The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis

Abstract

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box-type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP-triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs, suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP-induced signaling.

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

“…For this purpose, we used Exo70B2, a subunit of the exocyst complex, which is ubiquitinated by PUB22 (Stegmann et al, 2012). As shown previously, PUB22 was able to ubiquitinate Exo70B2 in vitro ( Figure 4D).…”

“…We previously reported that PUB22 is stabilized within minutes after activation of the immune response with the flagellin-derived immunogenic peptide flg22 (Stegmann et al, 2012). This suggested that immune signaling feeds into the activation of PUB22 to activate a negative regulatory loop.…”

“…Initial stabilization of PUB22 in response to PAMP perception can be detected as early as 5 min after treatment, largely precluding a transcriptional effect (Stegmann et al, 2012). We therefore hypothesized that protein accumulation may result from the inhibition of its degradation.…”

“…The stabilization of PUB22, which occurs within 5 min after flg22 elicitation and is transient in nature (Stegmann et al, 2012), suggested the involvement of phosphorylation. In addition, the prolonged MPK3 activity in pub22 pub23 pub24 after elicitation implied a PUB22-linked negative feedback loop (Trujillo et al, 2008).…”

“…Their function is required for the general dampening of signaling induced by PAMPs (Stegmann et al, 2012). Inactivation of the ligase triplet results in enhanced resistance toward pathogens, such as Pseudomonas syringae pv tomato DC3000 (Pst), Hyaloperonospora arabidopsidis, and the fungus Piriformospora indica (Jacobs et al, 2011;Stegmann et al, 2012).…”