ZmPP2C45 and ZmBELL4 suppress maize biochemical defense against insect herbivores

Summary Benzoxazinoids (BZX) are the most abundant defensive metabolites of maize ( Zea mays ). Genetic fine‐tuning of BZX metabolism holds the potential to enhance maize resistance against insect herbivory. Natural variation in BZX abundance has been associated with genetic polymorphism in ZmPP2C45. Here, we demonstrate that ZmPP2C45 encodes a nucleocytoplasmic‐localized protein phosphatase 2C. The total BZX content in maize leaves was elevated by more than threefold in Zmpp2c45 knockout lines, whereas overexpression of ZmPP2C45 had no effect. Insect herbivore growth was significantly hampered in the Zmpp2c45 mutants. Expression of BZX biosynthetic (BX) genes was upregulated in a Zmpp2c45 mutant. Comparative phosphoproteomic analyses, protein–protein interaction experiments, and ex situ dephosphorylation activity assays suggested that a homeodomain‐containing transcription factor, ZmBELL4, could be a potential target of ZmPP2C45. Dual luciferase assays and transient gene silencing in maize seedlings supported that ZmBELL4 suppressed BX gene expression dependent on its own phosphorylation state and reduced BZX content. Our findings reveal ZmPP2C45 and its putative molecular target, ZmBELL4, as two suppressors of BZX metabolism. These results shed light on a novel regulatory pathway of maize biochemical defense and present ZmPP2C45 as a promising candidate for genetic enhancement of maize insect resistance.