Fusarium oxysporum f. sp. vasinfectum Race 4 (Fov4) FNP1 , a Nonribosomal Peptide Synthetase Gene, Plays an Important Role in Cotton Fusarium Wilt

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is one of the most destructive early-season cotton diseases worldwide. The recent emergence of the highly virulent Fov race 4 (Fov4) and its aggressiveness have raised significant concerns for the U.S. cotton industry. Unlike predominant Fov races in U.S. cotton production, which require root-knot nematodes to cause damage, Fov4 is known to infect cotton independent of nematodes. However, the molecular mechanisms of Fov4 virulence in cotton are not clearly understood. Secondary metabolites are often identified as the culprits in pathogen virulence toward plant hosts. To investigate these factors in Fov4, we analyzed the genomes of Fov1 and Fov4 using Fungal antiSMASH and identified a Fov4-specific nonribosomal peptide synthetase gene, FNP1. To investigate its function, we generated an FNP1 knockout mutant using the CRISPR-Cas9 approach. Growth assays revealed that the mutant exhibited significantly attenuated hyphal production on media containing cotton roots as the sole carbon source, increased sensitivity to cell stress agents, and lagged spore germination. Furthermore, the mutant exhibited a defect in cotton root rot virulence and a significant decrease in fusaric acid production. Microscopic observation of the GFP-labeled FNP1 deletion mutant showed impeded infection progression in cotton roots compared with the wild type. RNA-seq analysis further revealed extensive transcriptional reprogramming in the FNP1 deletion mutant, supporting a regulatory role for FNP1 in fusaric acid biosynthesis and broader metabolic networks. Gene complementation restored the observed defects, confirming that FNP1 is critical for Fov4 virulence, hyphal development, fusaric acid production, and stress responses. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .