Mechanisms governing the global regulation of mycotoxin production and pathogenicity by Penicillium expansum in postharvest fruits

The original objectives of the study, as defined in the approved proposal, are: To characterize the relationship of CreA and LaeA in regulation of P T production To understand how PacC modulates P. expansumpathogenicity on apples To examine if other secondary metabolites are involved in virulence or P. expansumfitness To identify the signaling pathways leading to PAT synthesis Penicilliumexpansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxinpatulin (PAT) in colonized apple fruit tissue. Although PAT is produced by many Penicilliumspecies, the factors activating its biosynthesis were not clear. This research focused on host and fungal mechanisms of activation of LaeA (the global regulator of secondary metabolism), PacC (the global pH modulator) and CreA (the global carbon catabolite regulator) on PAT synthesis with intention to establish P. expansumas the model system for understanding mycotoxin synthesis in fruits. The overall goal of this proposal is to identify critical host and pathogen factors that mechanistically modulate P. expansumgenes and pathways to control activation of PAT production and virulence in host. Several fungal factors have been correlated with disease development in apples, including the production of PAT, acidification of apple tissue by the fungus, sugar content and the global regulator of secondary metabolism and development, LaeA. An increase in sucrose molarity in the culture medium from 15 to 175 mM negatively regulated laeAexpression and PAT accumulation, but, conversely, increased creAexpression, leading to the hypothesis that CreA could be involved in P. expansumPAT biosynthesis and virulence, possibly through the negative regulation of LaeA. We found evidence for CreAtranscriptional regulation of laeA, but this was not correlated with PAT production either in vitro or in vivo, thus suggesting that CreA regulation of PAT is independent of LaeA. Our finding that sucrose, a key ingredient of apple fruit, regulates PAT synthesis, probably through suppression of laeAexpression, suggests a potential interaction between CreA and LaeA, which may offer control therapies for future study. We have also identified that in addition to PAT gene cluster, CreA regulates other secondary metabolite clusters, including citrinin, andrastin, roquefortine and communesins, during pathogenesis or during normal fungal growth. Following creation of P. expansumpacCknockout strain, we investigated the involvement of the global pH regulator PacC in fungal pathogenicity. We demonstrated that disruption of the pH signaling transcription factor PacC significantly decreased the virulence of P. expansumon deciduous fruits. This phenotype is associated with an impairment in fungal growth, decreased accumulation of gluconic acid and reduced synthesis of pectolytic enzymes. We showed that glucose oxidase- encoding gene, which is essential for gluconic acid production and acidification during fruit colonization, was significantly down regulated in the ΔPepacCmutant, suggesting that gox is PacC- responsive gene. We have provided evidence that deletion of goxgene in P. expansumled to a reduction in virulence toward apple fruits, further indicating that GOX is a virulence factor of P. expansum, and its expression is regulated by PacC. It is also clear from the present data that PacC in P. expansumis a key factor for the biosynthesis of secondary metabolites, such as PAT. On the basis of RNA-sequencing (RNA-seq) analysis and physiological experimentation, the P. expansumΔlaeA, ΔcreAand ΔpacCmutants 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 these global regulatory factors and their downstream signalling pathways as promising targets for the development of strategies to fight against this post-harvest pathogen.