Foc4-Ap1, a putative aspartic protease of Fusarium oxysporum f. sp. cubense race 4, targets banana NAD(P)H-quinone oxidoreductase subunit S to promote virulence

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense race 4 (Foc4) is a major threat to banana production. Secreted proteins are central to Foc4-banana interactions, but many remain uncharacterized. A putative aspartic protease named Foc4- Ap1 was identified from the infection-induced transcriptomes. RT-qPCR showed the highest expression in conidia, reduced expression during early infection, and strong induction at 96 h post inoculation. We generated the ΔFoc4- Ap1 deletion strains and the complemented strain by homologous recombination. Loss of Foc4- Ap1 reduced vegetative growth and altered sensitivity to a cell wall-perturbing agent and osmotic stress, while complementation restored these phenotypes. In banana assays, ΔFoc4- Ap1 deletion strains caused delayed onset of wilt symptoms and reduced disease severity; complementation restored virulence to wild-type levels. Yeast signal sequence trap (YSST) assay confirmed secretion activity for the predicted signal peptide. In Nicotiana benthamiana , GFP fusions showed that Foc4-Ap1 Δsp localized mainly to the cytoplasm and nucleus, whereas full-length Foc4-Ap1 and the PR1 signal peptide-substituted construct were enriched at the cell periphery. Yeast two-hybrid screening followed by BiFC validated an interaction between Foc4-Ap1 and banana NAD(P)H-quinone oxidoreductase subunit S (Ma-NdhS). These results indicate that Foc4- Ap1 contributes to vegetative growth, stress responses, and full virulence, and reveal Ma-NdhS as a candidate host-interacting protein.