Functional Characterization of the Methyl Esterase MES4 in Hydrolytic Activity of Methylated Phytohormones in Tea Plants Based on Multi-Omics Analysis

This study integrated transcriptome and hormone profiling of <i>Colletotrichum camelliae</i>-infected tea leaves to identify the <i>methyl esterase</i> (<i>MES</i>) gene family. Genome-wide analysis identified 17 <i>MES</i> genes, with <i>CsMES4</i> as a crucial candidate localized in the nucleus and cell membrane. Through prokaryotic expression, purification, in vitro enzymatic assays, and transient silencing experiments in tea plants, we demonstrated that CsMES4 hydrolyze methyl salicylate (MeSA), methyl indole-3-acetate (MeIAA), and methyl jasmonate (MeJA) to produce salicylic acid, indole-3-acetic acid, and jasmonic acid, respectively. Transient silencing of <i>CsMES4</i> reduced these hormones and enlarged lesions, confirming its role in disease resistance. Key catalytic residues predicted by docking were validated by mutagenesis: the F60L mutation significantly decreased CsMES4's catalytic activity toward all three substrates, while the S127Y mutation enhanced its catalytic efficiency. This study highlights the pivotal role of CsMES4 in phytohormone metabolism and provides insights into its multifunctional catalytic mechanism in tea plants.