Minimal gene set from () pSymA required for efficient symbiosis with .

Reduction of N gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, () , using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete , , and modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.