Inhibition of Phytophthora infestans chitin synthase via cyclic peptide targeting for sustainable disease control

Phytophthora spp. are devastating plant pathogens causing significant economic losses in agriculture, but unlike true fungi, oomycetes have cell walls composed predominantly of cellulose and glucans, with little to no detectable chitin. Despite this, one to two putative chitin synthase (Chs) genes are present in Phytophthora spp. suggesting a functional role in its biology. In this study, we characterized the single chitin synthase of P. infestans (PiChs) to better understand its function and explore its potential as a target for novel fungicidal treatments. Unlike typical chitin synthases that generate long-chain chitin polymers, the recombinantly expressed PiChs predominantly synthesizes soluble chitooligosaccharides with degrees of polymerisation ranging from four to six. This finding provides a potential explanation for the undetectable chitin polymer in P. infestans cell wall analyses. Proteolytic processing was found to enhance PiChs activity, similar to observations in yeast and bacterial chitin synthases. PiChs exhibited sensitivity to Nikkomycin Z (NikZ), a well-characterized chitin synthase inhibitor, albeit with a higher IC₅₀ value compared to fungal heterologs. To enhance inhibition specificity, we developed a novel cyclic peptide inhibitor, named CS5, which exhibited stronger binding affinity and greater efficacy against PiChs compared to NikZ. Molecular docking, molecular dynamics simulations, and free energy calculations performed on both ligands revealed that CS5 competes with NikZ by binding to the same site on PiChs. In vitro and ex vivo assays confirmed that CS5 significantly impacted P. infestans growth, sporangial germination and host infection, supporting its potential as an alternative PiChs inhibitor. Our findings highlight the role of chitin synthase in P. infestans and its potential as a target for oomycete control. The development of tailored inhibitors such as CS5 presents an opportunity for sustainable disease management strategies, reducing reliance on broad-spectrum fungicides and addressing emerging resistance issues in agriculture.