Ammonia activates pacC and patulin accumulation in an acidic environment during apple colonization by Penicillium expansum

Abstract

Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin.

The pH‐responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum

“…We further found that the PacC 3 could be detected at pH 5.0-8.0 (Fig. 1D), confirming the result that the Pal pathway is already active at low pH (4.5-5.0) in P. expansum (Barad et al, 2016). In addition, the PacC 3 was not detected at pH 3.0-4.0 and the cleavage efficiency increased with the rise of ambient pH (Fig.…”

“…In A. nidulans , the full‐length PacC polypeptide is processed by two successive proteolytic cleavages and yields a shorter, functional PacC at alkaline pH (Orejas et al ., ). In P. expansum‐21 , EMSA and supershift analysis have showed that the PePacC is not fully protected against proteolytic signalling at pH 4.5 relative to pH 7.0 (Barad et al ., ). Here, pH shift experiments with Flag 3 ::PacC strain straightly showed the presence of three truncated versions of PePacC (Fig.…”

Pectin methyl esterases and rhamnogalacturonan hydrolases: weapons for successful Monilinia laxa infection in stone fruit?

“…This raises the possibility that regulation occurred through the zinc finger protein denoted PacC, involved in the regulation of pHcontrolled genes in filamentous fungi (Peñalva et al, 2008). PacC activates transcription of those pathogenicity factors depending on the environmental pH in several host-pathogen interactions (Alkan et al, 2013;Barad et al, 2016). All these differences among the members of RG-HYDs family would provide the fungus with versatile and complementary tools for degrading substrates under different conditions during degradation of the cell wall.…”

Molecular aspects in pathogen-fruit interactions: Virulence and resistance

“…The pacC gene has also been found in several other postharvest fungal pathogens and contributes to the full virulence of these pathogens, including Colletotrichum gloeosporioides (Alkan et al, 2013); P. expansum (Barad et al, 2014); and P. digitatum (Zhang et al, 2013). In P. expansum, ambient pH and pacC are also involved in the secondary metabolism and have been shown to affect the production of the mycotoxin patulin (Barad et al, 2015;Zong et al, 2015). Although there are conflicting reports on the possible role of this mycotoxin in pathogenesis (Ballester et al, 2015;Barad et.…”