Functional analysis of maize Plant Elicitor Peptide-mediated immune signaling. Functional analysis of maize Plant Elicitor Peptide-mediated immune signaling

Plant Elicitor Peptides (Peps) are conserved regulators of defense responses across diverse plant species and are multigene families in most species. However, the functional relevance of Peps as multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize implied specificity of function. To better define function of individual ZmPeps, activities of each peptide were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns. In addition to delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling. ZmPROPEP precursors were found to harbor multiple ZmPeps, a phenomenon not previously observed. In all, seven new ZmPeps were identified and together the family members were found to have specific activities defined by relative magnitude of response output rather than uniqueness. A striking correlation between ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses was observed, indicating that ZmPeps regulate immune output through rheostat-like regulation of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to ZmPep signaling function. Structural analysis also led to identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit activity of other ZmPeps, a regulatory mechanism not previously observed for this family. Using heterologous expression assays, endogenous gene expression patterns and analysis of CRISPR/Cas9 generated knockout plants, ZmPEPR1 was found to be the dominant receptor regulating ZmPep-mediated anti-herbivore defenses in planta. Overall design: Leaf 5 of V3 stage maize plants was excised and placed in glass vials containing either water or 5 micromolar solutions of ZmPeps; tissue was collected 12 hours post-treatment. For each treatment four biological replicates were generated. Microarray sample preparation and analysis was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) gene expression core.