New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB

Abstract

Penicillium expansum is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, P. expansum produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in Aspergillus species, and impact virulence of plant pathogenic fungi. Here we show that deletion of sntB in P. expansum results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis in vitro and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔsntB strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO₂ levels) to which P. expansum is commonly exposed during fruit storage, resulted in a significant reduction of sntB expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.

Keywords: Penicillium expansum; SntB; citrinin; epigenetic reader; epigenetic regulation; patulin; secondary metabolism; virulence.

FIGURE 1

Physiological analysis of the control, sntB deletion and complement strains of P. expansum Pe-21. (A) Growth phenotype of P. expansum strains monitored on four different media (GMM, Glucose Minimal Medium; CYA, Czapek Yeast Extract Agar; PDA, Potato Dextrose Agar; YES, Yeast Extract Sucrose Agar) for 72 h at 25°C. (B) Radial growth, (C) conidia production were monitored on GMM agar, and (D) germination rate of P. expansum strains was measured on GMM broth. One-way ANOVA differences were considered significant when p < 0.05. Different letters above the columns indicate statistically significant differences between the strains as determined using Tukey’s single-step multiple comparison test.

FIGURE 2

SntB-associated regulation of patulin and citrinin biosynthesis. The three Penicillium strains (control, ΔsntB, and sntBc) were grown on solidified YES media at 28°C. Patulin (A) and citrinin (B) levels, relative expressions of patulin (C) and citrinin cluster genes (D), and global transcription factors (E) were evaluated at 4 days post-inoculation. Error bars represent the standard error of the mean (SEM) across three technical replicates. One-way ANOVA differences were considered significant when p < 0.05. Different letters above the columns indicate statistically significant differences between the strains as determined using Tukey’s single-step multiple comparison test.

FIGURE 3

SntB is required for P. expansum pathogenicity and patulin production on apples. (A) Apples were inoculated with 10⁶ spores and incubated at 25°C in the dark for 5 days. (B) Histogram showing the rot diameter on apples inoculated with the three Penicillium strains (control, ΔsntB, sntBc). (C) Histogram displaying patulin production on apples analyzed by HPLC. Each strain was inoculated with 3 reps. One-way ANOVA differences were considered significant when p < 0.05. Different letters above the columns indicate statistically significant differences between the strains as determined using Tukey’s single-step multiple comparison test.

FIGURE 4

Effect of abiotic factors on sntB expression and mycotoxin accumulation. The three Penicillium strains (control, ΔsntB, and sntBc) were grown on YES agar media at 28°C for 48h in the dark and then subjected to different treatment conditions for additional 48 h. Control – 28°C incubation in the dark; 18–18°C incubation in the dark; 5–5°C incubation in the dark; light – 28°C incubation under continuous white fluorescent lighting; CO₂ – 28°C incubation at 20% CO₂ in the dark. (A) Total RNA was extracted and the relative expression of sntB was evaluated. (B) Patulin and (C) citrinin were extracted and subjected to HPLC analysis. Each strain was inoculated with 3 reps. One-way ANOVA differences were considered significant when p < 0.05. Different letters above the columns indicate statistically significant differences between the strains as determined using Tukey’s single-step multiple comparison test.

FIGURE 5

Effect of abiotic factors on relative expression of mycotoxin cluster genes. The three Penicillium strains (control, ΔsntB, and sntBc) were grown on YES agar media at 28°C for 48 h in the dark and then subjected to different treatment conditions as mentioned in Figure 4. RNA was extracted and the relative expression of patulin cluster genes (A), and citrinin cluster genes (B) was evaluated. Error bars represent the standard error of the mean (SEM) across three technical replicates. One-way ANOVA differences were considered significant when p < 0.05. Different letters above the columns indicate statistically significant differences between the strains as determined using Tukey’s single-step multiple comparison test.