Differential N-end Rule Degradation of RIN4/NOI Fragments Generated by the AvrRpt2 Effector Protease

Abstract

In plants, the protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both pattern-triggered immunity and effector-triggered immunity. RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates potentially unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RESISTANT TO P. SYRINGAE2. Hence, identifying the determinants of RIN4 degradation is key to understanding RESISTANT TO P. SYRINGAE2-mediated effector-triggered immunity, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI domain proteins by AvrRpt2 may contribute to pattern-triggered immunity regulation, the (in)stability of these proteolytic fragments and the determinants regulating their stability remain unexamined. Notably, a common feature of RIN4, and of many NOI domain protein fragments generated by AvrRpt2 cleavage, is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed the N-degron pathway). By contrast, several NOI domain protein fragments are genuine substrates of the N-degron pathway. The discovery of this set of substrates considerably expands the number of known proteins targeted for degradation by this ubiquitin-dependent pathway in plants. These results advance our current understanding of the role of AvrRpt2 in promoting bacterial virulence.

The Arg/N-Degron Pathway—A Potential Running Back in Fine-Tuning the Inflammatory Response?

“…For instance, the knockout of key components of the Arg/N-degron pathway in Arabidopsis thaliana prevents mounting an immune response to a variety of pathogens, which suggests that this pathway is involved in the degradation of proteins involved in the initiation of the immune response [ 64 ]. Conversely, the Arg/N-degron pathway is involved in the degradation of protein fragments cleaved by the Pseudomonas syringae protease effector AvrRpt2, and the removal of these fragments could help in the resolution of the immune response in the plant [ 65 ].…”

Cellular Control of Protein Turnover via the Modification of the Amino Terminus

“…This protease targets an important component of basal immunity, RIN4 [ 57 , 58 ]. Released fragments have the destabilizing neo-N-termini Asn and Asp [ 59 ] (see also Table 1 ). While these RIN fragments are metabolically unstable, they are apparently not stabilized in the prt6 N-degron ubiquitin ligase mutant.…”

The plant N‐degron pathways of ubiquitin‐mediated proteolysis

“…This is because following cleavage by Pst type III effector protein cysteine protease AvrRpt2 two cleavage products with Nt‐Asn or Nt‐Asp are revealed. A recent analysis suggested that these are not physiological substrates of these pathways, based on the lack of accumulation of these cleavage products in prt6 or ate1 ate2 protoplasts (Goslin et al ), though alternative internal degrons might also degrade these fragments. Based on the cleavage sites of AvrRpt2 several Arabidopsis nitrate‐induced (NOI) domain family proteins were identified as possible substrates, that were predicted to reveal secondary destabilizing residues Asp and Glu (Chisholm et al ).…”

“…Based on the cleavage sites of AvrRpt2 several Arabidopsis nitrate‐induced (NOI) domain family proteins were identified as possible substrates, that were predicted to reveal secondary destabilizing residues Asp and Glu (Chisholm et al ). Analysis of these 10 predicted cleavage sites in Arabidopsis protoplasts indicated that cleavage of NOI1, 6 and 11 may generate substrates of the ATE branch of the PRT6 pathway (Goslin et al ), and it will be important to understand the physiological relevance of this regulation in plant defense.…”