NPR1 is Instrumental in Priming for the Enhanced flg22-induced MPK3 and MPK6 Activation

Abstract

Pathogen-associated molecular patterns (PAMPs) activate mitogen-activated protein kinases (MAPKs), essential components of plant defense signaling. Salicylic acid (SA) is also central to plant resistance responses, but its specific role in regulation of MAPK activation is not completely defined. We have investigated the role of SA in PAMP-triggered MAPKs pathways in Arabidopsis SA-related mutants, specifically in the flg22-triggered activation of MPK3 and MPK6. cim6, sid2, and npr1 mutants exhibited wild-type-like flg22-triggered MAPKs activation, suggesting that impairment of SA signaling has no effect on the flg22-triggered MAPKs activation. Pretreatment with low concentrations of SA enhanced flg22-induced MPK3 and MPK6 activation in all seedlings except npr1, indicating that NPR1 is involved in SA-mediated priming that enhanced flg22-induced MAPKs activation.

Keywords: CIM6; MPK3; MPK6; NPR1; SID2; flg22 response; mitogen-activated protein kinases (MAPKs); priming; salicylic acid signaling.

Fig. 1

SA-related mutants exhibit WT-like flg22-induced phosphorylation of MPK6 and MPK3. WT plant and SA-related mutants (cim6, npr1 and sid2) were treated with SA (100 μM) for 24 h or flg22 (1 μM) for 10 min. Seedlings were then assayed for dual phosphorylation of the TEY motif in MPK3 and MPK6. Phosphorylated MAPKs corresponding to MPK3 and MPK6 is indicated. NT is representing control (H₂O) treated seedling. Activated MAPKs were detected by immunoblot using the antibody against Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology). Signal detection was performed using the enhanced chemiluminescence (ECL) western blotting detection kit (Bio-Rad). The experiment was performed three times with similar results. Prior to transfer to PVDF membrane, protein equal loading was checked by fluorescence image of Rubisco in the stain-free gels.

Fig. 2

The phosphorylation of MPK3 and MPK6 is enhanced in SA pretreated seedlings but npr1 showed reduction of potentiated dual phosphorylation. Arabidopsis seedlings were pre-incubated with SA (100 μM) or H₂O for 24 h before the treatment of flg22 (1 μM). Seedlings were then assayed for dual phosphorylation of the TEY motif in MPK3 and MPK6 10 minute after treatment with flg22. Activated MAPKs were detected by immunoblot blot using the antibody against Phospho-p44/42 MAPK (Erk1/2). The experiment was performed three times with similar results. Prior to transfer to PVDF membrane, protein equal loading was checked by fluorescence image of Rubisco in the stain-free gels.