UC San Diego

In plants, the precise regulation of cell survival or cell death decisions control effective immunity to different types of pathogens. Cellular damage in plants results in the enzymatic and non-enzymatic peroxidation of fatty acids known as oxylipins. Enzymatic oxylipin biosynthesis often begins with the lipase-based cleavage of linoleic (18:2) or linolenic acid (18:3) from membrane bound lipids. Fatty acids are then dioxygenated by lipoxygenases (LOXs) with regiospecificity at carbons 9 (9-LOX) or 13 (13-LOX) resulting in unstable fatty acid hydroperoxides. Specific oxylipins serve as direct antimicrobial defenses and plant signaling molecules regulating diverse processes such as development, stress acclimation and innate immune responses against pests and pathogens. A commonly studied enzymatic pathway for the 13-LOX 18:3 fatty acid product, termed 13(S)-hydroperoxylinolenic acid, is the sequential activity of 13-allene oxide synthase (13-AOS) and 13-allene oxide cyclase (13-AOC) to yield 12,13(S)- epoxylinolenic acid and finally the 18-carbon cyclopentenone termed 12-oxo-phytodienoic acid (12-OPDA). 12-OPDA is an essential precursor to the 12-carbon cyclopentanone termed jasmonic acid (JA), an essential precursor to the plant hormone JA-isoleucine that controls defense activation and reproduction. In maize (Zea mays) leaves, southern leaf blight (SLB; Cochliobolus heterostrophus) infection results in the additional accumulation of jasmonate-like postional isomers derived from the activity of 9-LOX. These molecules include 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), derived from 18:3 and 18:2 respectively, and the respective series of 14- and 12- carbon metabolites termed death acids (DA). The accumulation of 10-OPEA becomes wound-inducible in fungal-infected maize tissues, is phytotoxic and acts as a direct defense suppressing fungal growth [1]. Conceptually DAs are predicted to be derived from 9-LOX, 9-AOS and novel 9-AOC enzyme activities. Using forward genetics via metabolite based association mapping in maize, efforts in the Schmelz laboratory identified a novel 9-AOC, termed Death Acid Synthase (DAS), and confirmed catalytic activity using Agrobacterium-mediated Nicotiana benthamiana heterologous enzyme expression assays with key combinations of established LOX, AOS and 13-AOCs to generate appropriate substrates for comparisons. During my Masters thesis I used defined CRISPR/Cas9 maize 9-aoc mutants to biochemically characterize plants for the endogenous loss of DA production, examine for non-target effects on maize antibiotic biosynthesis and explore alterations in disease resistance to fungal pathogens. Results support the existence of a single functional copy 9-aoc in maize responsible for DA biosynthesis and additional biochemical immune layers that contribute to Fusarium graminearum resistance.