Fungal attack and host defence pathways unveiled in near‐avirulent interactions of Penicillium expansum creA mutants on apples

Abstract

Amongst the universal diseases affecting apples, blue mould caused by Penicillium expansum is a major concern, resulting in yield and quality losses as a result of the production of the mycotoxin patulin. Despite the characterization of the patulin biosynthetic gene cluster at both the molecular and chemical levels, the underlying regulation of patulin biosynthesis in P. expansum and the mechanisms of apple colonization remain largely obscure. Recent work has indicated that sucrose, a carbon catabolite repressive metabolite, is a critical factor in the regulation of patulin synthesis. Here, CreA, the global carbon catabolite regulator, was assessed for virulence both in vitro and in vivo. We showed that loss-of-function creA strains were nearly avirulent and did not produce patulin in apples. On the basis of RNA-sequencing (RNA-seq) analysis and physiological experimentation, these mutants were unable to successfully colonize apples for a multitude of potential mechanisms including, on the pathogen side, a decreased ability to produce proteolytic enzymes and to acidify the environment and impaired carbon/nitrogen metabolism and, on the host side, an increase in the oxidative defence pathways. Our study defines CreA and its downstream signalling pathways as promising targets for the development of strategies to fight against the development and virulence of this post-harvest pathogen.

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

“…In our recent work, a ku70 strain of P. expansum Pe-21 was generated using hygromycin as a selectable marker (Tannous et al, 2018). Here a new P. expansum ku70 was created to allow recycling of the hygromycin resistance gene that can be re-used as a selectable marker in future transformations.…”

“…To generate the different deletion and complement strains, the fungal transformation was performed following the polyethylene glycol-mediated protoplast transformation protocol described previously by Tannous et al (2018). All transformants were screened by PCR and further subjected to southern blot to confirm the single insertion of the deletion cassette using [α32P] dCTP (PerkinElmer, United States) to label the DNA probes, following manufacturer's instructions.…”

“…Golden Delicious, obtained from a local orchard, to evaluate the role of sntB in the virulence process of P. expansum. As described previously by Tannous et al (2018), apples were surface sterilized with 2% sodium hypochlorite solution and thoroughly rinsed with sterile distilled water. A single uniform 5 mm deep wound was made at the equator of each apple (put on its side) using sterile toothpicks.…”

“…Three technical replicates were performed for each strain, and the entire experiment was repeated twice for confirmation of the observed results. At the end of the incubation period, patulin was extracted from apples and quantified by HPLC as described earlier by Tannous et al (2018). Moreover, to eliminate the effect of variability between apples and to compare the fruit colonization, the three strains (control, sntB, and sntBC) were inoculated twice on the same apple.…”

“…Lastly, CreA, the carbon catabolite repressor has been reported to act as a positive regulator of both patulin and citrinin production (Tannous et al, 2018). In addition to their impact on secondary metabolism and some physiological traits, the loss of all these regulatory proteins resulted in attenuated virulence of P. expansum on apples (Kumar et al, 2016;El Hajj Assaf et al, 2018;Tannous et al, 2018).…”