LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Abstract

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose-responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175- and five-fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch-Pe-T01 strains to 15%-25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is-Pe-21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.

Keywords: Penicillium; mycotoxin; pathogenicity; patulin; sucrose.

Figure 1

Effect of sucrose concentration on patulin accumulation and gene expression. Solid Secondary Medium (SM) at initial pH 5.0, amended with 15–175 mm sucrose, was inoculated with 100 μL of a 10⁶ spore/mL suspension. Patulin (A), laeA relative expression (RE) (B) and creA RE (C) were evaluated at 3 days post‐inoculation. Five 10‐mm‐diameter discs were sampled from five independent culture plates. Average values ± standard errors of five replicates are reported. Experiments were repeated three times and the results of a single representative experiment are shown. DW, dry weight.

Figure 2

Relationship between laeA expression and patulin accumulation in the wild‐type (WT), mutants and complementary strains of the Israeli (Pe‐21) and Chinese (Pe‐T01) Penicillium expansum isolates. The fungus was grown in liquid medium for 48 h with shaking at 150 rpm and then transferred to liquid secondary medium (SM) at pH 5; 48 h later, the mycelia were collected and frozen. RNA was extracted and the relative expression (RE) of laeA (A,C) was analysed for the WT (Pe‐21), ΔlaeA1, ΔlaeA3, ΔlaeA5 and complementary strain (C‐1) (A), and for the WT (Pe‐T01), ΔlaeA1, ΔlaeA6, ΔlaeA18 and complementary strain (C‐3) (C). Solid SM medium at initial pH 5.0 amended with 50 mm sucrose was inoculated with 100 μL of a 10⁶ spore/mL suspension. Patulin accumulation in the Pe‐21 (B) and Pe‐T01 (D) strains was evaluated at 3 days post‐inoculation. Five 10‐mm‐diameter discs were sampled from five independent culture plates. Average values ± standard errors of five replicates are reported. Experiments were repeated three times and the results of a single representative experiment are shown. DW, dry weight.

Figure 3

Fungal growth and sporulation of the ΔlaeA mutants of the Israeli (Pe‐21) (A,B) and Chinese (Pe‐T01) (C,D) Penicillium expansum strains. Fungal growth and sporulation of the ΔlaeA mutants were measured on solid secondary media (SM) at initial pH 5.0 amended with 50 mm sucrose and inoculated with 100 μL of a 10⁶ spore/mL suspension. Evaluations were carried out at 3, 5 and 7 days post‐inoculation. Average values ± standard errors of three replicates are reported and the experiments were repeated twice; a single representative experiment is shown.

Figure 4

Relative expression of the 15 clustered patulin genes of Penicillium expansum. Expression of the genes involved in the biosynthesis of patulin was evaluated in the Israeli (Pe‐21, A) and Chinese (Pe‐T01, B) wild‐type (WT) strains and their ΔlaeA mutant strains. Primers used were from Li et al. (2015).

Figure 5

Colonization and patulin accumulation by the Israeli (Pe‐21) and Chinese (Pe‐T01) wild‐type (WT) and ΔlaeA strains of Penicillium expansum in apple. Apple fruits were harvested at 157 days after fruit set and the following harvests were each 1 week apart. Fruits were inoculated with WT (Pe‐21) and its ΔlaeA1, ΔlaeA3 and ΔlaeA5 strains, and WT (PeT01) and its ΔlaeA1, ΔlaeA6 and ΔlaeA18 strains, immediately after harvest, and patulin accumulation was evaluated 5 days later. Different letters represent significant differences between the WT and mutant strains. The decay area (mm²) of the WT strain was set as 100% for each separate fruit. The decay areas (mm²) of the mutant strains were compared with the WT and are presented as the percentage inhibition of colonization. Average values ± standard errors of five replicates are reported. FW, fresh weight.

Figure 6

Schematic representation of gene clusters in Penicillium expansum Israeli isolate Pe‐21. The direction of transcription is indicated by arrowheads. The genes and intergenic regions are drawn to scale. (A) The patulin cluster of P. expansum refers to the work of Tannous et al. (2014). (B) The communesin cluster refers to the study by Lin et al. (2015). (C) The citrinin cluster refers to the reports of Ballester et al. (2015) and He and Cox (2016). (D–H) The clusters refer to the prediction of secondary metabolism gene clusters in P. expansum by Ballester et al. (2015). (I) The andrastin cluster was predicted by looking for sequence similarity with the characterized andrastin cluster in P. chrysogenum (Matsuda et al., 2013) by MultiGeneBlast (Medema et al., 2013). (J) The roquefortine cluster refers to the work of Banani et al. (2016). (K) The siderophore cluster was predicted by looking for sequence similarity with the characterized siderophore cluster in Aspergillus fumigatus (Gründlinger et al., 2013) by MultiGeneBlast.

Figure 7

Semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) validation of secondary metabolite backbone genes in Penicillium expansum regulated by LaeA. Gene expression was examined from 3‐ and 5‐day‐old cultures of the Israeli isolate Pe‐21 and its mutant ΔlaeA3 grown on Czapek ‐ yeast extract (CY) medium (A) and apple purée–agar medium (APAM) (B). laeA expression was monitored under different experimental growth conditions. β‐Actin housekeeping gene expression was used to normalize mRNA levels. Pe‐21 genomic DNA was used as a template to validate the primer sets. All samples were analysed on 0.8% agarose gels stained with ethidium bromide. A full set of the expression of the 54 secondary metabolite genes is shown in Fig. S4 (see Supporting Information). WT, wild‐type.

Figure 8

Venn diagram showing the overlap of Penicillium expansum backbone gene expression profiles between five experimental conditions. Gene expression was examined on Czapek ‐ yeast extract (CY) medium (days 3 and 5 of culture), apple purée–agar medium (APAM) (days 3 and 5 of culture) and apples (data based on a previous study by Ballester et al., 2015). Bold genes are those down‐regulated by LaeA; genes designated by asterisks are up‐regulated by LaeA (on CY medium only); genes corresponding to the identified clusters are underlined. The assigned putative clusters are: PEXP_071900 (epipolythiodioxopiperazine‐like), PEXP_030510 (communesin), PEXP_005520 (citrinin), PEXP_029660 (loline‐like), PEXP_030140 (roquefortine‐like), PEXP_094460 (patulin), PEXP_063340 (andrastin‐like), PEXP_104890 (siderophore‐like), PEXP_058600 (epipolythiodioxopiperazine‐like), PEXP_093210 (monodictyphenone‐like). It should be noted that there are a few genes that are down‐regulated in one medium and up‐regulated in another medium.