Molecular mechanism of fungal effector PevD1-NRP interaction and discovery of a small-molecule disruptor

Verticillium wilt, caused by Verticillium dahliae, is a devastating vascular disease that severely affects cotton and other dicotyledonous crops worldwide. The fungal effector protein PevD1 promotes disease progression by interacting with the host asparagine-rich protein (NRP) and disrupting cryptochrome 2 (CRY2)-mediated signaling; however, the molecular basis of this interaction remains poorly understood. Here, we combined computational modeling, molecular interaction analysis, mutagenesis, and structural biology approaches to investigate the PevD1-NRP-CRY2 regulatory mechanism in Arabidopsis thaliana. AlphaFold-Multimer prediction and molecular dynamics (MD) simulations indicated that both PevD1 and the photolyase homology region of CRY2 interact with the death-associated domain of NRP through highly overlapping interfaces. Surface plasmon resonance and isothermal titration calorimetry analyses showed that PevD1 binds NRP with substantially higher affinity than CRY2 (K_D values of 0.19 μM and 4.34 μM, respectively). Site-directed mutagenesis further identified E108 and L111 of PevD1 as critical residues required for NRP recognition. We subsequently determined the crystal structure of PevD1 at 1.35 Å resolutions and performed structure-guided virtual screening targeting the predicted interaction interface. This led to the identification of compound A6, which binds PevD1, forms a key interaction with residue E108, and disrupts the PevD1-NRP interaction in biochemical assays. Together, these findings define the molecular mechanism underlying the PevD1-NRP-CRY2 interaction and establish a framework for the development of effector-targeting anti-virulence compounds against V. dahliae.