Summary
Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and rhizobial symbionts. In legumes, several secreted protein families have undergone expansions and play key roles in nodulation. Thus, identifying lineageâspecific expansions (LSEs) of nodulationâassociated genes can be a strategy to discover candidate gene families.
Using bioinformatic tools, we identified 13 LSEs of nodulationârelated secreted protein families, each unique to either Glycine, Arachis or Medicago lineages. In the Medicago lineage, noduleâspecific Polycystinâ1, Lipoxygenase, Alpha Toxin (PLAT) domain proteins (NPDs) expanded to five members. We examined NPD function using CRISPR/Cas9 multiplex genome editing to create Medicago truncatula NPD knockout lines, targeting one to five NPD genes.
Mutant lines with differing combinations of NPD gene inactivations had progressively smaller nodules, earlier onset of nodule senescence, or ineffective nodules compared to the wildâtype control. Doubleâ and tripleâknockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of a DHHCâtype zinc finger and an aspartyl protease gene, possible candidates for the observed disturbance of proper nodule function.
By postulating that gene family expansions can be used to detect candidate genes, we identified a family of noduleâspecific PLAT domain proteins and confirmed that they play a role in successful nodule formation.