Non-invasive imaging of defence responses in plants

The plasmids used in this study are available for non-commercial use through Addgene [https://www.addgene.org/browse/article/28247826/]. Plant lines will be made available under an MTA upon request. Metabolomics data are available at MetaboLights repository⁵⁰ under accession MTBLS13465. Raw imaging data is available at Figshare [https://doi.org/10.6084/m9.figshare.30911858]. Source data are provided with this paper.

Python code for processing and plotting data is available at Github [https://github.com/Perfus/BL_plant_hormones].

1. Peng, Y., Yang, J., Li, X. & Zhang, Y. Salicylic acid: biosynthesis and signaling. Annu. Rev. Plant Biol. 72, 761–791 (2021).

   Google Scholar 

2. Onkokesung, N. et al. Jasmonic acid and ethylene modulate local responses to wounding and simulated herbivory in Nicotiana attenuata leaves. Plant Physiol. 153, 785–798 (2010).

   Google Scholar 

3. Thaler, J. S., Humphrey, P. T. & Whiteman, N. K. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 17, 260–270 (2012).

   Google Scholar 

4. Gimenez-Ibanez, S., Zamarreño, A. M., García-Mina, J. M. & Solano, R. An evolutionarily ancient immune system governs the interactions between Pseudomonas syringae and an early-diverging land plant lineage. Curr. Biol. 29, 2270–2281.e4 (2019).

   Google Scholar 

5. Xu, H.-X. et al. A salivary effector enables whitefly to feed on host plants by eliciting salicylic acid-signaling pathway. Proc. Natl. Acad. Sci. USA 116, 490–495 (2019).

   Google Scholar 

6. Li, N., Han, X., Feng, D., Yuan, D. & Huang, L.-J. Signaling crosstalk between salicylic acid and ethylene/jasmonate in plant defense: Do we understand what they are whispering? Int. J. Mol. Sci. 20, 671 (2019).

   Google Scholar 

7. Hou, S. & Tsuda, K. Salicylic acid and jasmonic acid crosstalk in plant immunity. Essays Biochem. 66, 647–656 (2022).

   Google Scholar 

8. Waadt, R., Hsu, P.-K. & Schroeder, J. I. Abscisic acid and other plant hormones: methods to visualize distribution and signaling. Bioessays 37, 1338–1349 (2015).

   Google Scholar 

9. Isoda, R. et al. Sensors for the quantification, localization and analysis of the dynamics of plant hormones. Plant J. 105, 542–557 (2021).

   Google Scholar 

10. Engelberth, M. J. & Engelberth, J. Monitoring plant hormones during stress responses. J. Vis. Exp. 28, 1127 (2009).

    Google Scholar 

11. Kaku, T., Sugiura, K., Entani, T., Osabe, K. & Nagai, T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci. Rep. 11, 14994 (2021).

    Google Scholar 

12. Kusuma, S. H., Hattori, M. & Nagai, T. Autonomous multicolor bioluminescence imaging in bacteria, mammalian, and plant hosts. Proc. Natl. Acad. Sci. USA 121, e2406358121 (2024).

    Google Scholar 

13. Khakhar, A. et al. Building customizable auto-luminescent luciferase-based reporters in plants. eLife 9, e52786 (2020).

    Google Scholar 

14. Gregor, C. et al. Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system. Proc. Natl. Acad. Sci. USA 116, 26491–26496 (2019).

    Google Scholar 

15. Gregor, C., Gwosch, K. C., Sahl, S. J. & Hell, S. W. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc. Natl. Acad. Sci. USA 115, 962–967 (2018).

    Google Scholar 

16. Mitiouchkina, T. et al. Plants with genetically encoded autoluminescence. Nat. Biotechnol. 38, 944–946 (2020).

    Google Scholar 

17. Close, D. M., Xu, T., Sayler, G. S. & Ripp, S. In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals. Sensors 11, 180–206 (2011).

    Google Scholar 

18. Ge, J. et al. Integration of biological and information technologies to enhance plant autoluminescence. Plant Cell 36, 4703–4715 (2024).

    Google Scholar 

19. Kotlobay, A. A. et al. Genetically encodable bioluminescent system from fungi. Proc. Natl. Acad. Sci. USA 115, 12728–12732 (2018).

    Google Scholar 

20. Purtov, K. V. et al. The chemical basis of fungal bioluminescence. Angew. Chem. Int. Ed. Engl. 54, 8124–8128 (2015).

    Google Scholar 

21. Kaskova, Z. M. et al. Mechanism and color modulation of fungal bioluminescence. Sci. Adv. 3, e1602847 (2017).

    Google Scholar 

22. Zheng, P. et al. Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol. J. 21, 1671–1681 (2023).

    Google Scholar 

23. Shakhova, E. S. et al. An improved pathway for autonomous bioluminescence imaging in eukaryotes. Nat. Methods 21, 406–410 (2024).

    Google Scholar 

24. Palkina, K. A. et al. A hybrid pathway for self-sustained luminescence. Sci. Adv. 10, eadk1992 (2024).

    Google Scholar 

25. Moreno-Giménez, E., Selma, S., Calvache, C. & Orzáez, D. GB_SynP: A modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants. ACS Synth. Biol. 11, 3037–3048 (2022).

    Google Scholar 

26. Calvache, C., Vazquez-Vilar, M., Moreno-Giménez, E. & Orzaez, D. A quantitative autonomous bioluminescence reporter system with a wide dynamic range for plant synthetic biology. Plant Biotechnol. J. 22, 37–47 (2024).

    Google Scholar 

27. Montiel, G., Zarei, A., Körbes, A. P. & Memelink, J. The jasmonate-responsive element from the ORCA3 promoter from Catharanthus roseus is active in Arabidopsis and is controlled by the transcription factor AtMYC2. Plant Cell Physiol. 52, 578–587 (2011).

    Google Scholar 

28. Van Der Fits, L. & Memelink, J. The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J. 25, 43–53 (2001).

    Google Scholar 

29. Lehmann, S. et al. Novel markers for high-throughput protoplast-based analyses of phytohormone signaling. PLoS One 15, e0234154 (2020).

    Google Scholar 

30. Nagata, T. Hidden history of the tobacco BY-2 cell line. J. Plant Res. 136, 781–786 (2023).

    Google Scholar 

31. Li, J., Brader, G. & Palva, E. T. The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16, 319–331 (2004).

    Google Scholar 

32. Raman, V. et al. Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses. Sci. Rep. 9, 13503 (2019).

    Google Scholar 

33. Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227 (2005).

    Google Scholar 

34. Uppalapati, S. R. et al. The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. Mol. Plant Microbe Interact. 20, 955–DC3965 (2007).

    Google Scholar 

35. Smith-Becker, J. et al. Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems1. Plant Physiol. 116, 231–238 (1998).

    Google Scholar 

36. Gao, Y. et al. Pseudomonas syringae activates ZAT18 to inhibit salicylic acid accumulation by repressing EDS1 transcription for bacterial infection. N. Phytol. 233, 1274–1288 (2022).

    Google Scholar 

37. Zayed, M. S., Taha, E.-K. A., Hassan, M. M. & Elnabawy, E.-S. M. Enhance systemic resistance significantly reduces the silverleaf whitefly population and increases the yield of sweet pepper, Capsicum annuum L. var. annuum. Sustain. Sci. Pract. Policy 14, 6583 (2022).

    Google Scholar 

38. Murugan, M. & Dhandapani, N. Induced systemic resistance activates defense responses to interspecific insect infestations on tomato. J. Veg. Sci. 12, 43–62 (2007).

    Google Scholar 

39. Ruan, J. et al. Jasmonic acid signaling pathway in plants. Int. J. Mol. Sci. 20, 2479 (2019).

    Google Scholar 

40. Huang, W. E. et al. Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1. Plant J. 46, 1073–1083 (2006).

    Google Scholar 

41. Lebel, E. et al. Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis. Plant J. 16, 223–233 (1998).

    Google Scholar 

42. Sarrion-Perdigones, A. et al. GoldenBraid 2.0: A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).

    Google Scholar 

43. Fernandez-Moreno, J.-P. et al. A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters. Plant Biotechnol. J. 22, 1942–1956 (2024).

    Google Scholar 

44. Alonso, J. M. & Stepanova, A. N. Arabidopsis transformation with large bacterial artificial chromosomes. Methods Mol. Biol. 1062, 271–283 (2014).

    Google Scholar 

45. Lazo, G. R., Stein, P. A. & Ludwig, R. A. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9, 963–967 (1991).

    Google Scholar 

46. Nagata, T., Nemoto, Y. & Hasezawa, S. Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Int. Rev. Cytol. 132, 1–30 (1992).

    Google Scholar 

47. Gengenbach, B. B., Opdensteinen, P. & Buyel, J. F. Robot cookies – plant cell packs as an automated high-throughput screening platform based on transient expression. Front. Bioeng. Biotechnol. 8, 393 (2020).

    Google Scholar 

48. Clough, S. J. & Bent, A. F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Google Scholar 

49. Scikit-posthocs. PyPI https://pypi.org/project/scikit-posthocs/.

50. Yurekten, O. et al. MetaboLights: open data repository for metabolomics. Nucleic Acids Res. 52, D640–D646 (2024).

    Google Scholar 

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Author notes

1. These authors contributed equally: Anastasia V. Balakireva, Tatiana A. Karataeva, Michael Karampelias, Tatiana Yu Mitiouchkina, Viktor V. Morozov, Jan Petrášek, Alexander S. Mishin, Karen S. Sarkisyan.

Authors and Affiliations

1. Planta LLC, Moscow, Russia

   Anastasia V. Balakireva, Tatiana A. Karataeva, Tatiana Yu Mitiouchkina, Ekaterina S. Shakhova, Maxim M. Perfilov, Olga A. Belozerova, Kseniia A. Palkina, Liliia I. Fakhranurova, Nadezhda M. Markina, Evgenia N. Bugaeva, Galina M. Delnova, Vladimir V. Choob, Ilia V. Yampolsky & Alexander S. Mishin

2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

   Anastasia V. Balakireva, Tatiana A. Karataeva, Tatiana Yu Mitiouchkina, Viktor V. Morozov, Ekaterina S. Shakhova, Maxim M. Perfilov, Olga A. Belozerova, Sergey I. Kovalchuk, Kseniia A. Palkina, Liliia I. Fakhranurova, Nadezhda M. Markina, Dmitry A. Gorbachev, Ilia V. Yampolsky & Alexander S. Mishin

3. Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czechia

   Michael Karampelias, Jan Macháček, Nikola Drážná, Zuzana Vondráková, Karel Müller, Tetiana Kalachova & Jan Petrášek

4. Synthetic Biology Group, MRC London Institute of Medical Sciences, London, UK

   Aubin Fleiss & Karen S. Sarkisyan

5. Institute of Clinical Sciences, Faculty of Medicine, and Imperial College Centre for Engineering Biology, Imperial College London, London, UK

   Aubin Fleiss & Karen S. Sarkisyan

6. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA

   Josefina Patricia Fernandez-Moreno, Jose M. Alonso & Anna N. Stepanova

7. Departamento de Bioquímica y Biología Molecular, Campus Universitario de Rabanales y Campus de Excelencia Internacional Agroalimentario 3, Universidad de Córdoba, 14071, Cordoba, Spain

   Josefina Patricia Fernandez-Moreno

8. Botanical Garden of Lomonosov Moscow State University, Moscow, Russia

   Vladimir V. Choob

9. Pirogov Russian National Research Medical University, Moscow, Russia

   Ilia V. Yampolsky

10. Light Bio Inc., Ketchum, ID, USA

    Ilia V. Yampolsky & Karen S. Sarkisyan

Contributions

Performed experiments: T.A.K., E.S.S., K.A.P., T.Y.M., K.M., V.V.M., M.K., N.M.M., L.I.F., G.M.D., O.A.B., S.I.K., E.N.B., Z.V., K.M., T.K., J.M., N.D., J.P.F.M., and D.A.G. Planned experiments: A.V.B., T.A.K., E.S.S., K.A.P., T.Y.M., M.K., K.S.S., A.S.M., V.V.C., J.P.F.M., J.M.A., and A.N.S. Analysed data: M.M.P., A.V.B., A.F., K.S.S., A.S.M., V.V.C., J.P.F.M., J.M.A., A.N.S., and M.K. Proposed and directed the study: K.S.S., M.K., J.P., J.M.A., A.N.S., A.S.M., and I.V.Y. Reviewed the paper draft: all authors.

Corresponding authors

Correspondence to Jan Petrášek, Alexander S. Mishin or Karen S. Sarkisyan.

Competing interests

A.V.B., T.A.K., T.Y.M., E.S.S., M.M.P., O.A.B., K.A.P., L.I.F., N.M.M.,. E.N.B., G.M.D., V.V.C., I.V.Y., and A.S.M. are employed by Planta LLC (https://planta.bio/). K.S.S. and I.V.Y. are employed by Light Bio Inc. (https://light.bio/). Other authors don’t claim competing interests.

Peer review information

Nature Communications thanks Hao Du and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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