A conserved 5′ UTR element controls <i>LIN </i>expression to function asymmetrically with <i>LIN-Like</i>70together to direct symbiosome formation in <i>Medicago truncatula</i>

The formation of symbiosomes—organelle-like structures where rhizobia fix nitrogen—represents a major evolutionary innovation in legumes, yet the molecular mechanisms governing bacterial release from infection threads (ITs) remain poorly understood. Here, we identify lin-5, a hypomorphic allele of the Medicago truncatula Lumpy Infections (LIN) gene, that uncouples IT elongation from symbiosome formation. The lin-5 mutation is a 2.3-kb deletion in the promoter and 5' UTR that attenuates rather than abolishes LIN expression. Consequently, lin-5 supports normal IT development but delays rhizobial release, producing swollen ITs with thickened, rigid cell walls that prevent infection droplet formation. We discover a critical 41-bp cis-regulatory motif in the LIN 5' UTR—the Regulatory Element Associated with Symbiosome formation (REAS)—that is conserved exclusively in symbiosome-forming species and is essential for upregulating LIN above the threshold required for droplet formation. This regulatory element operates with the more ancient LIN paralog LIN-Like, which functions asymmetrically with LIN to support bacterial release. Thus, our findings reveal how gene duplication and the acquisition of symbiosis-specific cis-regulatory elements create a multilayered regulatory network that enabled the evolutionary innovation of symbiosome