Skip to main content
Download PDF

Protocol: a simple method for biosensor visualization of bacterial quorum sensing and quorum quenching interaction on Medicago roots

Plant Methods volume 18, Article number: 112 (2022) Cite this article

Abstract

Background

Defining interactions of bacteria in the rhizosphere (encompassing the area near and on the plant root) is important to understand how they affect plant health. Some rhizosphere bacteria, including plant growth promoting rhizobacteria (PGPR) engage in the intraspecies communication known as quorum sensing (QS). Many species of Gram-negative bacteria use extracellular autoinducer signal molecules called N-acyl homoserine lactones (AHLs) for QS. Other rhizobacteria species, including PGPRs, can interfere with or disrupt QS through quorum quenching (QQ). Current AHL biosensor assays used for screening and identifying QS and QQ bacteria interactions fail to account for the role of the plant root.

Methods

Medicago spp. seedlings germinated on Lullien agar were transferred to soft-agar plates containing the broad-range AHL biosensor Agrobacterium tumefaciens KYC55 and X-gal substrate. Cultures of QS and QQ bacteria as well as pure AHLs and a QQ enzyme were applied to the plant roots and incubated for 3 days.

Results

We show that this expanded use of an AHL biosensor successfully allowed for visualization of QS/QQ interactions localized at the plant root. KYC55 detected pure AHLs as well as AHLs from live bacteria cultures grown directly on the media. We also showed clear detection of QQ interactions occurring in the presence of the plant root.

Conclusions

Our novel tri-trophic system using an AHL biosensor is useful to study QS interspecies interactions in the rhizosphere.

Background

Dissecting the interactions within the rhizo-microbiome is a major focus for defining the activities of plant beneficial bacteria. Plant growth promoting rhizobacteria (PGPR) have long been studied individually and described by their specific plant growth promoting traits (e.g., antibiotic, siderophore or secondary metabolite production) [1, 2]. One feature of interest is the widespread bacterial intraspecies communication utilizing extracellular signal molecules known as quorum sensing (QS). Populations of bacterial cells coordinate community level activities in a conditionally responsive manner to produce extracellular autoinducer (AI) molecules. These AIs modulate transcription of genes (via transcriptional regulators) for behaviors ranging from motility, biofilm formation and virulence [3].

The most comprehensively studied family of QS AI signals are the N-acyl homoserine lactones (AHLs) produced by a variety of different Gram-negative bacteria [4]. AHL signal molecules are uniquely recognizable due to their differing N-acyl chain lengths, degrees of saturation, and various substitutions on the 3rd carbon [5]. AHL-based QS is recognized as having an influence on other species, families and kingdoms of organisms occupying the rhizosphere [6,7,8,9]. As such, QS is a valuable target for research on interactions in the rhizosphere.

In the rhizosphere, AHLs are ubiquitous, but generally short-lived [10]; genera of known AHL producing bacteria include Pseudomonas, Rhizobium and Sinorhizobium [11]. The transient nature of AHLs is likely attributable to enzymatic degradation mechanisms found in both AHL and non-AHL producing bacteria [12]. Quorum quenching (QQ) of AHL signal molecules was first identified in a Bacillus sp. through the lactonase enzyme AiiA [13] which cleaves the lactone ring. QQ has been shown to inhibit both the virulence of plant pathogens [14,15,16] and the QS controlled nodulation efficiency of symbiotic Sinorhizobium meliloti [17, 18]. The AHLs of plant pathogens have been proposed as specific targets through QQ as a biocontrol mechanism either through PGPR application or transgenic plant expression of lactonases [6, 19, 20].

QS and the critical role of AHL signaling in controlling bacterial activities is of great interest across the spectrum of health and environmental sciences. As such, constructs of bacteria which can sense and report the presence of AHLs (without producing endogenous AHLs) have been developed. AHL biosensors are useful for detection, localization, and relative quantification of AHLs in situ and in vivo. Markers implemented in these biosensors include naturally occurring products of QS as in the case for Chromobaterium violaceum CV026 (pigment violacein) and introduction of luxCDABE into Escherichia coli from Vibrio fisheri [21, 22]. Other AHL biosensors are constructed in hosts such as E. coli, S. melilotii and Agrobacterium tumefaciens through fusions of uidA (ß-glucuronidase), lacZ (β-galactosidase) and gfp to AHL response elements from a variety of different QS bacteria [23,24,25,26,27,28].

Biosensors have proved to be particularly useful in querying QS and QQ in the rhizosphere microbiome. Different groups have used biosensors to identify AHL producing bacteria in the rhizospheres of Avena (wild oats) Arachis hypogaea (peanut), and Populus deltoides (Eastern cottonwood tree) [29,30,31]. Biosensors are also used to identify specific AHLs produced by PGPR [32] and to screen root isolates for QQ activity [11, 33, 34]. A recent protocol from Begum et al. [35] presents a more streamlined approach to screening for AHLs by placing detached rice root samples collected from the field directly on agar containing C. violaceum CV026 and A. tumefaciens NTL1 AHL biosensors [35].

Using biosensors has several advantages in screening rhizosphere isolates for AHL production and degradation. As noted by Shaw et al. [36], AHL biosensors allow for rapid, sensitive detection of AHL molecules without the need for specialized equipment such as a mass spectrometer, spectrophotometer, or fluorescent microscope [36]. One disadvantage has been the requirement for multiple different biosensor strains to adequately encompass detection of the large range of different AHL molecules produced by rhizobacteria. Researchers have used combinations such as C. violaceum CV026 for short chain detection and either C. violaceum VIR07 [33] or A. tumefaciens NTLR4 for long chain detection [30, 32]. This approach increases the time and complexity of AHL biosensor protocols.

AHL biosensors are clearly a powerful tool for identification of QS bacteria and characterizing the types of AHLs produced. However, the full potential of this technique could be expanding by using AHL biosensors to observe interspecies interactions between different bacteria (e.g., QQ) and between QS bacteria and live plant roots. Recent work by [18] demonstrated QQ of S. meliloti AHLs by B. subtilis UD1022, likely through the lactonase enzyme YtnP [18]. Using the same bacterial interactions, we present a protocol using a single biosensor strain suitable for visually assessing the presence of QS and QQ bacteria on Medicago truncatula A17 plant roots. This technique represents a potentially valuable tool for observing QS and QQ bacterial interactions in the rhizosphere as it incorporates the presence of live plant roots.

Methods

Preparation of seeds and seedling growth

Seeds of M. truncatula A17 ‘Jemalong’ were scarified in sulfuric acid for 6 min and sterilized in 70% ethanol for 1 min and 3% bleach for 10 min, rinsing thoroughly between each solution. Seeds were resuspended in sterile water and placed on shaker at room temperature for 4 h, rinsing and replacing water every hour. After the final rinse seeds were resuspended in sterile water and placed in 4 °C for 48 h. Seeds were again rinsed and placed on sterile, empty 120 × 120 mm2 plates, sealed with Parafilm® (Bemis Company, Inc.) and germinated vertically in dark conditions for 24 h. Seedlings were transferred onto Lullien medium [37] agar (25 ml of 1.3% agar) 120 × 120 mm2 plates and sealed with micropore tape. The ‘root’ portion of the plates were wrapped in foil and placed vertically in a growth chamber (22 °C, 16:8 light/dark cycle) for 24 h. To reduce contamination, seed coats were gently removed with sterile tweezers; plates were resealed with micropore tape and grown for 2 more days.

Preparation of bacteria for inoculation

QS strain

All strains used in this study are listed in Table 1. Rm8530 carrying a plasmid resistant to spectinomycin was grown on TYC agar (5 g of tryptone, 3 g yeast extract, and 0.4 g of CaCl2/liter) with 50 µg ml−1 spectinomycin for 4 days at 28 °C. Colonies were re-suspended in 5 mL TYC with spectinomycin to OD600 ~ 0.5 and grown shaking for 3 h. Cells were washed once, resuspended in sterile water and diluted to OD600 = 0.2 in preparation for inoculation onto plant roots.

Table 1 Bacterial strains used in this study
Full size table

QQ strain

Spectinomycin resistance was introduced into the PGPR B. subtilis UD1022 (‘UD1022 ycbU’) through SPP1 phage transduction [38]. UD1022 ycbU was streaked onto LB agar plates with 100 µg ml−1 spectinomycin and grown 24 h at 37 °C. Colonies were re-suspended in 5 ml LB with 100 µg ml−1 spectinomycin and grown shaking for 3 h. Cells were washed once, resuspended in sterile water and diluted to OD600 = 1.0 in preparation to inoculate onto plant roots.

Biosensor strain

A. tumefaciens ‘KYC55’ pre-induced cells [39] were inoculated into minimal glutamate mannitol (MGM) [40] broth and grown shaking at 28 °C for 24 h. KYC55 cells were used to make X-gal soft agar following the protocol of Joelsson and Zhu [41] with the following modifications: addition of final concentration 0.5 µM AVG (2- aminoethoxyvinyl glycine, ethylene inhibitor, Sigma) and 50 µg ml−1 spectinomycin to MGM-based agar medium. AVG is a common additive to M. truncatula growth medium to prevent inhibition of nodule development by plant produced ethylene [42, 43]. Twenty-five ml KYC55 soft agar was aliquoted per 120 × 120 mm2 plate and allowed to dry 30 min.

Assembly of the biosensor growth plates

Three-day old M. truncatula seedlings were transferred from Lullien medium plates to freshly poured KYC55 with 50 µg ml−1 spectinomycin and 0.5 µM AVG soft agar plates (5 seedlings per plate). Ten microliter drops of UD1022 ycbU were pipetted 1 cm below the root tips (‘UD1022 and Rm8530’ treatments) and allowed to dry. Ten microliter drops of Rm8530-spec were pipetted onto appropriate treatments (‘Rm8530’ or ‘UD1022 and Rm8530’ treatments). Sterile water was used for ‘control’ treatments.

Lactonase and oxo-C16 AHL experiments

Purified UD1022 YtnP lactonase protein (UNC School of Medicine) was diluted in sterile phosphate buffered saline (PBS) (8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4/liter) to 100 µg ml−1. Aliquots were split with one set subjected to ‘heat treatment’ at 100 °C for 15 min to inactivate the enzyme activity. Ten microliters of ‘YtnP’ and ‘YtnP heat killed (HK)’ were pipetted 1 cm below the root tip. N-3-oxo-hexadecanoyl-L-Homoserine lactone (‘oxo-C16 AHL), Cayman Chemical, was selected as it is identified as one of a suite of AHLs produced by S. meliloti [44]. Oxo-C16 AHL was resuspended in methanol and diluted in sterile PBS to 10 µM. Ten microliter drops were pipetted 1 cm below the root tip on top of the dried drop of ‘YtnP’ or ‘YtnP HK’. All treatments included one plate with bacteria or ‘YtnP’ treatments only (without plants). Three replicate plates were included per treatment and experiments were repeated three times. Biosensor-plant treatment plates were incubated for 3 days in the growth chamber vertically as described in seedling growth. Six day after germination (DAG) seedlings on biosensor plates were documented through photography.

Results and discussion

This protocol presents a significant advancement in the application of an AHL biosensor due to its effectiveness in visualizing interspecies QS and QQ interactions on the plant root. QS is important in interspecies communication between plant and bacteria and QQ is a potential mechanism for plant pathogen biocontrol [6]. The goal of this protocol was to incorporate the antibiotic spectinomycin to control for the influence of contaminating bacteria as well as enabling the visualization of QS/QQ interactions on plant roots. The biosensor A. tumefaciens KYC55 has multiple AHL response elements, allowing for the detection of a broad range of AHLs. The plasmids of KYC55 encoding the multiple AHL response elements also confer antibiotic resistance for spectinomycin, gentamycin and tetracycline [39]. Though spectinomycin has been reported to bleach M. sativa cultured cells [45], in this protocol, plants were initially grown without the antibiotic to ensure vigor. We observed no deleterious effects of 50 µg ml−1 spectinomycin to Medicago spp. for the short length of time required to carry out the experiment. The addition of spectinomycin to the medium was tested without the presence of plants to validate the ability of the biosensor to respond similarly to QS signal molecules as without antibiotics (Additional file 1: Figure S1). Modifications of this method could include introducing gentamycin or tetracycline resistance to bacteria of interest due to the multi-antibiotic resistance of the KYC55 strain.

Application of the PGPR UD1022 and the symbiont Rm8530 to Medicago root zones on a substrate containing the biosensor KYC55 successfully visually reflected the presence of QS signals and QQ activity (Fig. 1). Further, this technique allowed for the visualization of YtnP lactonase activity in the presence of live M. truncatula roots (Fig. 2). Recent work by [46] describes differences in M. truncatula root nodulation in response to AHLs depending on whether the field-sourced seeds were sterilized with a suite of antibiotics [46]. The potential QQ of seed-borne bacteria in their system could be visualized through replicating such experiments on KYC55 as described in this protocol.

Fig. 1
figure 1

Three day after germination (DAG) M. truncatula and bacteria on KYC55 biosensor plates co-incubated with treatment for 3 days. Seedlings are 6 DAG in figures. a Control plants without bacteria b Rm8530 applied to root zone and KYC55 response to Rm8530 AHLs c KYC55 response indicating quorum quenching of Rm8530 by UD1022

Full size image
Fig. 2
figure 2

Six day after germination (DAG) M. truncatula seedlings and YtnP lactonase on KYC55 plates. a Rm8530 and 100 µg ml−1 heat killed YtnP lactonase b Rm8530 and 100 µg ml−1 YtnP lactonase

Full size image

Our protocol would be easily adapted to elucidate the QS and QQ interactions of rhizosphere bacteria on plant roots and for evaluating direct plant or root influence on QS bacteria. By omitting the antibiotic this protocol could be used to evaluate natural plant microbiomes from field grown plants. The advantage of using KYC55 is its ability to detect a broad range of AHLs with both short and long chain lengths as well as 3-oxo derivatives [39]. Previous works would often implement two different biosensor strains to fully profile the AHLs produced [30, 32, 33], whereas here, a larger spectrum of naturally occurring QS bacteria may be detected. This technique could also be used in to observe QS/QQ interactions of synthetic rhizo-microbiome consortia from mesocosm studies by designing the bacterial strains to have spectinomycin resistance.

Incorporating the whole plant in this assay allows for further research on both the response of plants or roots to QS bacteria or AHLs and to observe potential plant influences. Many studies over the past two decades have shown plants respond to AHLs, independent of the presence of bacteria, beneficial or otherwise [8]. Roots of Arabidopsis thaliana were elongated in the presence of short-chain AHLs [47]. Addition of long-chain AHLs significantly increased the number of symbiotic root nodules formed by the symbiotic mutualist S. meliloti on its legume host M. truncatula [48]. Plant responses to pathogens has also been shown to be mediated by AHLs. Plant defense mechanisms such as ROS and SA accumulation were found to be primed in A. thaliana pre-treated with long-chain and short-chain AHLs prior to pathogen challenge [8, 49, 50]. Applying this protocol into the experimental design for plant responses to AHLs could enhance the understanding of spatial and temporal interactions of AHLs with the plant root while also allowing for observations of root phenotypes.

Similarly, this protocol would be useful for observing potential plant effects such as QQ or quorum interference (QI) on QS rhizobacteria and their AHLs. Several AHL ‘mimic’ molecules have been described as having an influence on bacteria in the rhizosphere. The marine algae Delisea pulchra was shown to produce halogenated furones which can occupy the active site of LuxR protein AHL receptors and disrupt QS [51]. Calatrava-Morales et al. [52] reported positive responses of suite of AHL bioreporters (not including KYC55) to pea root extracts, possibly detecting substances which could competitively inhibit AHLs [52]. Substances such as L-canavanine found in alfalfa and legume root exudates and rosmarinic acid are also reported as AHL mimics inhibiting QS [53, 54]. Many other plant natural products and phytochemicals, including phytohormones have also been reported to interfere with bacterial QS [19, 55,56,57,58,59,60,61]. In this system, we observed no detectable plant derived quorum signals (Fig. 1a) or quenching of S. meliloti AHL signal (Additional file 2: Figure S2). Adaptation of other AHL biosensors, AHLs or plants could also be incorporated into this assay to evaluate plant root influences on QS signals or QS rhizobacteria. This method could also be suitable for evaluating transgenic plants expressing QQ enzymes [14, 62, 63], a technique proposed for control of QS plant pathogens.

Although the use of various mass spectroscopy and HPLC techniques [64,65,66] for AHL detection and identification is the gold standard, the relatively simple visual assays using AHL biosensors are most useful for screening and validation [67]. Here we demonstrate an effective protocol based on the foundations of AHL biosensor assays and could be applied toward multiple different lines of research. The comprehensive AHL responsiveness of the AHL biosensor KYC55 combined with the advantages of the low requirement for technical equipment and the ability to broadly observe interspecies interactions makes this protocol extremely useful for the active field of QS research in the rhizosphere.

Availability of data and materials

Original data and materials used in this work are freely available upon request.

References

  1. Gamalero E, Glick BR. Mechanisms used by plant growth-promoting bacteria. In: Maheshwari DK, editor. Bacteria in agrobiology: plant nutrient management. Berlin: Springer; 2011. p. 17–46.

    Chapter  Google Scholar 

  2. Kloepper JW, Leong J, Teintze M, Schroth MN. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature. 1980;286:885–6.

    Article  CAS  Google Scholar 

  3. Bassler BL, Miller MB. Quorum sensing. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F, editors. The Prokaryotes. Berlin: Springer; 2013. p. 495–509.

    Chapter  Google Scholar 

  4. Whiteley M, Diggle SP, Greenberg EP. Progress in and promise of bacterial quorum sensing research. Nature. 2017;551:313–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chernin LS. Quorum-sensing signals as mediators of PGPRs’ beneficial traits. In: Maheshwari DK, editor. Bacteria in agrobiology: plant nutrient management. Berlin: Springer; 2011. p. 209–36.

    Chapter  Google Scholar 

  6. Hartmann A, Klink S, Rothballer M. Importance of N-acyl-homoserine lactone-based quorum sensing and quorum quenching in pathogen control and plant growth promotion. Pathogens. 2021;10:1561.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mosquito S, Meng X, Devescovi G, Bertani I, Geller AM, Levy A, et al. LuxR solos in the plant endophyte Kosakonia sp. strain KO348. Appl Environ Microbiol. 2020;86:e00622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shrestha A, Schikora A. AHL-priming for enhanced resistance as a tool in sustainable agriculture. FEMS Microbiol Ecol. 2020. https://doi.org/10.1093/femsec/fiaa226.

    Article  PubMed  Google Scholar 

  9. Wellington S, Greenberg EP. Quorum sensing signal selectivity and the potential for interspecies cross talk. MBio. 2019. https://doi.org/10.1128/mBio.00146-19.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ortiz-Castro R, López-Bucio J. Review: phytostimulation and root architectural responses to quorum-sensing signals and related molecules from rhizobacteria. Plant Sci. 2019;284:135–42.

    Article  CAS  PubMed  Google Scholar 

  11. d’Angelo-Picard C, Faure D, Penot I, Dessaux Y. Diversity of N-acyl homoserine lactone-producing and-degrading bacteria in soil and tobacco rhizosphere. Environ Microbiol. 2005;7:1796–808.

    Article  PubMed  CAS  Google Scholar 

  12. Grandclément C, Tannières M, Moréra S, Dessaux Y, Faure D. Quorum quenching: role in nature and applied developments. FEMS Microbiol Rev. 2016;40:86–116.

    Article  PubMed  CAS  Google Scholar 

  13. Dong YH, Xu JL, Li XZ, Zhang LH. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci USA. 2000;97:3526–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature. 2001;411:813–7.

    Article  CAS  PubMed  Google Scholar 

  15. Li Q, Ni H, Meng S, He Y, Yu Z, Li L. Suppressing Erwinia carotovora pathogenicity by projecting N-acyl homoserine lactonase onto the surface of Pseudomonas putida cells. J Microbiol Biotechnol. 2011;21:1330–5.

    Article  CAS  PubMed  Google Scholar 

  16. Mahmoudi E, Naderi D, Venturi V. AiiA lactonase disrupts N-acylhomoserine lactone and attenuates quorum-sensing-related virulence in Pectobacterium carotovorum EMPCC. Ann Microbiol. 2013;63:691–7.

    Article  CAS  Google Scholar 

  17. Gao M, Chen H, Eberhard A, Gronquist MR, Robinson JB, Connolly M, et al. Effects of AiiA-mediated quorum quenching in Sinorhizobium meliloti on quorum-sensing signals, proteome patterns, and symbiotic interactions. Mol Plant Microbe Interact. 2007;20:843–56.

    Article  CAS  PubMed  Google Scholar 

  18. Rosier A, Beauregard PB, Bais HP. Quorum quenching activity of the PGPR Bacillus subtilis UD1022 alters nodulation efficiency of Sinorhizobium meliloti on Medicago truncatula. Front Microbiol. 2020;11: 596299.

    Article  PubMed  Google Scholar 

  19. Helman Y, Chernin L. Silencing the mob: disrupting quorum sensing as a means to fight plant disease. Mol Plant Pathol. 2015;16:316–29.

    Article  PubMed  Google Scholar 

  20. Uroz S, D’Angelo-Picard C, Carlier A, Elasri M, Sicot C, Petit A, et al. Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria. Microbiology. 2003;149:1981–9.

    Article  CAS  PubMed  Google Scholar 

  21. McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, et al. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology (Reading, Engl). 1997;143(12):3703–11.

    Article  CAS  Google Scholar 

  22. Winson MK, Swift S, Fish L, Throup JP, Jørgensen F, Chhabra SR, et al. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett. 1998;163:185–92.

    Article  CAS  PubMed  Google Scholar 

  23. Andersen JB, Heydorn A, Hentzer M, Eberl L, Geisenberger O, Christensen BB, et al. gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication. Appl Environ Microbiol. 2001;67:575–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cha C, Gao P, Chen YC, Shaw PD, Farrand SK. Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria. Mol Plant Microbe Interact. 1998;11:1119–29.

    Article  CAS  PubMed  Google Scholar 

  25. Farrand SK, Qin Y, Oger P. Quorum-sensing system of Agrobacterium plasmids: analysis and utility. Methods Enzymol. 2002;358:452–84.

    Article  CAS  PubMed  Google Scholar 

  26. Gao M, Benge A, Wu T-J, Javier R. Use of plasmid pVMG to make transcriptional ß-glucuronidase reporter gene fusions in the Rhizobium genome for monitoring the expression of rhizobial genes in vivo. Biol Proced Online. 2019;21:8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Llamas I, Keshavan N, González JE. Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones. Appl Environ Microbiol. 2004;70:3715–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Steidle A, Sigl K, Schuhegger R, Ihring A, Schmid M, Gantner S, et al. Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere. Appl Environ Microbiol. 2001;67:5761–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. DeAngelis KM, Lindow SE, Firestone MK. Bacterial quorum sensing and nitrogen cycling in rhizosphere soil. FEMS Microbiol Ecol. 2008;66:197–207.

    Article  CAS  PubMed  Google Scholar 

  30. Nievas F, Bogino P, Sorroche F, Giordano W. Detection, characterization, and biological effect of quorum-sensing signaling molecules in peanut-nodulating bradyrhizobia. Sensors (Basel). 2012;12:2851–73.

    Article  CAS  Google Scholar 

  31. Schaefer AL, Lappala CR, Morlen RP, Pelletier DA, Lu T-YS, Lankford PK, et al. LuxR- and luxI-type quorum-sensing circuits are prevalent in members of the Populus deltoides microbiome. Appl Environ Microbiol. 2013;79:5745–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sessitsch A, Coenye T, Sturz AV, Vandamme P, Barka EA, Salles JF, et al. Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. Int J Syst Evol Microbiol. 2005;55:1187–92.

    Article  CAS  PubMed  Google Scholar 

  33. Rashid R, Morohoshi T, Someya N, Ikeda T. Degradation of N-acylhomoserine lactone quorum sensing signaling molecules by potato root surface-associated Chryseobacterium strains. Microbes Environ. 2011;26:144–8.

    Article  PubMed  Google Scholar 

  34. Rodríguez M, Torres M, Blanco L, Béjar V, Sampedro I, Llamas I. Plant growth-promoting activity and quorum quenching-mediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6. Sci Rep. 2020;10:4121.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Begum JF, Tamilarasi M, Pushpakanth P, Balachandar D. A simple method for direct isolation of N-acyl-L-homoserine lactone mediated biofilm-forming rhizobacteria from roots. J Microbiol Methods. 2019;156:34–9.

    Article  CAS  PubMed  Google Scholar 

  36. Shaw PD, Ping G, Daly SL, Cha C, Cronan JE, Rinehart KL, et al. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci U S A. 1997;94:6036–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lullien V, Barker DG, de Lajudie P, Huguet T. Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol Biol. 1987;9:469–78.

    Article  CAS  PubMed  Google Scholar 

  38. Yasbin RE, Young FE. Transduction in Bacillus subtilis by bacteriophage SPP1. J Virol. 1974;14:1343–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhu J, Chai Y, Zhong Z, Li S, Winans SC. Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol. 2003;69:6949–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gurich N, González JE. Role of Quorum sensing in Sinorhizobium meliloti-alfalfa symbiosis. J Bacteriol. 2009;191:4372–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Joelsson AC, Zhu J. LacZ-based detection of acyl-homoserine lactone quorum-sensing signals. Curr Protoco Microbiol. 2006. https://doi.org/10.1002/9780471729259.mc01c02s3.

    Article  Google Scholar 

  42. Oldroyd GE, Engstrom EM, Long SR. Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula. Plant Cell. 2001;13:1835–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Peters NK, Crist-Estes DK. Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol. 1989;91:690–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Marketon MM, Gronquist MR, Eberhard A, González JE. Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones. J Bacteriol. 2002;184:5686–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dudas B, Jenes B, Kiss GB, Maliga P. Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa. Theor Appl Genet. 2012;125:1517–23.

    Article  CAS  PubMed  Google Scholar 

  46. Veliz-Vallejos DF, Kawasaki A, Mathesius U. The presence of plant-associated bacteria alters responses to N-acyl homoserine lactone quorum sensing signals that modulate nodulation in Medicago truncatula. Plants (Basel). 2020. https://doi.org/10.3390/plants9060777.

    Article  Google Scholar 

  47. von Rad U, Klein I, Dobrev PI, Kottova J, Zazimalova E, Fekete A, et al. Response of Arabidopsis thaliana to N-hexanoyl-DL-homoserine-lactone, a bacterial quorum sensing molecule produced in the rhizosphere. Planta. 2008;229:73–85.

    Article  CAS  Google Scholar 

  48. Veliz-Vallejos DF, van Noorden GE, Yuan M, Mathesius U. A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation. Front Plant Sci. 2014;5:551.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Liu F, Zhao Q, Jia Z, Song C, Huang Y, Ma H, et al. N-3-oxo-octanoyl-homoserine lactone-mediated priming of resistance to Pseudomonas syringae requires the salicylic acid signaling pathway in Arabidopsis thaliana. BMC Plant Biol. 2020;20:38.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Schenk ST, Hernández-Reyes C, Samans B, Stein E, Neumann C, Schikora M, et al. N-acyl-homoserinel primes plants for cell wall reinforcement and induces resistance to bacterial pathogens via the salicylic acid/oxylipin pathway. Plant Cell. 2014;26:2708–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, et al. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology (Reading, Engl). 2002;148:1119–27.

    Article  CAS  Google Scholar 

  52. Calatrava-Morales N, McIntosh M, Soto MJ. Regulation mediated by N-acyl homoserine lactone quorum sensing signals in the Rhizobium-legume symbiosis. Genes (Basel). 2018. https://doi.org/10.3390/genes9050263.

    Article  Google Scholar 

  53. Corral-Lugo A, Daddaoua A, Ortega A, Espinosa-Urgel M, Krell T. Rosmarinic acid is a homoserine lactone mimic produced by plants that activates a bacterial quorum-sensing regulator. Sci Signal. 2016. https://doi.org/10.1126/scisignal.aaa8271.

    Article  PubMed  Google Scholar 

  54. Keshavan ND, Chowdhary PK, Haines DC, González JE. l-canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti. J Bacteriol. 2005;187:8427–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Delalande L, Faure D, Raffoux A, Uroz S, D’Angelo-Picard C, Elasri M, et al. N-hexanoyl-L-homoserine lactone, a mediator of bacterial quorum-sensing regulation, exhibits plant-dependent stability and may be inactivated by germinating Lotus corniculatus seedlings. FEMS Microbiol Ecol. 2005;52:13–20.

    Article  CAS  PubMed  Google Scholar 

  56. Deryabin D, Galadzhieva A, Kosyan D, Duskaev G. Plant-derived inhibitors of AHL-mediated quorum sensing in bacteria: modes of action. Int J Mol Sci. 2019;20:E5588.

    Article  PubMed  CAS  Google Scholar 

  57. Joshi JR, Khazanov N, Khadka N, Charkowski AO, Burdman S, Carmi N, et al. Direct binding of salicylic acid to Pectobacterium N-acyl-homoserine lactone synthase. ACS Chem Biol. 2020;15:1883–91.

    Article  CAS  PubMed  Google Scholar 

  58. Koh C-L, Sam C-K, Yin W-F, Tan LY, Krishnan T, Chong YM, et al. Plant-derived natural products as sources of anti-quorum sensing compounds. Sensors (Basel). 2013;13:6217–28.

    Article  CAS  Google Scholar 

  59. Saral A, Kanekar S, Koul KK, Bhagyawant SS. Plant growth promoting bacteria induce anti-quorum-sensing substances in chickpea legume seedling bioassay. Physiol Mol Biol Plants. 2021;27:1577–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ta CAK, Arnason JT. Mini review of phytochemicals and plant taxa with activity as microbial biofilm and quorum sensing inhibitors. Molecules. 2015;21:E29.

    Article  PubMed  CAS  Google Scholar 

  61. Teplitski M, Robinson JB, Bauer WD. Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. Mol Plant Microbe Interact. 2000;13:637–48.

    Article  CAS  PubMed  Google Scholar 

  62. Ban H, Chai X, Lin Y, Zhou Y, Peng D, Zhou Y, et al. Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease. Plant Cell Rep. 2009;28:1847–55.

    Article  CAS  PubMed  Google Scholar 

  63. Zhang L-H. Quorum quenching and proactive host defense. Trends Plant Sci. 2003;8:238–44.

    Article  CAS  PubMed  Google Scholar 

  64. Cataldi TRI, Bianco G, Frommberger M, Schmitt-Kopplin P. Direct analysis of selected N-acyl-L-homoserine lactones by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2004;18:1341–4.

    Article  CAS  PubMed  Google Scholar 

  65. Gould TA, Herman J, Krank J, Murphy RC, Churchill MEA. Specificity of acyl-homoserine lactone synthases examined by mass spectrometry. J Bacteriol. 2006;188:773–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Kim Y-W, Sung C, Lee S, Kim K-J, Yang Y-H, Kim B-G, et al. MALDI-MS-based quantitative analysis for ketone containing homoserine lactones in Pseudomonas aeruginosa. Anal Chem Am Chem Soc. 2015;87:858–63.

    Article  CAS  Google Scholar 

  67. Zhang H-B, Wang L-H, Zhang L-H. Detection and analysis of quorum-quenching enzymes against acyl homoserine lactone quorum-sensing signals. Curr Protoc Microbiol. 2007. https://doi.org/10.1002/9780471729259.mc01c03s05.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

This manuscript is part of the dissertation ‘Interactions of the plant growth promoting rhizobacterium Bacillus subtilis UD1022 with Medicago root microbes’ submitted to the University of Delaware by Amanda Rosier for the fulfilment of a doctoral degree requirements. The authors would like to thank Jean-Sébastien Bourassa and Pascale B. Beauregard from the Département de biologie, Université de Sherbrooke, Canada for providing us B. subtilis UD1022 ycbU-spc-ImrB.

Funding

HPB acknowledges the support from Multistate Hatch grant from USDA. AR acknowledges support from USDA SARE.

Author information

Authors and Affiliations

  1. Department of Plant and Soil Sciences, University of Delaware, 311 AP Biopharma, 590 Avenue 1743, Newark, DE, 19713, USA

    Amanda Rosier & Harsh P. Bais

Authors
  1. Amanda Rosier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Harsh P. Bais
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed equally to the work presented. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Amanda Rosier.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Spectinomycin resistant bacteria tests of the KYC55 biosensor. Left plate: top row is Rm8530-spec, bottom row is Rm8530-spec with UD1022 ycbU. Right plate: top row is Rm8530-spec, middle row is Rm8530-spec with 100 µg ml-1 YtnP, bottom row is Rm8530-spec with 100 µg ml-1 heat killed YtnP.

Additional file 2: Figure S2.

Six day after germination (DAG) M. truncatula seedlings do not QQ oxo-C16 AHL. Left plate: top row is Rm8530 alone, bottom row is 10 µM oxo-C16 AHL alone. Right plate: M. truncatula with 10 µM oxo-C16 AHL.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rosier, A., Bais, H.P. Protocol: a simple method for biosensor visualization of bacterial quorum sensing and quorum quenching interaction on Medicago roots. Plant Methods 18, 112 (2022). https://doi.org/10.1186/s13007-022-00944-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13007-022-00944-5

Plant Methods

ISSN: 1746-4811