Stachydrine Catabolism Contributes to an Optimal Root Nodule Symbiosis Between <i>Sinorhizobium meliloti</i> and <i>Medicago sativa</i>

<i>Sinorhizobium meliloti</i> forms a robust N₂-fixing root-nodule symbiosis with <i>Medicago sativa</i>. We are interested in identifying the minimal symbiotic genome of the model strain <i>S</i>. <i>meliloti</i> Rm1021. This gene set refers to the minimal genetic determinants required to form a robust N₂-fixing symbiosis. Many symbiotic genes are located on the 1,354-kb pSymA megaplasmid of <i>S</i>. <i>meliloti</i> Rm1021. We recently constructed a minimalized pSymA, minSymA2.1, that lacked over 90% of the pSymA genes. Relative to the wild type, minSymA2.1 showed a reduction in <i>M</i>. <i>sativa</i> shoot biomass production and nodule size with an increase in total nodule number. Here, we show that the addition of either the stachydrine (<i>stc</i>) or trigonelline (<i>trc</i>) catabolism genes from pSymA to minSymA2.1 restores nodule size and total nodule number to levels indistinguishable from the wild type but does not restore reduced shoot biomass production. In the context of the complete Rm1021 genome, removing the <i>stc</i> genes reduced the nodule size and increased the total nodule number, whereas removal of the <i>trc</i> genes alone had no apparent effect. Together, these observations implicate stachydrine catabolism as an important determinant of root nodule symbiosis between <i>S</i>. <i>meliloti</i> and <i>M</i>. <i>sativa</i>, whereas trigonelline catabolism seems to contribute in a more conditional manner, in the context of the minimized genome. These findings highlight the minimal symbiotic genome as a tool for investigating the impact of individual genetic determinants in conferring an optimal symbiosis, factors whose impact, in the context of a complete genome, may be hidden or dampened due to redundancies. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.