Inhibition of chitin deacetylases to attenuate plant fungal diseases

Plant diseases caused by over 10,000 phytopathogenic fungi substantially impact agriculture and food security. Plants recognize fungal chitin-derived chitooligosaccharides (COs) as microbe-associated molecular patterns via cell-surface receptors to trigger immunity, including MAPK signaling and ROS bursts. Pathogenic fungi counteract this by secreting chitin deacetylases (CDAs) that convert chitin to chitosan, a poor substrate for plant chitinases, thereby dampening immune activation. Prior work identified VdPDA1 from Verticillium dahliae and Pst_13661 from Puccinia striiformis f. sp. tritici as virulence effectors that suppress CO-induced defenses. CDAs belong to CE4 metalloenzymes featuring a conserved NodB domain and an Asp-His-His metal-binding triad, but no structures of phytopathogenic fungal CDAs had been available, limiting structure-guided inhibitor development. This study aimed to determine representative phytopathogenic fungal CDA structures, validate the necessity of CDA enzymatic activity for virulence, and identify small-molecule inhibitors (notably BHA) to attenuate plant fungal diseases.

The study builds on evidence that fungal effectors targeting host immunity are promising anti-disease targets. CDAs from multiple pathogens (V. dahliae, Fusarium oxysporum, Puccinia striiformis f. sp. tritici, Blumeria graminis, Uromyces fabae, Colletotrichum graminicola, Pyricularia oryzae, Ustilago maydis) contribute to virulence by deacetylating chitin, reducing chitinase efficacy and CO release, thereby suppressing plant defense gene expression and ROS production. CE4-family deacetylases employ metal-assisted catalysis with an Asp-His-His triad and a proposed mechanism wherein a Zn2+-activated water is deprotonated by a catalytic Asp to attack the acetyl group, and a catalytic His protonates the leaving group, releasing acetate. Structures exist for some fungal CDAs (e.g., C/CDA from Colletotrichum lindemuthianum, AnCDA from Aspergillus nidulans, AngCDA from Aspergillus niger), but not for phytopathogenic counterparts, motivating structural analysis and inhibitor discovery targeting conserved catalytic features.

• Protein production and purification: Genes encoding VdPDA1 (WT and catalytic mutant D55A,H200/201A) and Pst_13661 were synthesized, codon-optimized, cloned into pPIC9, and expressed in Pichia pastoris GS115. Secreted proteins were purified via ammonium sulfate precipitation, desalting, and HisTrap HP affinity chromatography, achieving >95% purity.
• Crystallography: Proteins were crystallized by hanging-drop vapor diffusion. Apo structures were solved for VdPDA1 (2.64 Å) and Pst_13661 (1.96 Å). Complexes of Pst_13661 with BHA and derivatives (compounds 2 and 3) were crystallized and solved at 1.61–1.93 Å. Data were collected at SSRF beamlines, processed with HKL3000, and structures solved by molecular replacement (Phaser) using CiCDA (PDB 2IWO), refined with PHENIX; model building in Coot; validation by PROCHECK.
• Metal identification and dependence: X-ray fluorescence spectra indicated Ni2+ and Zn2+. Zn-SAD phasing on Pst_13661 identified Zn2+ in the active site. Metal dependence was assayed after chelation with dipicolinic acid and reconstitution with various metal chlorides; activity with Zn2+ exceeded Ni2+.
• Enzyme activity assay: Deacetylation of (GlcNAc)3 by 100 nM enzyme at 30°C for 10 min in 50 mM HEPES pH 7.5; free amines quantified via fluorescamine versus glucosamine standard. Triplicates were performed.
• Inhibitor screening and Ki determination: Nine compounds (Topscience) including benzohydroxamic acid (BHA) and derivatives were screened at 100 µM against VdPDA1 and Pst_13661 using a liquid-handling platform. Hits were titrated; Ki values determined via Dixon plots at three substrate concentrations (0.5, 0.2, 0.1 mM) using GraphPad Prism.
• Mutant construction and virulence assays: VdPDA1 deletion and complementation strains (WT or catalytic mutant D55A,H201A) were generated in V. dahliae V991 via Agrobacterium-mediated transformation, selected on hygromycin/G418. In vitro growth was compared on PDA.
• Plant infection and anti-virulence testing: Cotton seedlings were pre-treated with BHA or solvent, then inoculated with V. dahliae conidia (root-dip). Disease symptoms and fungal biomass were assessed at 30 dpi by qPCR (fungal DNA relative to cotton actin). Defense gene expression (GhMPK6, GhRbohD) in cotton roots at 36 hpi was measured by qRT-PCR.
• Spectrum testing: Additional CDAs (FoPDA1 from F. oxysporum, FgCDA from F. graminearum, RsCDA from R. solani) were cloned, expressed, and tested for BHA inhibition (Ki). Soybean hypocotyl infection assays used pre-treatment with BHA or water followed by inoculation with F. graminearum, F. oxysporum, or R. solani; fungal biomass quantified by qPCR at 48 hpi. Wheat cultivar Ming Xian 169 was pre-treated with BHA and inoculated with P. striiformis f. sp. tritici CYR32; disease indices were scored at 19, 21, and 23 dpi.
• Phylogenetics and conservation: Multiple sequence alignments (ClustalW) and phylogenetic trees (MEGA 3.0; maximum parsimony, 5000 bootstraps) were generated; active-site conservation mapped (ConSurf) across 247 phytopathogenic fungal CDAs.
• Statistics: Experiments employed biological and technical replicates; Student’s two-sided unpaired t-tests; data acquisition randomized and blinded where applicable.

• Structural biology: Crystal structures of two phytopathogenic fungal CDAs (VdPDA1 and Pst_13661) were solved (apo and inhibitor-bound). Both share a classic (β/α)8 fold with identical substrate-binding pockets and a conserved Asp-His-His metal-binding triad. Zn2+ is the catalytic metal as determined by Zn-SAD and activity assays.
• Catalytic mechanism: VdPDA1 catalytic residues (Asp55 as base, His201 as acid) and metal-binding triad (Asp56/His108/His112) are positioned consistent with CE4 mechanisms. Pst_13661 aligns closely (RMSD 0.43 Å over 32 Cα), indicating conserved active-site architecture.
• Virulence dependence on enzyme activity: VdPDA1 catalytic mutant (D55A,H201A) lost deacetylase activity. VdPDA1 deletion and catalytic-mutant complement strains showed normal in vitro growth but significantly reduced cotton wilt symptoms and fungal biomass in planta. Cotton defense genes GhMPK6 and GhRbohD were upregulated upon infection with deletion or catalytic-mutant strains but suppressed by WT or WT-complemented strains, demonstrating enzymatic activity is essential for full virulence.
• Inhibitor discovery: Benzohydroxamic acid (BHA) and three derivatives with a BHA moiety inhibited VdPDA1 and Pst_13661. BHA was most potent with Ki = 8.31 µM (VdPDA1) and 9.83 µM (Pst_13661). Other derivatives showed Ki values of 8.53–80.68 µM depending on enzyme and compound (Table 2).
• Binding mode: Co-crystal structures of Pst_13661 with BHA and derivatives showed hydroxamate bidentate chelation of Zn2+. BHA forms H-bonds with Tyr152 (backbone NH), catalytic His207 (imidazole), and Asp58 (carboxylate). The benzene ring occupies a hydrophobic/aromatic pocket (Leu205, Tyr152, Trp174).
• In planta efficacy and mode of action: BHA pre-treatment of cotton prevented wilt symptoms and reduced fungal biomass in a dose-dependent manner without affecting V. dahliae growth in vitro, indicating anti-virulence rather than fungicidal activity. BHA restored GhMPK6 and GhRbohD expression suppressed by WT V. dahliae, consistent with in vivo CDA inhibition.
• Broad-spectrum activity: Active-site residues are highly conserved among CDAs from V. dahliae, P. striiformis, F. oxysporum, F. graminearum, and R. solani. BHA inhibited FgCDA (Ki 20.73 µM), FoPDA1 (Ki 43.5 µM), and RsCDA (Ki 2.39 µM). Soybean hypocotyl infections by these pathogens showed fewer lesions and significantly reduced fungal biomass with BHA pre-treatment, while fungal morphology and growth remained normal. In wheat, BHA reduced disease indices after P. striiformis infection across multiple timepoints and doses.

The study demonstrates that the enzymatic activity of phytopathogenic fungal CDAs is necessary for immune evasion and full virulence, validating CDAs as anti-virulence targets. Solved structures reveal a conserved active site and Zn2+-dependent catalysis across phylogenetically distant pathogens, enabling structure-guided inhibitor design. BHA, a known metal-chelating pharmacophore, effectively binds the CDA Zn2+ center and interacts with catalytic residues, conferring inhibitory activity that translates to restoration of plant immune gene expression and attenuation of disease without impairing fungal growth. This anti-virulence mode suggests lower selective pressure for resistance and compatibility with beneficial fungi. The broad-spectrum efficacy across major crop pathogens underscores the translational potential of CDA inhibition for plant protection. Specificity against fungal versus insect CDAs further supports targeting feasibility. Remaining questions include potential off-target effects on other metalloproteins in planta and optimization for potency, stability, and delivery.

This work provides the first crystal structures of phytopathogenic fungal CDAs (VdPDA1 and Pst_13661), establishes that CDA catalytic activity is required for virulence, and identifies benzohydroxamic acid as a lead inhibitor that chelates the catalytic Zn2+ and attenuates fungal diseases in cotton, soybean, and wheat. The conserved active-site architecture across diverse pathogens supports CDA as a broad-spectrum anti-virulence target. Future research should optimize BHA-derived scaffolds for higher potency and selectivity, assess field efficacy and formulation, evaluate potential off-targets within plant metalloproteomes, characterize pharmacokinetics and environmental safety, and explore combination strategies with existing fungicides or biological controls.

• Off-target potential: As a generic metal chelator, BHA may inhibit other plant or fungal metalloproteins involved in immunity or metabolism; comprehensive off-target profiling is needed.
• Potency and selectivity: Ki values are in low-to-moderate micromolar range for several CDAs; medicinal chemistry optimization is required to improve potency and fungal CDA selectivity.
• In vivo scope: Efficacy demonstrated in controlled greenhouse assays; field performance, environmental stability, and formulation have not been evaluated.
• Resistance and durability: Although anti-virulence strategies may reduce resistance pressure, long-term evolution and variability among CDA isoforms or expression levels across pathogens remain to be tested.
• Safety and non-target effects: Broader impact on plant physiology, beneficial microbiota, and non-target organisms requires assessment.