Biochemical and Structural Characterization of RCR3-AVR2: A Model for Protease-Inhibitor Interactions at the Plant-Pathogen Interface.

The tomato apoplast is a molecular battle ground for proteases and inhibitors during plant-pathogen interactions. The interaction between structurally diverse pathogen-derived inhibitors (EPIC1, EPIC2B, AVR2 and RIP1) and their target proteases (RCR3, PIP1 and C14) is an ideal system to study molecular arms-races. First we generated a collection of 52 isoforms of proteases and inhibitors in the pFLAG-ATS expression vector for expression in Escherichia coli. We summarise the expression of both the proteases and inhibitors and show that the inhibitor proteins were produced successfully, unlike the proteases. Second we expressed and purified AVR2 on a large scale and show that purified AVR2 is capable of inhibiting RCR3 and triggering the Cf2-mediated hypersensitive response (HR) in tomato demonstrating that recombinant AVR2 is functional. We next implement biophysical and structural biology tools to elucidate the secondary and tertiary structure of AVR2. The major findings are that: (i) AVR2 is a beta protein determined by circular dichroism (CD) (ii) At apoplastic pH, AVR2 exerts a conformational change associated with RCR3 inhibition determined by CD and tyrosine fluorescent spectroscopy (iii) AVR2 is a potent inhibitor of papain determined by enzyme assay using BODIPY FL casein. Attempts to crystallize AVR2 with and without epitope tags and at different concentrations and conditions failed, possibly because AVR2 is a heavily charged basic protein. Next all 13 lysines and the N-terminus of epitope-tagged AVR2 were methylated but no crystals were obtained. This methylated AVR2 still triggers HR in Cf2 tomato plants. Preliminary NMR experiments of non-methylated AVR2 showed good resolution for future structure elucidation of AVR2. Third, we tested four different heterologous expression systems (plant, bacterium, insect and yeast) to generate high quantities of active and soluble RCR3. We conclude that yeast is the best expression system to produce high amounts of soluble proRCR3 and proRCR3 is fully converted into mature RCR3 in the presence of reducing agents and acidic pH buffer. Finally, we study the role of double cysteine (Cys24, Cys25) in the catalytic site of RCR3 which is common to PLCP subclass 6 of plant PLCPs. Using agroinfiltration in Nicotiana benthamiana we produced C24A, C25A and C24AC25A mutant RCR3 proteins and we discovered that (i) Cys25 but not Cys24 is the essential catalytic residue labelled by activity-based probe MV201. (ii) Surprisingly maturation of RCR3 does not require the catalytic Cys25, indicating that other endogeneous proteases activate RCR3. (iii) Proteolytically inactive RCR3 mutants triggers Cf-2-mediated HR in the presence of AVR2 and (iv) Interestingly, ascorbate enhances the activity of the C24A mutant but not wild-type RCR3, suggesting that Cys24 in RCR3 might have a role in sensing redox potential. The autocatalytic activation of proRCR3 produced in yeast combined with the maturation of the C25A mutant inplanta suggests that RCR3 can be activated by both intramolecular and intermolecular processing

Extracellular proteolytic cascade in tomato activates immune protease Rcr3

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 (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) 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 P. infestans 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 Nicotiana benthamiana 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