A CCaMK/Cyclops response element in the promoter ofL. japonicus Calcium-Binding Protein 1(CBP1) mediates transcriptional activation in root symbioses

SummaryEarly gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. TheLotus japonicusT-DNA insertion line T90, carrying a promoterlessuidA(GUS) gene in the promoter ofCalcium Binding Protein1(CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsiblecis-andtrans-acting factors, we subjected deletion/modification series ofCBP1promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrantGUSexpression. We identified onecis-regulatory element required forGUSexpression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack ofGUSexpression in T90whitemutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promotersNIN(RNS),RAM1(AM), andCBP1(AM and RNS), calling for yet-to-be identified specificity-conferring factors.