Elucidating the mechanisms of necrotrophic effectors in the Stagonospora nodorum-wheat pathosystem

Abstract

Stagonospora nodorum is a host-specific necrotrophic pathogen of wheat and the causal agent of Stagonospora nodorum blotch (SNB). Stagonospora nodorum secretes several unique effector proteins that induce chlorosis and necrosis on leaves of susceptible hosts. Several dominant genes in the host confer susceptibility to Stagonospora nodorum effectors in an inverse gene-for-gene manner. The mechanisms underlying effector-induced necrosis are yet to be fully elucidated. The aim of this study was to determine the host metabolite response to infiltration of purified effectors. Comprehensive metabolite profiling was used to analyse host primary and secondary metabolite changes after infiltration of the effectors SnToxA and SnTox3. SnToxA induced significant perturbations in primary metabolism leading to increases in tricarboxylic acid cycle metabolites and metabolites involved in detoxification of reactive oxygen species. SnToxA also induced host tryptophan metabolism causing accumulation of the secondary metabolites Serotonin and 6-Methoxy-2-Benzoxazolinone (MBOA). Growth and pathogenicity assays on Stagonospora nodorum revealed MBOA reduced in vitro growth at 0.3 mM and completely inhibited spore germination at 3 mM. Application of 1 mM exogenous Serotonin inhibited asexual sporulation of Stagonospora nodorum, resulting in a 10-fold reduction in the number of spores produced in vitro and in vivo. Although no visible difference in the number or appearance of pycnidia was observed, microscopy revealed that serotonin disrupted the development and maturation of the spores within pycnidia. Metabolite profiling of Stagonospora nodorum exposed to exogenous serotonin revealed a significant reduction in the disaccharide trehalose, which has been shown to be required for sporulation of Stagonospora nodorum. Relative quantitation of serotonin in infected plants demonstrated a significant difference between plants with varying effector sensitivities. Plants harbouring multiple susceptibility loci accumulated 10-fold less serotonin than those only sensitive to SnToxA. The primary metabolite response of wheat to SnTox3 was similar to that induced by SnToxA. In contrast, SnTox3 induced different host secondary metabolite responses including the phenylpropanoid pathway evident by accumulation of putatively identified feruloylquinic acid and chlorogenic acid. SnTox3 also caused accumulation of the putatively identified cyanogenic glycoside dhurrin. The tryptophan-derived metabolites induced by SnToxA were not observed in SnTox3-infiltrated wheat. Next generation sequencing was utilised to investigate gene expression of Stagonospora nodorum during the initial stages of infection on a susceptible and non-susceptible cultivar. There were no obvious differences in hyphal proliferation or plant cell death at 8 to 48 hpi observed by trypan blue viability staining and microscopy. Symptom development during infection on the non-susceptible cultivar was arrested four to five days post infection at which point effector-induced necrosis was observed on the susceptible cultivar. Remarkably, Stagonospora nodorum was reisolated from within leaves of non-susceptible plants at three weeks post infection in the absence of effector-induced plant cell death. Although the onset of spore germination on leaves was observed at six hours post infection, by eight hours a substantial induction of gene expression had occurred exclusively on susceptible plants. All three characterized Stagonospora nodorum effectors were expressed at significantly higher levels on the susceptible cultivar. Additionally, several further candidate effector genes were proposed. Show more