As outlined earlier, increasing evidence suggests that removal of negative regulatory components may play an important role in the execution of plant HR cell death. In the case of proteinaceous factors, this could be achieved by proteases with high substrate specificity or alternatively by specific targeting of such proteins to the ubiquitin-proteasome degradation pathway.
It is well established that activation of regulatory protease cascades is required for the initiation of PCD in animal cells (Gratter, 2000). The most prominent family of proteases involved in this process carries a cysteine in their catalytic site and cleaves specifically after an aspartate residue; therefore, these proteases are called caspases (cysteine dependent aspartate-specific proteases). Caspases are (auto)activated from an inactive zymogen after binding of an extracellular signal molecule to its cognate receptor or as a consequence of intracellular stresses. After activation, the initiator caspases process additional downstream effector procaspases, which finally lead to the execution of cell death by removal of suppressors of apoptosis (Aravind et al., 1999; Grutter, 2000). In plants, proteases with significant sequence similarity to caspases have not yet been identified, nor are such proteins encoded in the complete Arabidopsis genome (Estelle, 2001). However, by using specific peptide substrates and selective protease inhibitors, cas-paselike activities have recently been identified in plants. For example, caspaselike activities have recently been detected in tobacco tissue undergoing a virus-induced HR, as well as in the cytosol of embryonic barley cells and in the giant cells of the algae Chara corallina (Korthout et al., 2000; Lam and del Pozo, 2000). Another feature of PCD in animal cells is the specific cleavage of poly (ADP-ribose) polymerase (PARP) by caspase-3. Corresponding with the activation of caspase-3-like activity, PARP degradation has been observed in cultured tobacco cells upon heat treatment (Estelle, 2001).
Although data suggesting a participation of caspaselike proteases in regulation of cell death in plants are still indirect, increasing evidence suggests that regulatory proteins are specifically targeted to the ubiquitin/proteasome degradation pathway. In this pathway, ubiquitin becomes covalently attached to the protein destined for degradation by an ATP-dependent reaction cascade comprising three enzymes or enzyme complexes (Callis and Vierstra, 2000). E1 (ubiquitin-activating enzyme) catalyzes the formation of an activated ubiquitin that is subsequently transferred to the cysteinyl sulfhydryl group of a second enzyme called E2 (ubiquitin-conjugating enzyme). E2s represent a large family of proteins with at least 36 isoforms in Arabidopsis (Callis and Vierstra, 2000). The transfer of ubiquitin to its target protein requires a third specificity-conferring protein or protein complex called E3 (ubiquitin ligase). The highly diverse E3s have been grouped into four classes, including the SKP1-Cullin-F-box (SCF) E3 ligase subtype, which is named after the three yeast protein subunits: suppressor of kinetochore protein 1 (Skp1p), cell-division cycle 53 (Cullin), and F-box proteins (Callis and Vierstra, 2000). Following monoubiquitination, subsequent attachment of additional ubiquitin units to the primary ubiquitin residue leads to the formation of poly-
ubiquitinated proteins, which are then targeted for degradation by the 26S proteasome.
A direct link between resistance and protein ubiquitination has recently been discovered in barley and Arabidopsis. As outlined earlier, race-specific resistance of barley to powdery mildew that is mediated by some Mla alleles, such as Mla6 and Mla12, requires the additional components Rarl and Rar2. To elucidate the molecular function of RAR1 in plants, the Arabidopsis homologue AtRAR1 was used as bait in a yeast two-hybrid screen to search for interacting proteins. Thereby, two interacting proteins were identified, AtSGT1a and AtSGT1b, which shared extensive sequence similarity to each other and to the yeast protein SGT1 (Azevedo et al., 2002). In yeast, SGT1 is an essential regulator of the cell cycle. Its function could be complemented by both AtSGT1a and AtSGT1b, suggesting that these proteins are functional orthologs of yeast SGT1. In addition, yeast SGT1 is associated with the kinetochore complex and the SCF-type (Skp1-Cullin-F-box) E3 ubiquitin ligase (Kitagawa et al., 1999). In barley, immunoprecipitation experiments demonstrated that SGT1 interacts not only with either RAR1 and SCF subunits, but also with two subunits of the COP9 signalosome, which are closely related to the lid complex of the 26S proteasome (Karniol and Chamovitz, 2000; Azevedo et al., 2002). Thus one possible role of plant SGT1 could be to target resistance-regulating proteins for polyubiquitin-ation and subsequent degradation by the 26S proteasome.
In Arabidopsis, mutational screens independently identified ortho-logs of barley RAR1 and SGT1 as components of resistance specified by multiple resistance genes of the CC-NB-LRR type (e.g., RPM1, RPS2, RPP8) and TIR-NB-LRR type (e.g., RPP5, RPS4), recognizing different bacterial (Pseudomonas syringae) and oomycete (Per-onospora parasitica) pathogens (Austin et al., 2002; Muskett et al., 2002). The sgtlb mutation suppressed resistance against Perono-spora parasitica mediated by the RPP5 gene, but not as much as the rpp5 null mutation, and the rarl null mutant likewise partially suppressed RPP5-mediated resistance (Austin et al., 2002). The sgtlb/rarl double mutant exhibited an additive effect of both genes in compromising RPP5-mediated resistance with substantially delayed plant HR cell death and whole-cell ROI accumulation (Austin et al., 2002; Muskett et al., 2002). This finding is in agreement with the results ob tained by Azevedo and colleagues (2002), who demonstrated a physical interaction between RAR1 and SGT1 and that SGT1 exists in at least two pools (SGT1/RAR1 and SGT1/SCF) with presumably different functions. Collectively, these and additional findings by Tor and colleagues (2002) demonstrated that RAR1 and SGT1 are convergence points of defense signaling conferred by several R genes in different plants, and that both have partially combined and distinct roles in resistance, one of which is presumably related to ubiquiti-nation of still-unknown targets.
In addition to ubiquitin, several other ubiquitin-like polypeptide tags such as SUMO (small ubiquitin-like modifiers), RUB (related to ubiquitin), and APG12 (autophagy-defective-12) have been identified in plants. Similar to ubiquitin, these alternative modifiers are attached to -lysyl groups of target proteins, thereby influencing their structure, location, and turnover (Vierstra and Callis, 1999). Evidence for the involvement of the ubiquitin-like modifier SUMO-1 in disease resistance was recently obtained for the interaction between Nicotiana benthamiana and the bacterial pathogen Xanthomonas campestris (Orth et al., 2000). The bacterial avirulence gene product AvrBsT induces HR cell death and shares significant similarity with the YopJ protein of the human pathogen Yersinia pestis, which inhibits the host immune response. These YopJ family members were shown to act as cysteine proteases, specifically removing SUMO-1 residues from its protein conjugates (Orth et al., 2000). Protease-in-active variants of AvrBsT, generated by site-directed mutagenesis, were defective in HR induction. These results indicate the importance of ubiquitin-like protein conjugation and deconjugation in regulation of defense-related signaling pathways leading to HR cell death.
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