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Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen  Pyrenophora tritici-repentis , which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems:  Escherichia coli  and  Pichia pastoris . The  E. coli  SHue system was demonstrated to be superior to  P. pastoris  in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from  P. pastoris  induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 μg/ml of ToxB was obtained using the SHue system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 μg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and  Arabidopsis thaliana . A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of  P. tritici-repentis  effectors in host and non-host interactions.

frontiers in microbiology

Heterologous Expression of the Pyrenophora tritici-repentis Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops