Plants benefit from interactions with symbiotic microorganisms, with enhancements ranging from improved growth to activation of induced systemic resistance (ISR) against a broad range of pathogens. While pathogen-triggered systemic acquired resistance (SAR) is well understood with multiple signal molecules identified, ISR mobile signals remain unknown. Jasmonic acid (JA), a 13-lipoxygenase (LOX)-derived oxylipin, and ethylene (ET) have long been established as the main phytohormone regulators of ISR, although conclusive evidence for these two molecules as ISR mobile signals is lacking. However, there is increasing evidence that other oxylipin signals, especially those derived from 9-LOX activity, have roles in ISR. For instance, the maize 9-LOX, LOX3, has been identified as a negative regulator of ISR, with lox3 mutants displaying constitutive ISR against a broad range of pathogens. The objective of this study is to identify oxylipin biosynthesis genes and specific molecules that govern ISR. Maize wild-type and near-isogenic mutants disrupted for several LOX, JA biosynthesis, and ET biosynthesis and ISR-positive and -negative mutants of the beneficial fungus Trichoderma virens were used to identify key oxylipin regulators of ISR by metabolite and transcriptome profiling. Both the JA-producing 13-LOX, LOX10, and the 9-LOX, LOX12, were overexpressed in lox3 roots and found to be required for T. virens-induced ISR. T. virens-colonized lox10 and lox12 mutants became more susceptible to infection by Colletotrichum graminicola, causal agent of anthracnose leaf blight, leading to an induced systemic susceptibility (ISS) phenotype. Oxylipin profiling of xylem sap from maize exhibiting different defense responses after treatment with mutant or WT T. virens identified JA precursor, 12-oxo-phytodienoic acid (12-OPDA), and a 9-LOX-derived ?-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (KODA) as molecular signals involved in induction of ISR. Treatment with 12-OPDA or KODA enhanced resistance against infection in a dose-dependent effect, confirming signaling roles. Surprisingly, T. virens-induced ISR in either JA- or ET-deficient mutants, suggesting neither were required for ISR. Transcriptome analysis of T. virens-treated maize revealed upregulation of 12-OPDA biosynthesis and response genes, but downregulation of subsequent JA biosynthesis genes and JA response genes. These results show that OPDA and KODA, but not JA, are required for activation of T. virens-induced ISR in maize.
