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 efficacy of the nitrogenase system nifH promoter (P nifH ) 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 P nifH -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 P nifH -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 P nifH -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. IMPORTANCE The legume-rhizobium symbiosis is central to sustainable agriculture through its capacity for biological nitrogen fixation, yet tools for real-time, quantitative visualization of this interaction remain limited. Here, we demonstrate that the nifH promoter (P nifH ) effectively drives expression of superfolder yellow (sfYFP) and cyan (sfCFP) fluorescent proteins in both determinate ( Lotus japonicus-Mesorhizobium japonicum ) and indeterminate ( Pisum sativum-Rhizobium leguminosarum ) nodules. These reporters enable robust, reproducible fluorescence without impairing rhizobial competitiveness, supporting multiplexed imaging and quantitative nodulation analyses. By contrast, red fluorescent proteins exhibited host-dependent variability, underscoring the need for improved red fluorophores. Integration of validated promoter-reporter constructs with a deep learning-based image analysis pipeline establishes a scalable framework for high-throughput assessment of nodule occupancy and symbiotic dynamics. This work provides a practical molecular and computational toolkit for dissecting plant-microbe mutualisms across diverse host systems.
Gautam et al. (Fri,) studied this question.