Fig. 2: Enzymatic activity of VdPDA1 is essential for the full virulence of V. dahliae.
From: Inhibition of chitin deacetylases to attenuate plant fungal diseases
a Mutagenesis analysis confirmed the importance of the predicted catalytic residues for enzymatic activity. Coomassie blue-stained SDS-PAGE analysis of purified VdPDA1 and VdPDA1D55A, H201A (inset panel). M represents protein molecular weight marker. Time-dependent deacetylation of (GlcNAc)3 by VdPDA1 and VdPDA1D55A, H201A, determined via labeling the resulting free amine using fluorescamine and quantification using a glucosamine standard. All measurements were carried out in triplicate. b Schematic description of the VdPDA1 mutants. Trpc: a promoter of Aspergillus nidulans trpC gene; hpt: hygromycin phosphotransferase; NPTII: neomycin phosphotransferase II; PN: a native promoter of VdPDA1. c There were no growth differences among VdPDA1 mutant colonies grown on PDA medium. d Disease symptoms on cotton plants at 30 dpi with wild type (WT) or VdPDA1 mutants. e Enzymatic activity is essential for the full virulence function of VdPAD1. The fungal relative biomass on cotton plants at 30 dpi with the WT, the knockout mutant (VdΔpda1), and the complementary transformants (VdΔpda1/cPDA1D55A, H201A, VdΔpda1/cPDA1) was determined by qPCR, using genomic DNA as a template. f, g Expression of the defense response genes (GhMPK6 and GhRbohD) that was repressed by VdPDA1 during infection by V. dahliae. The expression patterns of GhMPK6 (f) and GhRbohD (g) in cotton roots at 36 hpi with the WT, the knockout mutant (VdΔpda1), and the complementary transformants (VdΔpda1/cPDA1D55A, H201A, VdΔpda1/cPDA1) were determined by qRT-PCR using cDNA as a template. Three independent replicates were used for measurement, and each experiment consisted of 3 cotton samples per V. dahliae infection. Bars in e–g represent mean values, whiskers represent mean ± SEM, and black circles represent each replicate value (n = 3). Statistical significance was determined by Student’s two-sided unpaired t-test. Source data are provided as a Source Data file.
