Plant Biology and Biogeochemistry Department, Risø National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark
The origin of de novo synthesized proteins in the interface between pea and a Rhizobium sp. in root nodules has been determined. The symbiotic interface is defined as the peribacteroid space including proteins associated with either the symbiosome membrane or the bacteroid outer membrane. Two approaches have been used to study the origin of proteins in the symbiotic interface. First, to determine the localization of de novo synthesized plant-produced proteins in the symbiosomes, an in vitro protein translocation assay was established. To produce plant proteins poly A+ RNA was isolated from root nodules followed by in vitro translation in the presence of [35S]methionine. Subsequently, purified symbiosomes were incubated with the [35S]methionine-labeled plant proteins. The symbiosomes were subfractionated and de novo synthesized plant proteins in the different fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These import studies demonstrate the presence of plant proteins in the symbiosome membrane, peribacteroid space, and bacteroid membrane pellet. In contrast, no proteins of plant origin were detected in the bacteroid cytosol. Second, the presence of de novo synthesized bacteroid proteins in the interface was examined by incubation of symbiosomes isolated under micro-aerobic or aerobic conditions with [35S]methionine. Analysis of symbiosome compartments by SDS-PAGE and phosphor image revealed that proteins of bacteroid origin are primarily detected in the bacteroid cytosol and bacteroid membrane pellet. However, a few bacteroid-produced proteins are also observed in the symbiosome membrane. Together these data demonstrate that the majority of de novo synthesized proteins in the pea-Rhizobium sp. symbiotic interface are of plant origin.