DgDef1, a nodule-specific defensin-like peptide from the actinorhizal plant Datisca glomerata, induces membrane disruption and transcriptome changes in Sinorhizobium meliloti 1021

Several types of cysteine-rich peptides are involved in the interaction between plants and microorganisms, e.g., anti-bacterial and anti-fungal defensins, characterized by eight conserved cysteine residues, or nodule-specific cysteine-rich peptides that manipulate microsymbiont development in legume nodules, containing five to six conserved cysteine residues. Nodule-specific defensins are found in all actinorhizal plants of Fagales, Rosales as well as Cucurbitales, and a defensin from Alnus glutinosa nodules was shown to affect membrane integrity of a compatible Frankia strain and cause leakage of amino acids (Carro et al., 2015). Nodules of Datisca glomerata (Datiscaceae. Cucurbitales) contain a gene family encoding nodule-specific defensins with an acidic C-terminal domain. The member expressed at the highest levels, DgDef1, was expressed in young infected cells and, transiently, above the nodule lobe meristem. Neither the complete protein excluding the N-terminal signal peptide produced in Pichia pastoris, nor the synthetically produced cysteine-rich domain had not effect on the growth of Gram-positive Streptomyces coelicolor. However, the cysteine-rich domain alone had a cytotoxic effect on Gram-negative E. coli K-12 and on Sinorhizobium meliloti 1021 with an ID50 of 20.8 µM. RNAseq analysis of Sm1021 cultures treated with this domain, compared to an untreated control, showed a strong effect on the expression of genes encoding transporters, e.g., a dicarboxylate uptake system, consistent with the fact that treated Sm1021 cells had leaky membrane as denoted by propidium iodide staining. However, the expression of the cell cycle regulator ctrA was not affected, suggesting that DgDef1 did not affect the ploidity of Sm1021. Phylogenetic analysis of the cysteine-rich domains showed that legume NCRs and actinorhizal nodule-specific defensins go back to a common ancestor.