Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD

Abstract

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.

RALF–FERONIA Signaling: Linking Plant Immune Response with Cell Growth

“…ROP2 has been reported to interact with the ECD of RBOHD, which is likely to regulate ROS levels ( Li et al., 2015 ). Recently, PBL13 (a homolog of RIPK) has been reported to interact with RBOHD to regulate NADPH oxidase-induced ROS production ( Lee et al., 2020 ). Thus, further studies are needed to determine whether Cr RLK1L/FER uses the ROP (e.g., ROP2) pathway to directly modulate ROS production, thus affecting cell polar growth and immune responses.…”

Plant plasma membrane‐resident receptors: Surveillance for infections and coordination for growth and development

“…Additionally, the peptide PLANT PEPTIDE CONTAINING SULFATED TYROSINE 1 (PSY1) also regulates the cell division and expansion in Arabidopsis , after being distinguished by an LRR‐RLK named as PSY1 RECEPTOR (PSY1R) (Amano et al, 2007). Moreover, the LRR‐RLKs ERECTA, ERECTA‐LIKE 1 (ERL1), and ERL2 play a role in stomatal development, reproductive organ development, and trigger cell differentiation, upon recognition of EPIDERMAL PATTERNING FACTOR 1 (EPF1) and EPF2 (Shpak et al, 2004; Lee et al, 2012; Shpak, 2013). A recent study identified another peptide, CLAVATA3/ESR‐RELATED 9/10 (CLE9/10), which also plays a role in stomatal development upon being recognized by HAESA‐LIKE 1 (HSL1) (Qian et al, 2018).…”

“…As reactive molecules, ROS are able to oxidize cellular components restricting their ability to function properly (Mittler et al, 2004). Thus, ROS levels require tight regulation to prevent cell damage due to excessive ROS production (Lee et al, 2020). In Arabidopsis, plasma membrane-resident RBOHD is mainly responsible for ROS burst upon MAMP perception (Torres et al, 2002;Wang et al, 2020b).…”

The phagocytosis oxidase/Bem1p (PB1) domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

“…We also detected the clathrin-related components HAP13 and AP4M, and flg22-inducible DRM components such as remorin and SPFH proteins by co-immunoprecipitation with RBOHD (Table S1). In addition, the E3 ubiquitin ligase PIRE, which interacts with both PBL13 and RBOHD, ubiquitinates RBOHD and decreases its abundance, possibly through endocytosis of RBOHD and vacuolar degradation (Lin et al, 2015;Lee et al, 2020a). Thus it is worth investigating the molecular relationship between PB1CP and PIRE for the endocytosis of RBOHD.…”

“…Similar to PRRs, RBOHD is also endocytosed under salt stress (Hao et al, 2014), suggesting the involvement of endocytosis in down-regulating RBOHD activity. Interestingly, PBL13, an RLCK, phosphorylates the C-terminal domain (CD) of RBOHD, which triggers the ubiquitination of RBOHD by PIRE (PBL13-INTERACTING RING DOMAIN E3 LIGASE) (Lee et al, 2020a).…”