Document Type
Article
Publication Title
Applied and Environmental Microbiology
Abstract
The legume-rhizobium symbiosis is a cornerstone of sustainable agriculture due to its ability to facilitate biological nitrogen fixation. Still, real-time visualization and quantification of this interaction remain technically challenging, especially across different host backgrounds. In this study, we systematically evaluate the efficacyefficacyefficacyof the nitrogenase system nifH promoter (PnifH) in driving expression of distinct fluorescent reporters; superfolder yellow fluorescent protein (sfYFP), superfolder cyan fluorescent protein (sfCFP), and various red fluorescent proteins (RFPs) within root nodules of determinate (Lotus japonicus-Mesorhizobium japonicum) and indeterminate (Pisum sativum-Rhizobium leguminosarum) systems. We show that PnifH-driven sfYFP and sfCFP yield strong, uniform, and reproducible fluorescence in nodules of both systems, facilitating reliable quantification of nodulation traits and strain occupancy. In contrast, RFPs including monomeric (mScarlet-I, mRFP1, mARs1) and multimeric (AzamiRed1.0) variants exhibited weak or inconsistent signals in pea. Notably, fluorescent labeling did not impair rhizobial competitiveness for root nodule occupancy, and PnifH-driven sfYFP and sfCFP reporters enabled robust multiplexed imaging in single-root and split-root assays. In the lotus, mScarlet-I worked robustly and facilitated a tripartite strain labeling system. Complementing our molecular toolkit, we established a deep learning-based analytical pipeline for high-throughput, automated quantification of nodulation traits, validated against standard ImageJ analysis. Altogether, our results identify PnifH-driven sfYFP and sfCFP as robust, broadly applicable reporters for legume-rhizobium symbiosis studies, while highlighting the need for optimized red fluorophores in some contexts. The integration of validated promoter-reporter constructs with state-of-the-art computational approaches provides a scalable framework for dissecting the spatial and competitive dynamics of plant-microbe mutualisms.
DOI
https://doi.org/10.1128/aem.02154-25
Publication Date
1-16-2026
Recommended Citation
Gautam CK, Senanayake G, Pease AB, Salem MA, Rabia AH, Prüß BM, Geddes BA. 2026. Host-specific fluorescence dynamics in legume-rhizobium symbiosis during nodulation. Appl Environ Microbiol 92:e02154-25. https://doi.org/10.1128/aem.02154-25