In order to effectively manipulate rhizobium–legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available -proline auxotrophs has limited our understanding of the role of -proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described -proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The auxotroph displayed reduced symbiotic capability with alfalfa () due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover () that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that -proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.


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