Modular Bacterial Tag: a versatile bacterial tag for tracking of near-isogenic bacteria in plant microbiota

Published in Microbiology and Plant Science
Modular Bacterial Tag: a versatile bacterial tag for tracking of near-isogenic bacteria in plant microbiota
Like

Share this post

Choose a social network to share with, or copy the shortened URL to share elsewhere

This is a representation of how your post may appear on social media. The actual post will vary between social networks

The sum and diversity of all microorganisms colonizing a plant is referred to as the plant microbiota. When grown in the same soil and environment, the composition of the microbiota varies between different plant species and genotypes, indicative of co-diversification of the microbiota with its plant hosts2,3. Members of the microbiota form commensal relationships with and provide beneficial services to the plant host8, including mobilization of nutrients from soil for uptake by roots9,10, indirect pathogen protection11-13, and abiotic stress tolerance3,14.

Beneficial activities are often strain-specific and depend on the presence or absence of only a few gene products. To determine the composition of the plant microbiota, microbial marker genes characterized by alternating hypervariable and conserved regions are commonly sequenced and natural nucleotide polymorphisms are employed to define different microbial taxa.

However, microbial community profiling based on marker gene sequences is insufficient for resolving community structures to a level that distinguishes near-isogenic strains with differential activities. Further, as in natural environments plants are colonized by a multitude of different microorganisms simultaneously, the establishment of the microbiota is not only dependent on microbe–host, but also microbe–microbe interactions. Thus, while attempting to identify genetic determinants that render a bacterial strain a robust member of the root microbiota using strain-specific genetic variations and mutant libraries, we started to approach the problem of how the identified genetic determinants could later be characterized in complex microbial communities, such as those found in natural environments. Thereby, we can estimate the contribution of a particular gene encoded by a member of the microbiota not only to the colonization efficiency in general but also to the prevalence of this strain in competition with other microbes on roots.

We developed the Modular Bacterial Tag (MoBacTag) toolkit with the basic idea of labelling near-isogenic strains of interest by chromosomally integrating an DNA barcode as an artificial marker gene sequence that is co-amplified during community profiling. We decided to include four different antibiotic resistance cassettes, as strains of the Arabidopsis culture collection (At-SPHERE23) naturally encode diverse antibiotic resistances and we wished to design a toolkit that would be applicable for taxonomically diverse bacteria. As root zones differ not only in their physical properties, but also in their exudates and immune responses24, we further decided to include transcription units for fluorescent proteins that allow correlation of abundances of near-isogenic strains in community profiling with strain-specific colonization patterns. To achieve these goals, we opted for GoldenGate-based modular cloning that enabled us to combine different antibiotic cassettes and fluorescent markers and allows high flexibility for easy extensions or replacement of specific expression cassettes, antibiotic resistance markers or primer binding sites.

When the first MoBacTag vectors were assembled, corresponding author Paul Schulze-Lefert, co-first author Julien Thouin and I sat down and thought about how to test the tool in a community profiling experiment. We discussed two options for a proof-of-principle experiment using either a flagellum mutant, as motility is required for efficient root colonization25, or mutants of the plant growth-promoting rhizobacterium (PGPR) Pseudomonas capeferrum (P. capeferrum) WCS358, WCS358::pqqF and WCS358::cyoB, recently identified by the group of Roeland Berendsen, that are impaired in immunosuppression and root colonization26. PqqF and cyoB commonly function in the production of gluconic acid and its derivative 2-keto gluconic acid, which suppress plant immunity by lowering extracellular pH locally26. Suppression of host immune responses by pathogenic microbes invariably leads to increased pathogen proliferation27 raising the question of whether other microbiota members also benefit indirectly from compromised host defenses. Thus, we decided to use the immunosuppressive P. capeferrum mutants for our proof-of-principle experiment to potentially gain new insights into how immunosuppression changes a bacterial community on A. thaliana roots.

We were able to label P. capeferrum wild type and the cyoB and pqqF mutants and recapitulated the colonization defects of both mutants using the DNA barcode-specific reads during community profiling, thus demonstrating the functionality of our MoBacTag toolkit. Moreover, we observed a reduction of total bacterial load on A. thaliana roots specifically upon co-colonization with the pqqF mutant revealing distinct activities of pqqF and cyoB in a microbial community context. As the pqqF mutant is impaired in the biosynthesis of PQQ, a co-factor of several dehydrogenases including the glucose dehydrogenase involved in gluconic acid production, we propose that the overall reduced microbial load on roots in the presence of the pqqF mutant results from depletion of extracellular PQQ, which might serve as a common good during root microbiota establishment and is shared between microbiota members. Thus, by using the MoBacTags we were not only able to recapitulate published data for our proof-of-principle experiment but also unexpectedly observed a phenotype specific to the pqqF immunosuppressive mutant, which would have been difficult to detect using conventional colonization estimates of near-isogenic strains.

We will deposit the MoBacTag toolkit with AddGene to allow colleagues to label their favorite members of the plant microbiota.

Thanks to Neysan Donnelly and Paul Schulze-Lefert for feedback on this article.


References:

1              Massoni, J., Bortfeld-Miller, M., Widmer, A. & Vorholt, J. A. Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation. Proceedings of the National Academy of Sciences of the United States of America 118 (2021). https://doi.org:10.1073/pnas.2100150118

2              Wippel, K. et al. Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota. Nat Microbiol 6, 1150-1162 (2021). https://doi.org:10.1038/s41564-021-00941-9

3              Fitzpatrick, C. R. et al. Assembly and ecological function of the root microbiome across angiosperm plant species. Proceedings of the National Academy of Sciences of the United States of America 115, E1157-E1165 (2018). https://doi.org:10.1073/pnas.1717617115

4              Vorholt, J. A., Vogel, C., Carlstrom, C. I. & Muller, D. B. Establishing Causality: Opportunities of Synthetic Communities for Plant Microbiome Research. Cell Host Microbe 22, 142-155 (2017). https://doi.org:10.1016/j.chom.2017.07.004

5              Ma, K. W., Ordon, J. & Schulze-Lefert, P. Gnotobiotic Plant Systems for Reconstitution and Functional Studies of the Root Microbiota. Curr Protoc 2, e362 (2022). https://doi.org:10.1002/cpz1.362

6              Kremer, J. M. et al. Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research. Nat Protoc 16, 2450-2470 (2021). https://doi.org:10.1038/s41596-021-00504-6

7              Carlstrom, C. I. et al. Synthetic microbiota reveal priority effects and keystone strains in the Arabidopsis phyllosphere. Nat Ecol Evol 3, 1445-1454 (2019). https://doi.org:10.1038/s41559-019-0994-z

8              Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T. & Singh, B. K. Plant-microbiome interactions: from community assembly to plant health. Nat Rev Microbiol 18, 607-621 (2020). https://doi.org:10.1038/s41579-020-0412-1

9              Castrillo, G. et al. Root microbiota drive direct integration of phosphate stress and immunity. Nature 543, 513-518 (2017). https://doi.org:10.1038/nature21417

10           Harbort, C. J. et al. Root-Secreted Coumarins and the Microbiota Interact to Improve Iron Nutrition in Arabidopsis. Cell Host Microbe (2020). https://doi.org:10.1016/j.chom.2020.09.006

11           Berendsen, R. L., Pieterse, C. M. & Bakker, P. A. The rhizosphere microbiome and plant health. Trends in plant science 17, 478-486 (2012). https://doi.org:10.1016/j.tplants.2012.04.001

12           Duran, P. et al. Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival. Cell 175, 973-983 e914 (2018). https://doi.org:10.1016/j.cell.2018.10.020

13           Carrion, V. J. et al. Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome. Science (New York, N.Y 366, 606-612 (2019). https://doi.org:10.1126/science.aaw9285

14           Santos-Medellin, C. et al. Prolonged drought imparts lasting compositional changes to the rice root microbiome. Nat Plants 7, 1065-1077 (2021). https://doi.org:10.1038/s41477-021-00967-1

15           Hemmerle, L. et al. Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ. Nature Communications 13 (2022). https://doi.org:10.1038/s41467-022-30469-3

16           Berendsen, R. L. et al. Disease-induced assemblage of a plant-beneficial bacterial consortium. ISME J 12, 1496-1507 (2018). https://doi.org:10.1038/s41396-018-0093-1

17           Voges, M., Bai, Y., Schulze-Lefert, P. & Sattely, E. S. Plant-derived coumarins shape the composition of an Arabidopsis synthetic root microbiome. Proceedings of the National Academy of Sciences of the United States of America 116, 12558-12565 (2019). https://doi.org:10.1073/pnas.1820691116

18           Huang, A. C. et al. A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science (New York, N.Y 364 (2019). https://doi.org:10.1126/science.aau6389

19           Hu, L. et al. Root exudate metabolites drive plant-soil feedbacks on growth and defense by shaping the rhizosphere microbiota. Nat Commun 9, 2738 (2018). https://doi.org:10.1038/s41467-018-05122-7

20           Getzke, F. et al. Cofunctioning of bacterial exometabolites drives root microbiota establishment. Proceedings of the National Academy of Sciences of the United States of America 120, e2221508120 (2023). https://doi.org:10.1073/pnas.2221508120

21           Snelders, N. C. et al. Microbiome manipulation by a soil-borne fungal plant pathogen using effector proteins. Nat Plants 6, 1365-1374 (2020). https://doi.org:10.1038/s41477-020-00799-5

22           Snelders, N. C., Petti, G. C., van den Berg, G. C. M., Seidl, M. F. & Thomma, B. An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation. Proceedings of the National Academy of Sciences of the United States of America 118 (2021). https://doi.org:10.1073/pnas.2110968118

23           Bai, Y. et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528, 364-369 (2015). https://doi.org:10.1038/nature16192

24           Tsai, H. H., Wang, J., Geldner, N. & Zhou, F. Spatiotemporal control of root immune responses during microbial colonization. Current opinion in plant biology 74, 102369 (2023). https://doi.org:10.1016/j.pbi.2023.102369

25           Cole, B. J. et al. Genome-wide identification of bacterial plant colonization genes. PLoS biology 15, e2002860 (2017). https://doi.org:10.1371/journal.pbio.2002860

26           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 e3914 (2019). https://doi.org:10.1016/j.cub.2019.09.015

27           Ngou, B. P. M., Ding, P. T. & Jones, J. D. G. Thirty years of resistance: Zig-zag through the plant immune system. The Plant cell 34, 1447-1478 (2022). https://doi.org:10.1093/plcell/koac041

Please sign in or register for FREE

If you are a registered user on Research Communities by Springer Nature, please sign in

Subscribe to the Topic

Plant Immunity
Life Sciences > Biological Sciences > Plant Science > Plant Immunity
Bacterial Synthetic Biology
Life Sciences > Biological Sciences > Microbiology > Bacteria > Bacterial Synthetic Biology
Bacterial Genetics
Life Sciences > Biological Sciences > Microbiology > Bacteria > Bacterial Genetics