The actinorhizal plantDatisca glomerata(Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbioticFrankiaclade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed inD.glomeratanodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels,DgDef1, was characterized. Promoter-GUSstudies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity.In vitrostudies withE.coliandSinorhizobium meliloti1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality forS.meliloti1021 at 20.8 μM. Analysis of theS.meliloti1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.