Application of Yeast-Two Hybrid Assay to Chemical Genomic Screens: A High-Throughput System to Identify Novel Molecules Modulating Plant Hormone Receptor Complexes

Abstract

Phytohormones are endogenous signalling molecules that regulate plant development, adaptation to the environment, and survival. Upon internal or external stimuli, hormones are quickly accumulated and perceived, which in turn activates specific signalling cascades regulating the appropriate physiological responses. In the last decade, great advances in understanding plant hormone perception mechanisms have been achieved. Among different methodological approaches, yeast-two hybrid (Y2H) assays played a pivotal role in the identification and analysis of plant hormone perception complexes. The Y2H assay is a rapid and straightforward technique that can be easily employed to identify small molecules directly modulating plant hormone perception complexes in a high-throughput manner. However, an Y2H chemical screen tends to isolate false positive molecules, and therefore a secondary in planta screen is required to confirm the genuine bioactivity of putative positive hits. This two-step screening approach can substantially save time and manual labor. This chapter focuses on the prospects of Y2H-based chemical genomic high-throughput screens applied to plant hormone perception complexes. Specifically, the method employed to carry out a chemical genomic screen to identify agonist and antagonist molecules of the phytohormone jasmonic acid in its conjugated form jasmonic acid–isoleucine (JA-Ile) is described. An easy in planta confirmation assay is also illustrated. However, this methodology can be easily extended to detect novel chemical compounds perturbing additional plant hormone receptor complexes. Finally, the high-throughput approach described here can also be implemented for the identification of molecules interfering with protein–protein interaction of plant complexes other than hormone receptors.