Uncovering the Differed Susceptibility of Fusarium\noxysporum (<i>Fo32931</i> and <i>FocII5</i>) to Fungicide Phenamacril: From Computational and\nExperimental Perspectives

<i>Fo32931</i> and <i>FoCII5</i> are two subtypes\nof Fusarium oxysporum (Fo), a pathogenic\nfilamentous fungus. Phenamacril (PHA), a <i>Fusarium</i>-specific fungicide that targets myosin I, exhibits significant hyphal\ngrowth inhibition in <i>Fo32931</i> but shows weak resistance\nin <i>FocII5</i>, despite only two amino acid differences\nin the PHA-binding pocket of myosin I. In this study, we aim to elucidate\nthe molecular basis for the differential sensitivity ofF. oxysporum myosin I variants (FoMyoI<sup>32931</sup> and FoMyoI<sup>cII5</sup>) to phenamacril through computational\nmethods and biochemical validation. The results suggest that phenamacril\nfunctions as an allosteric inhibitor for FoMyoI<sup>32931</sup>, inhibiting\nthe large oscillation of the converter lever domain (CLD) upon ATP\nbinding and promoting the opening of the outer cleft, further impairing\nprotein function. PHA significantly reduced the coupling between the\nCLD, especially the converter, and the catalytic center, diminishing\nthe response of the CLD to the motor domain in FoMyoI<sup>32931</sup>. From the residue mutation experiment, we found that the S418T substitution\nin FoMyoI<sup>cII5</sup> is the key to the reduced phenamacril sensitivity\nof <i>FocII5</i>. According to the microscale thermophoresis\n(MST) assay and pocket conformation analysis, the S418T mutation disturbs\nthe orientation of pocket residues, especially Lys537, leading to\na looser pocket and reduced interaction between Lys537 and phenamacril,\nwhich lowers the binding affinity of FoMyoI<sup>cII5</sup> for phenamacril.\nThese findings provide deeper insights into the reasons for the lower\nsensitivity of <i>FoCII5</i> to phenamacril from both molecular\nand structural perspectives and will also guide the design of novel\ninhibitors against resistant <i>Fusarium</i> spp., like <i>FoCII5</i>.