Prediction and Validation of New Targets of NNPCN in Inhibiting Rhizoctonia Solani Based on Molecular Docking, Dynamics, and Biotechnology

Abstract

The antifungal targets of the new fungicide N-(naphthalen-1-yl)-phenazine-1-carboxamide (NNPCN) are still incomplete, limiting its application. To identify potential new targets of NNPCN and facilitate target hunting, a suite of techniques was employed to conduct experiments on Rhizoctonia solani. Nine potential targets were identified, exhibiting strong binding affinity to NNPCN, as indicated by binding free energies below −100.000 kJ/mol. Notably, pectin lyase, glycosyl hydrolase, fumarate transporter, and cytochrome monooxygenase showed exceptionally strong binding. The mRNA expression analysis revealed significant downregulation in certain target genes: E3 ubiquitin ligase (AG1IA_02506), aldehyde dehydrogenase (AG1IA_03762), fumarate transporter (AG1IA_03944), and pectin lyase (AG1IA_03046) decreased by 42%, 66%, 83%, and 69%, respectively, while other key genes were upregulated. Pectin lyase protein was obtained through prokaryotic expression at 0.4 mg/mL concentration. A novel thiobarbituric acid test system verified pectin lyase as a potential NNPCN target, with the enzyme activity multiple being only 0.169 after NNPCN treatment. These findings enhance our understanding of NNPCN’s mode of action and could guide its improved application.