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 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 (KD 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 resolution 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.