Ubiquitination during Plant Immune Signaling

Abstract

Plant responses to pathogens depend on the rapid and effective coordination of microbial perception and downstream signal transduction events. Detection of pathogen invasion starts by the recognition of conserved microbial molecules called pathogen-associated molecular patterns (PAMPs), mainly by plant membrane-associated extracellular receptors, which results in PAMP-triggered immunity (PTI). Using a type III secretion system, plant pathogenic bacteria are able to inject type III effectors (T3Es) directly inside host cells, thereby overcoming PTI and favoring bacterial growth. Recognition of T3Es by plant resistance (R) proteins leads to effector-triggered immunity (ETI), a more efficient form of resistance that is regularly associated with the development of hypersensitive cell death (HR) at the site of pathogen penetration (Jones and Dangl, 2006). In addition, the onset of the HR typically triggers systemic acquired resistance (SAR), an inducible form of plant defense that spreads resistance to systemic tissues through mobilization of salicylic acid (SA)-mediated defenses and confers broad-spectrum immunity to secondary infection (Spoel and Dong, 2012). Plant hormones are crucial systemic signals that strongly influence the level of plant resistance. Indeed, significant changes in hormone levels and hormonal cross talk occur in plant cells interacting with microbes and are essential to the efficient integration of biotic stress cues (Pieterse et al., 2009). The intricate molecular mechanisms that govern plant immune responses engage a high degree of proteomic plasticity to which posttranslational protein modification through ubiquitination contributes crucially. Ubiquitin is a small (8.5 kD) and highly conserved protein modifier that, covalently linked to target proteins, leads to their proteasomal degradation or to other fates including relocalization or endocytosis. Typically, proteins modified by sequential linkage of multiple ubiquitin residues via the ubiquitin residue Lys-48 are targeted for degradation by the 26S proteasome, a highly conserved proteolytic complex composed of two subparticles (Smalle and Vierstra, 2004): (1) the barrel-shaped 20S proteasome that is a stack of two outer rings formed by seven α-subunits (α1–α7) and two inner rings of seven β-subunits (β1–β7) enclosing a cavity with the active sites for protein degradation and (2) the 19S regulatory particles that are attached at both ends of the 20S cylinder and recognize the protein targeted for degradation. The ubiquitin-26S proteasome system (UPS) involves the sequential action of three enzymes, namely E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase), to ultimately ligate one or more ubiquitin molecules to specific target proteins (Vierstra, 2009). Ubiquitin is first activated for transfer by the E1 enzyme and activated ubiquitin is then transferred to a Cys residue in the E2. The ubiquitin-E2 intermediate generally serves as the proximal ubiquitin donor, using the E3 to identify the target and catalyze ubiquitin transfer. E3 enzymes are key factors that determine substrate specificity and are classified into four main subfamilies depending on their subunit composition and mechanism of action: Homologous to E6-associated protein Carboxyl Terminus (HECT), Really Interesting New Gene (RING), U-box, and cullin-RING ligases (CRLs; Vierstra, 2009). HECT proteins are single polypeptides that, unlike other E3 ligases, form a thioester intermediate with ubiquitin before ubiquitination of the target (Downes et al., 2003). RING and U-box proteins are structurally related single polypeptides that respectively use zinc chelation and hydrogen bonds/salt bridges to transfer ubiquitin from the E2-ubiquitin intermediate to the substrate (Stone et al., 2005; Yee and Goring, 2009). CRLs are multisubunit E3 ligases that contain a cullin, a RING-BOX1 that binds to ubiquitin, and a variable module for target recognition (Vierstra, 2009). The modular S-phase Kinase-associated Protein1 (SKP1)/CULLIN1 (CUL1)/F-Box (SCF) group is the best-characterized CRL. Arabidopsis (Arabidopsis thaliana) SKP1-like proteins are known as Arabidopsis SKP1-like (ASK). In SCF complexes, CUL1 acts as a molecular scaffold by interacting at its C terminus with RING-BOX1 (which is linked to the E2-ubiquitin intermediate) and at the N terminus with SKP1/ASK (which is linked to the F-box protein, responsible for recruiting the target) thereby promoting the transfer of ubiquitin from the E2 to the target (Vierstra, 2009). Here, we discuss the involvement of different UPS-related components during the regulation of plant immune responses, paying special attention to the well-characterized family of E3 ubiquitin ligases. Strikingly, targeted protein turnover through the UPS is a shared feature by most hormone signaling pathways (Santner and Estelle, 2009; Vierstra, 2009). Due to space limitations, we refer the reader to recently published reviews for