Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that mediate cellular responses to various biotic and abiotic signals in plant pathogenic fungi. Here, we identified the orthologs of two MAPK genes (CcHog1 and CcSlt2) and assessed their functions by gene deletion and transcriptional analysis methods in Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants. We found that the MAPKs shared convergent and distinct roles in fungal development, stress responses and virulence. For example, CcHog1, CcSlt2 and CcPmk1 were all involved in conidiation and response to stresses, including hyperosmotic pressure, cell wall inhibition agents and H2O2, but only CcPmk1 and CcSlt2 were required for hyphal growth and fungal pathogenicity. RNA-Seq data showed that numerous hyperosmosis and cell wall related genes significantly reduced their expression levels in ΔCcHog1 and ΔCcSlt2, respectively. Importantly, RNA and ribosome related processes were significantly enriched in the up-regulated genes of ΔCcSlt2, while they were significantly enriched in the down-regulated genes of ΔCcPmk1. Moreover, some secondary metabolite biosynthesis core genes (20/68) and two gene clusters were regulated by at least one among CcPmk1, CcHog1 and CcSlt2. Importantly, some virulence-associated genes were significantly down-regulated in ΔCcPmk1 and/or ΔCcSlt2, such as candidate effector genes. Collectively, these results suggest that the similar and distinct phenotypes of each MAPKs mutant may result from the transcriptional regulation of a series common or specific downstream genes, which provide a better understanding of the regulation network of MAPKs in C. chrysosperma.
Cytospora chrysosperma is a destructive plant pathogenic fungus, which causes canker disease on numerous woody plants. However, knowledge concerning the interaction between C. chrysosperma and its host remains limited. Secondary metabolites produced by phytopathogens often play important roles in their virulence. Terpene cyclases (TC), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are the key components for the synthesis of secondary metabolites. Here, we characterized the functions of a putative terpene type secondary metabolite biosynthetic core gene CcPtc1 in C. chrysosperma, which was significantly up-regulated in the early stages of infection. Importantly, deletion of CcPtc1 greatly reduced fungal virulence to the poplar twigs and they also showed significantly reduced fungal growth and conidiation compared with the wild-type (WT) strain. Furthermore, toxicity test of the crude extraction from each strain showed that the toxicity of crude extraction secreted by ΔCcPtc1 were strongly compromised in comparison with the WT strain. Subsequently, the untargeted metabolomics analyses between ΔCcPtc1 mutant and WT strain were conducted, which revealed 193 significantly different abundant metabolites (DAMs) inΔCcPtc1 mutant compared to the WT strain, including 90 significantly downregulated metabolites and 103 significantly up-regulated metabolites, respectively. Among them, four key metabolic pathways that reported to be important for fungal virulence were enriched, including pantothenate and coenzyme A (CoA) biosynthesis. Moreover, we also detected significant alterations in a series of terpenoids, among which (+)-ar-turmerone, pulegone, ethyl chrysanthemumate, and genipin were significantly down-regulated, while cuminaldehyde and (±)-abscisic acid were significantly up-regulated. In conclusion, our results demonstrated that CcPtc1 acts as a virulence-related secondary metabolism factor and provides new insights into the pathogenesis of C. chrysosperma.
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