P. japonicum and also induces calcium signaling. I plan to determine whether selective inhibitors for TOR disrupt DMBQ-induced calcium signaling or the DMBQ-induced cell cycle. I will then determine the activity of TOR following DMBQ-treatment using antibodies and anti-phospho antibodies for the conserved TOR target S6K1.
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