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Figure 4
Roles of AfLkhA in protease production and biofilm formation. (A) Halo formed at edge of colonies of WT, ?AflkhA, and C?AflkhA strains. Each strains were point-inoculated on Czapek Dox medium containing 1% skim milk and incubated at 30°C. (B) Quantification of proteolytic activity. Supernatants were obtained from Czapek Dox broth (pH 7.3) containing 1% skim milk powder for 3 days at 30°C. Protein concentrations were calculated with a BCA Protein Assay Kit. Protease activity was measured using an azocasein assay. The experiments included six replicates (n = 6). ***P < 0.001. (C) Expression pattern of alkaline protease alp1 gene. Total RNA was extracted from mycelial balls incubated in Czapek Dox broth. RT-qPCR analysis was performed using 18S rRNA gene as an internal control. *P < 0.05. (D) Crystal violet staining assay. Biofilm formation was measured after 16 hrs of growth in GMM. The biofilms were stained with 0.01% crystal violet and dissolved in 30% acetic acid solution. The amounts of dye were measured by spectrophotometry at 550 nm (n = 10). ***P < 0.001. (E) Relative expression patterns of ?-1,3-glucan synthase (ags1), hydrophobins rodA, rodB, a ribonuclease (aspf1), and a GAG synthase (gt4c). Total RNA was extracted from biofilm cultures. RT-qPCR analysis was performed using 18S rRNA gene as an internal control. The x-axis shows a list of genes. The y-axis indicate the relative mRNA abundance of the genes in ?AflkhA strain compared to WT. The values were transformed and presented as Log10. ***P < 0.001.