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. 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.

植物激发肽（Plant Elicitor Peptides，Peps）是一类在多种植物中保守的防御反应调控因子，且在多数植物中属于多基因家族。然而，Peps作为多基因家族的功能相关性在很大程度上仍未明确。尽管拟南芥的Peps在功能上大多呈现冗余性，但此前针对玉米中Pep诱导反应的研究表明其功能具有特异性。为了更明确单个玉米源植物激发肽（ZmPeps）的功能，本研究通过检测防御相关植物激素、特殊代谢物以及全局基因表达模式的变化，分析了每种肽的活性。除了阐明单个ZmPep以及玉米植物激发肽受体（ZmPEPR）的活性外，这些实验还为Peps信号通路提供了诸多新见解。研究发现玉米源植物激发肽前体（ZmPROPEP）可携带多个ZmPeps，这是此前未被报道过的现象。共计鉴定出7种全新的ZmPeps，且该家族成员的活性特异性并非源于序列唯一性，而是由反应输出的相对强度所决定。研究观察到ZmPep诱导的茉莉酸（jasmonic acid）与乙烯（ethylene）水平变化，与诱导的防御反应强度之间存在显著相关性，这表明ZmPeps通过类似变阻器的方式调控植物激素水平，进而调节免疫输出。肽结构-功能研究以及配体-受体建模分析揭示了对ZmPep信号功能至关重要的结构特征。结构分析还鉴定出ZmPep5a作为一种潜在的拮抗肽，可竞争性抑制其他ZmPeps的活性，这是该家族此前未被发现的调控机制。通过异源表达实验、内源基因表达模式分析以及成簇规律间隔短回文重复序列相关蛋白9（CRISPR/Cas9）介导的敲除植株分析，研究发现ZmPEPR1是调控植物体内ZmPep介导的抗草食动物防御反应的主要受体。将V3期玉米植株的第5片叶片剪下，置于装有蒸馏水或5微摩尔浓度ZmPeps溶液的玻璃管中；于处理后12小时收集组织样本。每个处理组设置4个生物学重复。基因芯片（microarray）样本的制备与分析由佛罗里达大学生物技术研究跨学科中心（Interdisciplinary Center for Biotechnology Research，ICBR）的基因表达核心实验室完成。