Mitogen-activated protein kinase (MAPK)-mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme-substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104 -up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.itogen-activated protein kinase (MAPK) cascades transduce external signals into cellular responses in eukaryotes (1). In plants, MAPKs orthologous to the Arabidopsis MPK3, MPK4, and MPK6 are activated by various stimuli including flg22, a bacterial flagellin-derived peptide that acts as a pathogen-associated molecular pattern (PAMP) (2-5). These three MAPKs control defense positively (MPK3/MPK6) (3, 6) or negatively (MPK4) (7).Many phytohormones have been shown to affect defense responses; but most progress has been made in regard to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) (8). The tobacco MPK6 ortholog is activated by SA (9) and the Arabidopsis mpk4 mutant has elevated SA levels and enhanced pathogen resistance (7). Genetic evidence linking ET to MAPK signaling is also suggested by the negative regulator of the ET response, Constitutive Triple Response 1 (CTR1), a Raf-like kinase that was recently shown to control MPK3/6 activation via MKK9 (MAPK kinase 9) (10). Both JA and the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), activate MPK6 in Arabidopsis (11, 12) but not in tobacco (13). Although responses may differ between plant species, the activation of MPK6 by ET/ACC is highly debated (14). In another report, ACC did not activate MPK6, but ET biosynthesis was positively regulated by MPK6 through posttranslational stabilization of the rate-limiting ACC synthase (ACS) isoforms, ACS2 and ACS6 (14,15).In addition to the cytoplasmic ACSs, MAPKs also target nuclear proteins (10,16,17); this may occur either after MAPK nuclear translocation following activation (18,19) or as preformed nuclear protein complexes (20). The latter would imply movement of the upstream MKKs into the nucleus to modify the MAPKs or, alternatively, that the activated MAPKs enter the nucleus to displace the inactive MAPK from preformed complexes. Examples of nuclear targets include the MPK4 substrates, MKS1 and two MKS1-interactors of the WRKY transcription factor family, WRKY25 and WRKY33 ...