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Development of a protein-luciferase-based high-throughput screening system to monitor degradation of Jasmonate ZIM-domain family proteins

Hirotaka Ishida: Graduate School of Environment and Information Sciences, Yokohama National University

<div>We previously demonstrated that the system using Arabidopsis seedlings harboring a promoter-reporter fusion gene, such as luciferase (LUC) reporter fusion, is suitable for monitoring of regulated gene expression of pathogen responsive genes such as <em>PR1, PDF1.2,</em> and <em>VSP1</em>. Using the system we could successfully carry out the screening and evaluation of bioactive compounds capable of inducing the expression of defense-related genes involved in systemic acquired resistance or induced systemic resistance. However, the system provides us with indirect evidence for the resulting gene expression and the processes involved in the signaling pathway are not necessarily clear. On the other hand, protein degradation involved in a particular signaling pathway can be a suitable marker for specific regulation of the signaling process. Using transgenic plants expressing LUC-protein fusion, we are able to monitor the protein degradation process as the decay of luciferase activity <em>in vivo</em>. To monitor defense response against necrotrophic pathogens mediated by jasmonates (JA)<em>,</em> we attempted to observe the dynamics of Jasmonate ZIM-domain family proteins (JAZ) which are rapidly degradated in the presence of jasmonyl-L-isoleucine. Using transgenic Arabidopsis seedlings expressing JAZ-LUC fusion proteins, we could observe a rapid decay of the bioluminesence <em>in planta</em> in response to JA treatment. The system allows us to monitor JAZ-mediated signaling response within less than one hour and to perform high-throughput screening for JA agonists using 384-well plates.</div>