A select and resequence approach reveals strain-specific effects of medicago nodule-specific PLAT-domain genes

Genetic studies of legume symbiosis with nitrogen-fixing rhizobial bacteria have traditionally focused on nodule and nitrogenfixation phenotypes when hosts are inoculated with a single rhizobial strain. These approaches overlook the potential effect of host genes on rhizobial fitness (i.e. how many rhizobia are released from host nodules) and strain-specific effects of host genes (i.e. genome × genome interactions). Using Medicago truncatula mutants in the recently described nodule-specific PLAT domain (NPD) gene family, we show how inoculating plants with a mixed inoculum of 68 rhizobial strains (Ensifer meliloti) via a selectand- resequence approach can be used to efficiently assay host mutants for strain-specific effects of late-acting host genes on interacting bacteria. The deletion of a single NPD gene (npd2) or all five members of the NPD gene family (npd1-5) differentially altered the frequency of rhizobial strains in nodules even though npd2 mutants had no visible nodule morphology or N-fixation phenotype. Also, npd1-5 nodules were less diverse and had larger populations of colony-forming rhizobia despite their smaller size. Lastly, NPD mutations disrupt a positive correlation between strain fitness and wild-type host biomass. These changes indicate that the effects of NPD proteins are strain dependent and that NPD family members are not redundant with regard to their effects on rhizobial strains. Association analyses of the rhizobial strains in the mixed inoculation indicate that rhizobial genes involved in chromosome segregation, cell division, GABA metabolism, efflux systems, and stress tolerance play an important role in the strain-specific effects of NPD genes.