Cleavage of a pathogen apoplastic protein by plant subtilases activates immunity

Abstract

19The plant apoplast is a harsh environment in which hydrolytic enzymes, 20 especially proteases, accumulate during pathogen infection. However, the 21 defense functions of most apoplastic proteases remains largely elusive. Here, we 22 show that a newly identified small cysteine-rich secreted protein PC2 from the 23 potato late blight pathogen Phytophthora infestans induces immunity in Solanum 24 plant species only after cleavage by plant apoplastic subtilisin-like proteases, 25 such as tomato P69B. A minimal 61-amino-acid core peptide carrying two key 26 cysteines and widely conserved among most oomycete species is sufficient for 27 2 PC2 activity. Kazal-like protease inhibitors, such as EPI1 produced by P. 28 infestans can prevent PC2 cleavage and dampen PC2 elicited host immunity. 29 This study reveals that cleavage of pathogen proteins to release immunogenic 30 peptides is an important function of apoplastic proteases but that pathogens 31 interfere with these functions using protease inhibitor effectors. 32 83 colonization, adapted microbial pathogens deliver protease inhibitors into the 84 host apoplast to counteract host defense. For example, fungal apoplastic effector 85 Pit2 secreted by U. maydis inhibits a set of apoplastic maize cysteine proteases 86 implicated in maize defense and contributes to the virulence of U. maydis in 87 4 maize(Mueller et al., 2013; Misas Villamil et al., 2019). In Phytophthora, a well-88 characterized group of intercellular proteins are the extracellular cystatin-like 89 protease inhibitors (EPIC). P. infestans EPIC1 binds to tomato PLCP Rcr3 and 90 C14 and inhibits their activity in the tomato apoplast(Kaschani et al., 2010). 91EPIC2B is a more robust cysteine protease inhibitor with stronger inhibitory 92 activity against Rcr3 and Pip1(Dong et al., 2014). Beside Phytophthora EPIC2B 93 and fungal Avr2, Gr-Vap1 from cyst nematode and Cip1 from Pseudomonas 94 bacteria pathogen also target these tomato proteases (Misas Villamil et al., 2019), 95 indicating that different pathogens have independently evolved divergent 96 effectors to inhibit plant protease during co-evolution. In addition, P. infestans 97 secretes Kazal-like serine protease inhibitors, including the EPI1 and EPI10 98 proteins that inhibit tomato subtilase P69B (Tian et al., 2004; Tian et al., 2005). 99More recently, a study on the pathogenicity of Huanglongbing uncovered the 100 Candidatus Liberibacter asiaticus effector SDE1 that directly interacts with and 101 inhibits citrus PLCP activity (Clark et al., 2018). In another case, the cyst 102 nematode Heterodera schachtii effector protein 4E02 repurposes a PLCP from 103 its role in defense by targeting it to distinct plant cell compartments without 104 inhibibiting its activity (Pogorelko et al., 2019). These studies illustrate a 105 concerted interaction between the host proteases and the pathogen protease 106 inhibitors, which dictates the success of pathogen colonization. 107

The front line of defence: a meta-analysis of apoplastic proteases in plant immunity

“…We divide these mechanisms into four groups: antimicrobial activity, immune hydrolase activation, damage-associated molecular pattern (DAMP) release, and effector perception. A single protease may act on multiple substrates and hence perform multiple functions, as is the case for tomato P69B ( Paulus et al , 2020 ; Wang et al , 2020 ).…”

“…The second protease involved in effector perception utilizes a very different mechanism. The Cys-rich secreted protein PC2 produced by P. infestans is cleaved by apoplastic subtilases including P69B, triggering an immune response including cell death, the accumulation of ROS, and the up-regulation of defence-related genes ( Wang et al , 2020 ). This cleavage is essential for the PC2-triggered immune response and presumably produces a small peptide that is recognized at the cell surface.…”

“…This cleavage is essential for the PC2-triggered immune response and presumably produces a small peptide that is recognized at the cell surface. Interestingly, P. infestans is able to inhibit PC2 cleavage and therefore cell death by producing Ser protease inhibitors such as EPI1 ( Wang et al , 2020 ).…”