Early nodulin-like protein MtENODL29 inhibits nodule senescence in Medicago truncatula

The early nodulin-like (ENODL) subfamily, part of the phytocyanin, arabinogalactan protein, and nodulin-like families, is involved in plant growth and stress resistance. However, its role in symbiotic nodulation remains poorly understood. In barrel medic (Medicago truncatula), we found MtENODL29 was strongly activated at the late stages of nodule development, particularly in the infection zone of nodules. Both RNA interference (RNAi) and mutation of MtENODL29 caused a considerable reduction in nodule numbers, an increase in cysteine protease activity, a dramatic decrease in leghemoglobin content, and signs of premature senescence in nodule cells, suggesting that disruption of MtENODL29 accelerates nodule aging. Transcriptome analysis of 7-dpi (day post inoculation) inoculated roots and 28-dpi nodules in enodl29 mutants showed significant downregulation of symbiotic genes, accompanied by differential expression of genes associated with lipid metabolism and transport. MtENODL29 mutation also negatively impacted plant growth and development. MtENODL29 bound to MtnsLTP (nonspecific lipid transfer protein), MtKCR (very-long-chain 3-oxoacyl-CoA reductase), and MtSec61γ (gamma subunit of the translocase complex Sec61) through its ALR (arabinogalactan protein-like region) domain. MtENODL29 co-localized with these proteins in the plasma membrane and endoplasmic reticulum. Notably, MtnsLTP showed high expression in the nodules, similar to MtENODL29, while MtKCR and MtSec61γ were also highly expressed in the leaves and stems. These results suggest that MtENODL29 participates in membrane lipid modification and transport by interacting with MtnsLTP, MtKCR, and MtSec61γ, facilitating the formation of symbiosome membranes as alfalfa rhizobium (Sinorhizobium meliloti) strain 1021 are released into nodule cells. Moreover, MtENODL29 influences plant growth, highlighting its role in coordinating plant development and symbiosis.