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Abstract
Fusarium verticillioides and Sarocladium zeae naturally co-inhabit maize kernels and cycle in an evolutionary arms race to maintain their ecological niches. As an endophyte or pathogen, F. verticillioides is a prolific fumonisin producer and food safety threat. As a protective endophyte of maize, S. zeae produces pyrrocidines A and B, which inhibit the growth of F. verticillioides and block fumonisin biosynthesis. Previous work found FvZBD1 (FVEG_00314), a putative enoyl reductase located next to the fumonisin biosynthetic gene cluster, and FvABC3 (FVEG_11089), an efflux pump, to be highly induced by pyrrocidine. Here, pyrrocidine dose-response assays revealed a potent synergy between pyrrocidines A and B, where they functioned synergistically to inhibit F. verticillioides growth. Further, results provided evidence that FvZBD1 confers partial tolerance to pyrrocidine A. This work also showed that FvABC3 has functional specificity to pyrrocidine B, conferring resistance. Thus, pyrrocidine A and B show different target specificity (FvZBD1 or FvABC3) and synergistic action. Remarkably, coupled with transcriptomics and targeted and untargeted metabolomics, additional investigation discovered that FvZBD1 rapidly reduces pyrrocidine A into pyrrocidine B for efflux by FvABC3. Unexpectedly, fusaric acid and fusarubin production were triggered by pyrrocidines, exemplifying the metabolic arms race between F. verticillioides and S. zeae. Intriguingly, pyrrocidine treatments also triggered the accumulation of putative itaconic acid, succinic acid, and fumaric acid – TCA cycle intermediates, distinctly associated with cycle inhibition – indicating that pyrrocidine inhibits the TCA cycle and electron transport chain, decreasing ATP synthesis, and inducing mitochondrial dysregulation. Importantly, this work determined the mechanisms of pyrrocidine tolerance in F. verticillioides (biotransformation and efflux), and the core role of FvZBD1. Further, these results support that pyrrocidine inhibits F. verticillioides growth by disrupting energy metabolism, and fumonisin biosynthesis through itaconic acid-based aconitic acid depletion. These findings help inform the optimization of maximally efficacious S. zeae strains for eliminating F. verticillioides colonization and fumonisin contamination in maize, and advance our understanding of the role of secondary metabolites in fungal competition, communication, and mycotoxin control. A seed-delivered S. zeae biocontrol strain that produces both pyrrocidine A and B may effectively prevent F. verticillioides growth and mycotoxin production in the field.