Comparative analysis of nodulation-related small secreted peptides across legume species

Abstract

Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and their rhizobial symbiotic partners. Rapid evolution of nodulation-related signaling peptide families among legume lineages, including family expansion, can lead to novel functional traits such as differences in infection efficiency or nodule viability. A computational workflow was developed to identify nodulation-related gene families encoding small signaling peptides that have undergone lineage-specific expansions (LSEs). This workflow used RNA-seq data from nodule, leaf, flower and root tissues from five legume species to obtain an initial pool of candidate gene families, followed by iterative clustering and expansion of the list of candidates. After curation, 13 nodulation-related LSEs were identified, each one specific to either Glycine, Arachis or Medicago lineages. Four family expansions were especially notable, including a nodulin family which expanded independently in both soybean (Glycine max) and common bean (Phaseolus vulgaris). In the diploid peanut relative, Arachis duranensis, a family of cysteine-rich peptides with nodule enhanced expression expanded to ~100 members, while in the Medicago truncatula lineage, the LEED..PEED gene family arose de novo after the Pisum-Medicago split, expanded to thirteen genes, and became fixed in relatively short evolutionary time. Also in the Medicago lineage, a set of nodule-specific PLAT domain proteins (NPDs) expanded to five nodule-specific members through tandem duplication. Because NPDs represent a recently discovered component in nodulation, we examined their function in further detail. Using a CRISPR/Cas9 multiplex genome editing approach, an overlapping set of M. truncatula NPD knockout lines were generated, targeting one through five NPD genes. Mutant lines with differing combinations of NPD gene inactivations had progressively earlier onset of nodule senescence, smaller size, or ineffective nodules compared to the wild type control. Two triple-knockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of two proteolysis genes, possible candidates for the observed breakdown of proper nodule function. Studies of lineage-specific expansions are useful to discover new candidate gene families linked to phylogeny-restricted traits. Applying a bioinformatic approach to identify LSEs of nodulation-associated genes, we identified a new family of nodule-specific PLAT domain peptides and confirmed that they play a role in successful nodule formation.