# Dual-Transcriptome Dissection of the Mechanisms Underlying Alfalfa Phenotypic Differences Induced by Two Rhizobial Isolates

## TL;DR

This study explores how two different rhizobial strains affect alfalfa nodule development and function by analyzing gene expression patterns in both the plant and bacteria.

## Contribution

The study provides a dual-transcriptome analysis revealing coordinated gene expression differences in alfalfa and rhizobia that underlie distinct symbiotic phenotypes.

## Key findings

- WE2-induced nodules show higher expression of genes related to transport and microaerobic conditions in the host.
- WE2 rhizobia exhibit increased expression of genes for nitrogen fixation and respiration compared to WWL2.
- The study proposes a working model linking host and bacterial gene expression to symbiotic function differences.

## Abstract

Different rhizobial strains can lead to distinct symbiotic phenotypes in alfalfa, yet molecular differences at the mature nodule stage remain unclear. Here, we analyzed 21-day post-inoculation (dpi) nodules induced by strains WE2 and WWL2. We measured nitrogenase activity (acetylene reduction assay, ARA) and performed dual RNA-seq to compare gene expression in both the alfalfa host and the rhizobia. On the host side, WE2-induced nodules showed higher expression of mature nodule marker genes (ENOD93 and leghemoglobin (Lb) genes) and higher expression of genes encoding SWEET transporters and amino acid and peptide transporters. Host differentially expressed genes were enriched in pathways related to transmembrane transport, redox and heme-related functions, and processes linked to maintaining microaerobic conditions. On the rhizobial side, WE2 nodules showed higher expression of genes involved in microaerobic respiration and nitrogen fixation (e.g., nif/fix and key respiratory chain genes), whereas WWL2 nodules showed higher expression of genes linked to transport, chemotaxis/motility, and environmental information processing. Together, these host and rhizobia expression patterns suggest coordinated differences between host pathways related to resource supply and microaerobic conditions and rhizobial expression programs for respiration and nitrogen fixation. Based on these associations, we propose a working model and provide candidate genes and pathways for functional validation and inoculant screening.