Structure-function relationship of the type III-secreted protein NopE1 of Bradyrhizobium japonicum

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Previous studies showed that the type III secretion system of Bradyrhizobium japonicum affects symbiosis with different host plants. We identified two highly similar secreted proteins, NopE1 and NopE2, which are responsible for a nodulation-promoting effect on soybean and Macroptilium atropurpureum. Interestingly, both proteins exhibit self-cleavage. Because nodulation assays and in vitro analysis showed that both proteins have the same or a very similar function, we will concentrate on the in vivo activity of NopE1. For localization of NopE1 in nodule cells, reporter gene fusions with the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis CyaA protein, differential fractionation and immunological techniques will be used. This will also reveal if the fusion protein is present as fulllength protein or in cleaved form. To further examine if autocleavage is necessary for in vivo activity, a NopE1-NopE2 double-mutant will be complemented with the wildtype gene and a mutant derivative, which encodes a non-cleavable NopE1 protein. For autocleavage of NopE1 calcium ions are required. Because calcium is a wellstudied signalling molecule in plants, the calcium-binding activity of NopE1 and its part in protein function is of interest. Therefore, the putative calcium-binding sites, which form EF hand-like calcium-binding domains, will be mutagenised. The calciumbinding capacity of wild-type and modified proteins will be compared. Derivatives that are unable to bind calcium will be used for complementation of the nopE1-nopE2 double-mutant. These data will provide a new insight into the function of the novel effector protein NopE1.