Fluorescently Labeled Sinorhizobium sp. and Strain Specific Markers to Study Symbiotic Nitrogen Fixation in Medicago truncatula

Legumes are essential for sustainable agriculture due to their symbiotic association with soil bacteria collectively called rhizobia. Through symbiotic nitrogen fixation (SNF), legumes acquire fixed nitrogen in the form of usable ammonium (NH4+). A genetic model that is widely used to investigate SNF is Medicago truncatula, a relative to alfalfa. As a result of its association with the nitrogen-fixing symbiont Sinorhizobium sp., specialized new organs (root nodules) develop on its root system where rhizobia fix N2. SNF is a complex process that involves regulation of thousands of host and bacterial genes, and a proxy for effective nitrogen fixation is the accumulation of pink-colored leghemoglobin inside nodules. Using a pangenomics approach, strain-specific markers for Sinorhizobium meliloti (Rm41, M162, T073, M10, M270, and M30), S. medicae (A321, M161, and WSM419), and S. fredii (USDA205 and USDA207) were identified and validated by duplex PCR and Sanger sequencing. For S. meliloti 1021, one strain-specific Single-Nucleotide Polymorphism (SNP) marker was identified. To track infections and evaluate the symbiotic phenotype of WT M. truncatula R108 upon infection with various Sinorhizobium sp, the twelve strains were labeled with constitutively expressing fluorescent proteins (green, mCherry, cyan, and yellow). Whether wild-type (WT) or expressing fluorescent proteins, S. melilotistrains M10, Rm41, and T073 induced large pink functional nodules. In contrast, both WT and fluorescently tagged S. meliloti M30 and M270, S. medicae WSM419, and A321 induced defective nodules. The symbiotic phenotypes induced by S. meliloti M162 and Sm1021, S. medicae M161, and S. fredii USDA205 and USDA207 were negatively impacted by the constitutive expression of fluorescent proteins. We report that the parBDE partitioning system in the fluorescent protein expression plasmids impacts symbiosis with S. meliloti M162 and Sm1021. These findings suggest that genetic factors and yet to be determined host strain specificity mechanisms may explain the unusual phenotypes observed.