Plumbagin targets dihydroorotase in Magnaporthe oryzae to inhibit infection and shows antifungal activity against multiple phytopathogenic fungi

Plant-pathogenic fungi threaten both crop yield and quality. This, together with the increasing resistance of plant-pathogenic fungi to existing fungicides, underscores the need to develop environmentally sound fungicides with novel modes of action. In this study, we systematically evaluated the Antifungal activity of plumbagin and its inhibition of dihydroorotase (DHOase), a key enzyme in the de novo pyrimidine biosynthesis pathway of major plant-pathogenic fungi. Our results showed that plumbagin significantly inhibited DHOase activity in Magnaporthe oryzae, Fusarium graminearum and Fusarium oxysporum, markedly reducing the pathogenicity of these three fungi. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) analyses confirmed specific binding of plumbagin to MoPyr4. Molecular docking combined with site-directed mutagenesis further reveals a distinct binding mode between plumbagin and MoPyr4. Residues R90, N178, and H320 in MoPyr4 are critical for plumbagin binding. Transcriptomic analysis indicated that plumbagin significantly affected metabolic processes, particularly energy-related pathways, in M. oryzae. Our findings identify DHOase as an attractive molecular target for controlling plant-pathogenic fungi and highlight plumbagin as a promising lead compound for developing DHOase-targeting fungicides. This research holds immense significance for the potential application of novel 1,4-naphthoquinone derivatives in combating plant-pathogenic fungi.