Nb RD21 protease controls receptor kinase homeostasis in Nicotiana benthamiana

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana ( Nb RD21) is suppressed during infection with Pseudomonas syringae . Here, we studied the role of Nb RD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express Nb RD21 and candidate substrates. Genome edited lines lacking Nb RD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae . Shot gun proteomics revealed an increased accumulation of ∼20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that Nb RD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.