Abstract
Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because of low bacterial biomass and interference from plant RNA. To overcome this, we developed a randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) combined with chassis-independent recombinase-assisted genome engineering (CRAGE). Using Pseudomonas simiae WCS417 as a model rhizobacterium, this method enabled targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during Arabidopsis root colonization. Our analysis revealed temporally resolved transcriptional regulation, including those associated with cell growth, chemotaxis, plant immune suppression, biofilm formation, and stress responses, reflecting the coordinated physiological adaptation to the root environment. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor is crucial for evading plant immune systems. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.
Data availability
All sequencing data generated in this study have been deposited in the NCBI Short Read Archive under accession code PRJNA1221951. The genome-scale datasets are provided as Supplementary Data 1–6. Source data are provided with this paper.
Code availability
Custom scripts used for PI-seq analysis are available on GitHub at https://github.com/tomoyahonda/CRAGE-RB-PI-seq and archived on Zenodo at https://doi.org/10.5281/zenodo.18462023.
References
Zhalnina, K. et al. Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly. Nat. Microbiol. 3, 470–480 (2018).
McLaughlin, S., Zhalnina, K., Kosina, S., Northen, T. R. & Sasse, J. The core metabolome and root exudation dynamics of three phylogenetically distinct plant species. Nat. Commun. 14, 1649 (2023).
Jansson, J. K., McClure, R. & Egbert, R. G. Soil microbiome engineering for sustainability in a changing environment. Nat. Biotechnol. 1–13, https://doi.org/10.1038/s41587-023-01932-3 (2023).
Haney, C. H., Samuel, B. S., Bush, J. & Ausubel, F. M. Associations with rhizosphere bacteria can confer an adaptive advantage to plants. Nat. Plants 1, 15051 (2015).
Wang, N. R. et al. Commensal pseudomonas fluorescens strains protect arabidopsis from closely related pseudomonas pathogens in a colonization-dependent manner. mBio 13, e02892–21 (2022).
Acharya, S. M. et al. Fine scale sampling reveals early differentiation of rhizosphere microbiome from bulk soil in young Brachypodium plant roots. ISME Commun. 3, 1–9 (2023).
Huang, A. C. et al. A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science 364, eaau6389 (2019).
Levy, A. et al. Genomic features of bacterial adaptation to plants. Nat. Genet. 50, 138–150 (2018).
Lundberg, D. S. et al. Defining the core Arabidopsis thaliana root microbiome. Nature 488, 86–90 (2012).
Lebeis, S. L. et al. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Science 349, 860–864 (2015).
Pieterse, C. M. J. et al. Pseudomonas simiae WCS417: star track of a model beneficial rhizobacterium. Plant Soil 461, 245–263 (2021).
Berendsen, R. L. et al. Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417. BMC Genomics 16, 539 (2015).
Van Wees, S. C., Van der Ent, S. & Pieterse, C. M. Plant immune responses triggered by beneficial microbes. Curr. Opin. Plant Biol. 11, 443–448 (2008).
Yu, K. et al. Rhizosphere-associated pseudomonas suppress local root immune responses by gluconic acid-mediated lowering of environmental pH. Curr. Biol. 29, 3913–3920.e4 (2019).
Cole, B. J. et al. Genome-wide identification of bacterial plant colonization genes. PLOS Biol. 15, e2002860 (2017).
Stringlis, I. A. et al. Root transcriptional dynamics induced by beneficial rhizobacteria and microbial immune elicitors reveal signatures of adaptation to mutualists. Plant J. 93, 166–180 (2018).
Bjornson, M., Pimprikar, P., Nürnberger, T. & Zipfel, C. The transcriptional landscape of Arabidopsis thaliana pattern-triggered immunity. Nat. Plants 7, 579–586 (2021).
Verbon, E. H. et al. Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions. Mol. Plant 16, 1160–1177 (2023).
Humphrys, M. S. et al. Simultaneous transcriptional profiling of bacteria and their host cells. PLOS One 8, e80597 (2013).
Marsh, J. W., Humphrys, M. S. & Myers, G. S. A. A laboratory methodology for dual RNA-sequencing of bacteria and their host cells in vitro. Front. Microbiol. 8, 1830(2017).
Westermann, A. J., Barquist, L. & Vogel, J. Resolving host–pathogen interactions by dual RNA-seq. PLOS Pathog 13, e1006033 (2017).
Donaldson, G. P. et al. Spatially distinct physiology of Bacteroides fragilis within the proximal colon of gnotobiotic mice. Nat. Microbiol. 5, 746–756 (2020).
Andrés-Barrao, C. et al. Coordinated bacterial and plant sulfur metabolism in Enterobacter sp. SA187–induced plant salt stress tolerance. Proc. Natl. Acad. Sci. 118, e2107417118 (2021).
Vannier, N. et al. Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota. Nat. Commun. 14, 8274 (2023).
Kumar, N. et al. Efficient enrichment of bacterial mRNA from host-bacteria total RNA samples. Sci. Rep. 6, 34850 (2016).
Nobori, T. et al. Transcriptome landscape of a bacterial pathogen under plant immunity. Proc. Natl. Acad. Sci. 115, E3055–E3064 (2018).
Roux, B. et al. An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 77, 817–837 (2014).
Ma, Q., Bücking, H., Gonzalez Hernandez, J. L. & Subramanian, S. Single-cell RNA sequencing of plant-associated bacterial communities. Front. Microbiol. 10, 2452 (2019).
Johns, N. I. et al. Metagenomic mining of regulatory elements enables programmable species-selective gene expression. Nat. Methods 15, 323–329 (2018).
Jones, E. M. et al. A scalable, multiplexed assay for decoding GPCR-ligand interactions with RNA sequencing. Cell Syst. 8, 254–260.e6 (2019).
Crook, N., Ferreiro, A., Condiotte, Z. & Dantas, G. Transcript barcoding illuminates the expression level of synthetic constructs in E. coli nissle residing in the mammalian gut. ACS Synth. Biol. 9, 1010–1021 (2020).
Jores, T. et al. Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. Nat. Plants, https://doi.org/10.1038/s41477-021-00932-y (2021).
Wang, G. et al. CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nat. Microbiol. 4, 2498–2510 (2019).
Wang, B. et al. CRAGE-duet facilitates modular assembly of biological systems for studying plant–microbe interactions. ACS Synth. Biol. 9, 2610–2615 (2020).
Cho, B.-K. et al. The transcription unit architecture of the Escherichia coli genome. Nat. Biotechnol. 27, 1043–1049 (2009).
Wurtzel, O. et al. The single-nucleotide resolution transcriptome of pseudomonas aeruginosa grown in body temperature. PLOS Pathog 8, e1002945 (2012).
Filiatrault, M. J. et al. Genome-wide identification of transcriptional start sites in the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. PLOS ONE 6, e29335 (2011).
Salgado, H., Moreno-Hagelsieb, G., Smith, T. F. & Collado-Vides, J. Operons in Escherichia coli: genomic analyses and predictions. Proc. Natl. Acad. Sci. 97, 6652–6657 (2000).
Tjaden, B. A computational system for identifying operons based on RNA-seq data. Methods 176, 62–70 (2020).
Lugtenberg, B. J. J., Dekkers, L. & Bloemberg, G. V. Molecular determinants of rhizosphere colonization by pseudomonas. Annu. Rev. Phytopathol. 39, 461–490 (2001).
Muñoz-Elías, E. J. & McKinney, J. D. Carbon metabolism of intracellular bacteria. Cell. Microbiol. 8, 10–22 (2006).
Chen, H., Shiroguchi, K., Ge, H. & Xie, X. S. Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli. Mol. Syst. Biol. 11, 781 (2015).
Waters, L. S. & Storz, G. Regulatory RNAs in bacteria. Cell 136, 615–628 (2009).
Winnen, B., Hvorup, R. N. & Saier, M. H. The tripartite tricarboxylate transporter (TTT) family. Res. Microbiol. 154, 457–465 (2003).
Brocker, M., Schaffer, S., Mack, C. & Bott, M. Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA. J. Bacteriol. 191, 3869 (2009).
Finkel, O. M. et al. A single bacterial genus maintains root growth in a complex microbiome. Nature 587, 103–108 (2020).
De Weger, L. A. et al. Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots. J. Bacteriol. 169, 2769–2773 (1987).
Sivakumar, R. et al. Evaluation of INSeq To Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization. G3 GenesGenomesGenetics 9, 651–661 (2019).
Pankievicz, V. C. S. et al. RNA-seq transcriptional profiling of Herbaspirillum seropedicae colonizing wheat (Triticum aestivum) roots. Plant Mol. Biol. 90, 589–603 (2016).
Nobori, T. et al. Dissecting the cotranscriptome landscape of plants and their microbiota. EMBO Rep. 23, e55380 (2022).
WOJTASZEK, P. Oxidative burst: an early plant response to pathogen infection. Biochem. J. 322, 681–692 (1997).
Ebel, J. & Mithöfer, A. Early events in the elicitation of plant defence. Planta 206, 335–348 (1998).
Danhorn, T. & Fuqua, C. Biofilm formation by plant-associated bacteria. Annu. Rev. Microbiol. 61, 401–422 (2007).
Rudrappa, T., Biedrzycki, M. L. & Bais, H. P. Causes and consequences of plant-associated biofilms. FEMS Microbiol. Ecol. 64, 153–166 (2008).
Callewaert, L. et al. A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria. PLOS Pathog. 4, e1000019 (2008).
Yum, S. et al. Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria. Biochem. Biophys. Res. Commun. 378, 244–248 (2009).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Liu, X. et al. Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis. eLife 3, e01990 (2014).
Eitzen, K., Sengupta, P., Kroll, S., Kemen, E. & Doehlemann, G. A fungal member of the Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a GH25 lysozyme. eLife 10, e65306 (2021).
Lee, K.-M. et al. A genetic screen reveals novel targets to render pseudomonas aeruginosa sensitive to lysozyme and cell wall-targeting antibiotics. Front. Cell. Infect. Microbiol. 7, 59 (2017).
Torrens, G. et al. Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system. PLOS One 12, e0181932 (2017).
Flores-Cruz, Z. & Allen, C. Ralstonia solanacearum encounters an oxidative environment during tomato infection. Mol. Plant Microbe Interactions 22, 773–782 (2009).
Phelan, J. P., Bourgeois, J. S., McCarthy, J. E. & Hu, L. T. A putative xanthine dehydrogenase is critical for Borrelia burgdorferi survival in ticks and mice. Microbiology 169, 001286 (2023).
Sasse, J. et al. Root morphology and exudate availability are shaped by particle size and chemistry in Brachypodium distachyon. Plant Direct 4, e00207 (2020).
Palluk, S. et al. De novo DNA synthesis using polymerase-nucleotide conjugates. Nat. Biotechnol. 36, 645–650 (2018).
Plesa, C., Sidore, A. M., Lubock, N. B., Zhang, D. & Kosuri, S. Multiplexed gene synthesis in emulsions for exploring protein functional landscapes. Science 359, 343–347 (2018).
Sharma, C. M. et al. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464, 250–255 (2010).
Adiconis, X. et al. Comprehensive comparative analysis of 5′-end RNA-sequencing methods. Nat. Methods 15, 505–511 (2018).
Yu, K. et al. Transcriptome signatures in Pseudomonas simiae WCS417 shed light on role of root-secreted coumarins in arabidopsis-mutualist communication. Microorganisms 9, 575 (2021).
Canarini, A., Kaiser, C., Merchant, A., Richter, A. & Wanek, W. Root exudation of primary metabolites: mechanisms and their roles in plant responses to environmental stimuli. Front. Plant Sci. 10, 157 (2019).
Gliese, N., Khodaverdi, V., Schobert, M. & Görisch, H. AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933. Microbiology 150, 1851–1857 (2004).
Choi, O. et al. Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16. Plant Physiol. 146, 657–668 (2008).
Carreño-López, R., Alatorre-Cruz, J. M. & Marín-Cevada, V. Pyrroloquinoline quinone (PQQ): role in plant-microbe interactions. In Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms: Discovery and Applications (eds Singh, H. B., Keswani, C., Reddy, M. S., Sansinenea, E. & García-Estrada, C.) 169–184 (Springer, Singapore, 2019). https://doi.org/10.1007/978-981-13-5862-3_9.
Wang, X. et al. Elucidation of genes enhancing natural product biosynthesis through co-evolution analysis. Nat. Metab. 1–14, https://doi.org/10.1038/s42255-024-01024-9 (2024).
Boo, A. et al. Synthetic microbe-to-plant communication channels. Nat. Commun. 15, 1817 (2024).
Pini, F. et al. Bacterial biosensors for in vivo spatiotemporal mapping of root secretion. Plant Physiol. 174, 1289–1306 (2017).
Haskett, T. L. et al. Engineered plant control of associative nitrogen fixation. Proc. Natl. Acad. Sci. 119, e2117465119 (2022).
Toju, H. et al. Core microbiomes for sustainable agroecosystems. Nat. Plants 4, 247–257 (2018).
Law, C. W. et al. RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR. F1000Research 5, ISCB Comm J–1408 (2018).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47–e47 (2015).
Chen, I.-M. A. et al. IMG/M v.5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. Nucleic Acids Res. 47, D666–D677 (2019).
Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Acknowledgements
We thank Zhiying Zhao, Robert Evans, William Kauffman, Jan-Fang Cheng, Benjamin Cole, Yuko Yoshinaga, Vlastimil Novak, and Jeffery Dangle for helpful discussions and technical suggestions. We also thank QB3 Genomics Center at UC Berkeley and Azenta Life Sciences for sequencing. T.H. acknowledges a JSPS overseas research fellowship from the Japan Society for the Promotion of Science. Lawrence Berkeley National Laboratory is managed by University of California for DOE under contract number DE-AC02-05CH11231 (T.H. S.Y., D.M., L.B., Y.Y.). Argonne National Laboratory is managed by UChicago Argonne, LLC for DOE under contract number DE-AC02-06CH11357 (GB). This work was funded by the secure biosystem design (S.Y., G.B., Y.Y.) and the bioimaging programs (T.H., G.B., Y.Y.) of the U.S. Department of Energy, Office of Biological and Environmental Research under Contract No. DE-AC02-05CH11231 and DE-AC02-06CH11357. This work (proposal: 10.46936/10.25585/60001279), conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231 (T.H., D.M., L.B., Y.Y.). This project was also supported in part by the U. S. Department of Energy, Office of Science, through the Biomolecular Characterization and Imaging Sciences Program, Office of Biological and Environmental Research, under FWP 39156 (T.H., S.Y., Y.Y., G.B.). Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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T.H. and Y.Y. designed the study. T.H. performed the experiments, with contribution by S.Y., who helped prepare sequencing libraries and characterize mutant phenotypes. T.H. developed the computational pipeline. D.M. performed protein structural analysis. L.B. contributed to a plasmid construction and provided DAP-seq data. E.C. and S.Y. developed and performed Raman microscopy experiments. Y.Y. and G.B. supervised the study and acquired funding. T.H. and Y.Y. wrote the manuscript. All authors reviewed and approved the final version.
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Lawrence Berkeley National Laboratory has filed an international patent application related to high-throughput characterization of bacterial promoters from their host environments on behalf of the Regents of the University of California, on which T.H. and Y.Y. are the named inventors (PCT/US2023/031771). Y.Y. is a co-founder and has financial interest in Quorum Bio, Inc. The remaining authors declare no competing interests.
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Honda, T., Yu, S., Mai, D. et al. CRAGE-RB-PI-seq reveals transcriptional dynamics of plant-associated bacteria during root colonization. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69903-1
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DOI: https://doi.org/10.1038/s41467-026-69903-1
