A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase‐mediated oxidative burst are two independent signaling events in plant immunity

Abstract

Summary Plant recognition of pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin-derived flg22 triggers rapid activation of mitogen-activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). Arabidopsis has at least four PAMP/pathogen-responsive MAPKs: MPK3, MPK6, MPK4, and MPK11. It was speculated that these MAPKs may function downstream of ROS in plant immunity because of their activation by exogenously added H2O2. MPK3/MPK6 or their orthologs in other plant species were also reported to be involved in ROS burst from the plant respiratory burst oxidase homologue (Rboh) of human neutrophil gp91phox. However, detailed genetic analysis is lacking. Using a chemical genetic approach, we generated another conditional loss-of-function mpk3 mpk6 double mutant. Together with the conditionally rescued mpk3 mpk6 double mutant reported previously, we demonstrate that flg22-triggered ROS burst is independent of MPK3/MPK6. In Arabidopsis mutant lacking a functional AtRbohD, flg22-induced ROS burst was completely blocked. However, the activation of MPK3/MPK6 was not affected. Based on these results, we conclude that the rapid ROS burst and MPK3/MPK6 activation are two independent early signaling events downstream of FLS2 in plant immunity. We also found that MPK4 negatively impacts the flg22-induced ROS burst. In addition, salicylic acid-pretreatment enhances AtRbohD-mediated ROS burst, which is again independent of MPK3/MPK6 based on the analysis of mpk3 mpk6 double mutant. The establishment of a mpk3 mpk6 double mutant system using the chemical genetic approach offers us a powerful tool to investigate the function of MPK3/MPK6 in plant defense signaling pathway.

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

“…Consistent with previous reports using BTH and/or SA (Beckers et al, 2009;Sato et al, 2010;Xu et al, 2014), BTH treatment did not stimulate MAPK activity or ROS accumulation in our growth conditions within 10 or 30 min, respectively (Supplemental Figures 4A and 4B). Even after 24 h, there was no MAPK activation (Beckers et al, 2009) or ROS accumulation ( Figure 1J).…”

“…Given the observation that within 1 d after BTH treatment the levels of PRRs are increased, previous studies showing SA-mediated potentiation of flg22 responses (Sato et al, 2010;Xu et al, 2014) can probably be explained by higher levels of PAMP receptors. This agrees well with the observation that overexpression of FLS2 confers increased responsiveness to flg22 (Gómez-Gómez and Boller, 2000).…”

“…Additionally, flg22-induced accumulation of ROS in the wild type was enhanced by 24 h of BTH pretreatment ( Figure 1J). However, unlike in the acd6-1 plants, in these conditions, SA or BTH treatment did not affect the timing of the initiation of flg22-induced ROS production (Sato et al, 2010;Xu et al, 2014) (Figure 1K). acd6-1 sid2-1 and acd6-1 plants responded to flg22 with the same timing of ROS production ( Figure 1H, right panel).…”

“…SA enhances flg22-triggered ROS accumulation (Sato et al, 2010;Xu et al, 2014). Since acd6-1 accumulates high levels of SA (Vanacker et al, 2001), it seemed possible that SA signaling might affect the responses of acd6-1 plants to flg22.…”

Retraction

“…Together, these results suggest that IOS1 is required for full MPK activation, but not for the ROS burst after MAMP perception. This observation is consistent with these responses being uncoupled (Segonzac et al, 2011;Xu et al, 2014a). However, since we do not provide a kinetic analysis, we cannot exclude a slower or faster MPK response in ios1 mutants or OE lines, respectively.…”