The type III--secreted proteins NopE1 and NopE2 of Bradyrhizobium japonicum contain a repeated domain of unknown function (DUF1521), which is present in a few uncharacterized proteins. A nopE1/nopE2 double mutant strain exhibited higher nodulation efficiency on Vigna radiata KPS2 than the wild type or single nopE1 or nopE2 mutants. This indicates that both proteins are effectors that functionally overlap. To test translocation into the plant cell compartment during symbiosis, NopE1 and NopE2 were fused with adenylate cyclase (cya) as reporter. A fusion with the full-length proteins or N-terminal peptides resulted in increased cAMP levels in nodules, indicating translocation. Purified NopE1 exhibited self-cleavage in the presence of Ca2+. Two identical cleavage sites (GD'PHVD) were identified inside the DUF1521 domains. The C-terminal cleavage site was analyzed by alanine scanning. Protein variants in which aspartate or proline next to the cleavage sites was substituted displayed no cleavage. A noncleavable protein was obtained by exchange of the aspartate residues preceding both cleavage sites. Complementation analysis with the noncleavable NopE1 variant did not restore wild-type phenotype on Vigna radiata KPS2, indicating a physiological role of NopE1 cleavage in effector function.
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