Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signalresponsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB.

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Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. There are many reports about complementary, synergistic and overlapping functions of NO and ROS in the defence responses. Recent advances provide the molecular mechanisms of NO and oxidative bursts in the defence responses. NOA1 (NO ASSOCIATED1; formerly named NOS1) and membrane-bound NADPH oxidase are believed to participate in the radical burst. Here we describe that two mitogenactivated protein kinase (MAPK) cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NADPH oxidase at the transcriptional level in Nicotiana benthamiana. On the other hand, NOA1-mediated NO burst is regulated by the MEK2-SIPK cascade. Furthermore, we discuss the activation of NADPH oxidase by the calcium-dependent protein kinase (CDPK) through the direct phosphorylation of its N-terminal region.