Extracellular proteolytic cascade in tomato activates immune protease Rcr3

Abstract

<jats:p>Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (<jats:italic>Phytophthora infestans</jats:italic>) and in gene-for-gene resistance against the fungal pathogen <jats:italic>Cladosporium fulvum (</jats:italic>syn. <jats:italic>Passalora fulva)</jats:italic>. Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of <jats:italic>P. infestans</jats:italic> can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in <jats:italic>Nicotiana benthamiana</jats:italic> requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.</jats:p>

Cleavage of a pathogen apoplastic protein by plant subtilases activates host immunity

“…To address whether PC2 cleaved by P69B is required for PC2‐induced cell death activity, we grew the tomato asP69B line, a previously reported P69B ‐silenced tomato line (Paulus et al ., 2020). Here, we used qRT‐PCR to examine P69B expression and confirmed the silencing of P69B in the asP69B line (Fig.…”

“…7a). Recent discoveries suggest that P69B is an aspartic acid‐specific protease (Paulus et al ., 2020), and we therefore individually mutated aspartic acid into alanine in the putative cleavage region. Neither the D105A nor the D112A mutation led to clear cell death reduction.…”

“…The N. benthamiana and tomato subtilases were described by Paulus et al . (2020), and the potato subtilases were previously described by Norero et al . (2016).…”

Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

“…Still, some assumptions can be made based on studies of their homologues in other plant species including Arabidopsis thaliana and N. tabacum . The A. thaliana SBT most similar to NbSBT1 (recently also named NbSBT5.2 [35]) is AtSBT5.2, with whom it shares 58% sequence identity. As noted for NbSBT1, AtSBT5.2 is capable of autocatalytic processing [36].…”

Interrogating Plant-Microbe Interactions with Chemical Tools: Click Chemistry Reagents for Metabolic Labeling and Activity-Based Probes

“…Proteases are also highly implicated in plant immune responses and defense against pathogens. A recent study by Paulus et al used two different ABPs to examine the activation of Rcr3, a secreted papain-like cysteine protease (PLCP) involved in plant immune response, by the subtilisin class of serine proteases [ 45 ]. DCG-04, an ABP for cysteine proteases and PLCPs, was used on substitution mutants of Rcr3 to determine which active site cysteine residues were essential for catalytic activity; C154 was identified as a key residue, while C153 was not required for protease activity.…”