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Quantitative characterization of the interactions between LRT2, Hsp90, and OsIAA11 that regulate the development of lateral roots in rice plants

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2024-09-05
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Abstract

Auxin-responsive Aux/IAA proteins generally serve as transcriptional repressors, preventing the transcription of genes needed for plant development. To activate these pathways, Aux/IAA proteins must be ubiquitinated by making a tripartite interaction with auxin and their cognate E3 ubiquitin ligase, followed by degradation through the 26S proteasome. In rice, the Aux/IAA protein OsIAA11 represses the initiation of lateral root development in seedlings. As only the cis isomer of the OsIAA11 degron can bind to the TIR1 E3 ligase, the cyclophilin isomerase LRT2 is required to provide rapid cis-trans exchange, allowing the entire population of OsIAA11 to be removed quickly. The molecular chaperone heat shock protein 90 (Hsp90) is also involved in this pathway through a direct interaction with the E3 ligase TIR1.Using nuclear magnetic resonance (NMR), we found that the binding and catalysis steps of OsIAA11 isomerization by LRT2 can be separated by single residue mutations of the enzyme, allowing for the tuning of individual kinetic parameters within the system. The mutation LRT2-P125K weakens the interaction between isomerase and substrate while maintaining on-enzyme catalysis, resulting in slower overall isomerization. The mutations LRT2-W128A and LRT2-H133Q showed greatly reduced binding to and catalysis of OsIAA11. Mathematical models of the degradation system revealed the necessity of negative transcriptional regulation to recreate the kinds of damped oscillations in mRNA levels seen in response to auxin within rice roots. The involvement of Hsp90 in lateral root development was demonstrated whereby the inhibition of Hsp90 significantly reduced the elongation of lateral roots in rice seedlings, while the addition of auxin partly rescued this phenotype. The mutation LRT2-K89E greatly reduced the binding between LRT2 and Hsp90. Moreover, this mutation was still able to bind to the OsIAA11 substrate but showed no ability to catalyze isomerization of the degron, identifying it as a binding competent but isomerization incompetent mutation. Finally, two additional binding partners, SGT1 and IPK2, were shown to interact with other Aux/IAA response proteins in rice through pulldowns and NMR experiments. Overall, these studies provide LRT2 mutants for phenotypic studies and expand the auxin regulatory circuit through identification of the LRT2:Hsp90 interaction as an important component.

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162 pages

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2023-08

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Auxin; Hsp90; LRT2; Modeling; NMR; OsIAA11

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Nicholson, Linda

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Oswald, Robert
Kawate, Toshimitsu

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Biochemistry, Molecular and Cell Biology

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Ph. D., Biochemistry, Molecular and Cell Biology

Degree Level

Doctor of Philosophy

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Government Document

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Attribution-NonCommercial-ShareAlike 4.0 International

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dissertation or thesis

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