Enhanced late blight resistance by engineering an EpiC2B-insensitive immune protease

Papain-like immune proteases (PLCPs) are promising engineering targets for crop protection, given their significant roles in plant immunity for key crops such as tomato, maize and citrus (Misas-Villamil et al., 2016). The wide range of pathogen-secreted PLCP inhibitors highlights the importance of these proteases in defending against various pathogens. Depletion of the apoplastic immune PLCP Phytophthora-inhibited protease 1 (Pip1) from tomato, for instance, causes hyper-susceptibility to bacterial, fungal and oomycete tomato pathogens (Ilyas et al., 2015). Immunity by Pip1 in wild-type tomato is, however, suboptimal since Pip1 is suppressed during infection by diverse pathogen-secreted inhibitors, such as the cystatin-like EpiC2B from the oomycete late blight pathogen Phytophthora infestans (Tian et al., 2007). Here, we tested whether we could increase Pip1-based immunity against late blight by engineering Pip1 into an EpiC2B-insensitive protease. To guide Pip1 mutagenesis, we generated a structural model of the EpiC2B-Pip1 complex using AlphaFold-Multimer (Evans et al., 2022). This structural model represents a classic interaction between the tripartite wedge of cystatin (EpiC2B) in the substrate binding groove of papain (Pip1). This model indicated that engineering Pip1 to prevent inhibition without affecting Pip1 substrate specificity is possible because the interaction surface of Pip1 with EpiC2B is larger than the substrate binding groove (Figure 1a).