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 US cotton production, which require root-knot nematodes to cause damage, Fov4 is known to infect cotton independent of nematodes. However, 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 (NRPS) gene FNP1. To investigate its function, we generated FNP1 knock-out mutant using CRISPR-Cas9 approach. Growth assays revealed that the mutants exhibit significantly attenuated hyphal production on media containing cotton roots as the sole carbon source, increased sensitivity to cell stress agents, as well as lagged spore germination. Furthermore, the mutant exhibited defect in cotton root rot virulence and significant decrease in Fusaric acid production. Microscopic observation of GFP-labeled FNP1 deletion mutant showed impeded infection progression in cotton roots compared to the wild type (WT). RNA-seq analysis further revealed extensive transcriptional reprogramming in 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.
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