The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis

Abstract

Symbiotic nitrogen fixation by rhizobia in legume root nodules is a key source of nitrogen for sustainable agriculture. Genetic approaches have revealed important roles for only a few of the thousands of plant genes expressed during nodule development and symbiotic nitrogen fixation. Previously, we isolated .100 nodulation and nitrogen fixation mutants from a population of Tnt1-insertion mutants of Medigaco truncatula. Using Tnt1 as a tag to identify genetic lesions in these mutants, we discovered that insertions in a M. truncatula nodule-specific polycystin-1, lipoxygenase, a-toxin (PLAT) domain-encoding gene, MtNPD1, resulted in development of ineffective nodules. Early stages of nodule development and colonization by the nitrogen-fixing bacterium Sinorhizobium meliloti appeared to be normal in the npd1 mutant. However, npd1 nodules ceased to grow after a few days, resulting in abnormally small, ineffective nodules. Rhizobia that colonized developing npd1 nodules did not differentiate completely into nitrogen-fixing bacteroids and quickly degraded. MtNPD1 expression was low in roots but increased significantly in developing nodules 4 d postinoculation, and expression accompanied invading rhizobia in the nodule infection zone and into the distal nitrogen fixation zone. A functional MtNPD1:GFP fusion protein localized in the space surrounding symbiosomes in infected cells. When ectopically expressed in tobacco (Nicotiana tabacum) leaves, MtNPD1 colocalized with vacuoles and the endoplasmic reticulum. MtNPD1 belongs to a cluster of five nodule-specific single PLAT domain-encoding genes, with apparent nonredundant functions.

A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes

“…Our finding of strain-specific effects of NPD2 is consistent with the observation of strain-specific effects of NPD1 detected based on nodule phenotypes in four single-strain inoculations (Pislariu et al, 2019). The npd1 mutant forms small white nodules with Sm1021 but pink nodules with Rm41.…”

“…For instance, two nodule-specific Cys-rich peptides have been identified as regulators of strain specificity after bacteria enter host cells (Tirichine et al, 2000;Wang et al, 2018), and a late-acting nodulin in Lotus japonicus, an aspartic peptidase, is necessary for infection with Mesorhizobium loti strain TONO, but not M. loti strain MAFF303099 (Yamaya-Ito et al, 2018). Recently, the phenotypic effects of NPD1, a member of the nodule-specific polycystin-1, lipoxygenase, alpha toxin (PLAT) domain (NPD) gene family studied here, were shown to be strain specific in Medicago truncatula (Pislariu et al, 2019). Assaying strainspecific phenotypic effects, when surveying many strains, is empirically difficult because pairwise host and strain experiments (i.e.…”

“…In npd1 hosts, root infection and nodule initiation appear to progress normally, but rhizobia fail to differentiate into nitrogen-fixing bacteroids once inside host cells, leading to early onset of nodule senescence. Pislariu et al (2019) also found the effects of npd1 to be strain specific. Two of four strains used for phenotyping produced small white ineffective nodules, one strain formed brown nodules hypothesized to result from a buildup of phenolics due to a host immune response, and one formed large, pink nodules that led to bigger plants than the wild type.…”

“…S4B). USDA 1264 formed large exclusively brown nodules (possibly filled with phenolics, see Pislariu et al [2019]) that released an intermediate number of colony-forming bacteria.…”

“…Characterization of sequential NPD knockouts, conducted with a single rhizobia strain (E. meliloti Sm1021), revealed that NPD disruption can produce quantitative changes in symbiotic phenotypes, including plant size, number of pink nodules, time of nodule senescence, and the area of the fixation zone in nodules (Trujillo et al, 2019). To date, only the NPD1 gene has been functionally characterized in detail (Pislariu et al, 2019). In npd1 hosts, root infection and nodule initiation appear to progress normally, but rhizobia fail to differentiate into nitrogen-fixing bacteroids once inside host cells, leading to early onset of nodule senescence.…”