Visualizing bacterial quorum sensing and quorum quenching interaction on Medicago roots

doi:10.21203/rs.3.rs-1742677/v1

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Visualizing bacterial quorum sensing and quorum quenching interaction on Medicago roots

https://doi.org/10.21203/rs.3.rs-1742677/v1

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Background

Defining interactions of bacteria in the rhizosphere (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 use the extracellular autoinducer signal molecule N-acyl homoserine lactone (AHL) and other rhizobacteria, including PGPR, can interfere or disrupt QS through quorum quenching (QQ). Traditional 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’s agar are 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 QQ protein are 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.

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 as individuals 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 intraspecies communication utilizing extracellular signal molecules known as quorum sensing (QS). Populations of bacterial cells coordinate in a conditionally responsive manner to produce extracellular ‘autoinducer’ molecules which modulate transcription of genes (via transcriptional regulators) for activities ranging from motility, biofilm formation and virulence [3].

The most comprehensively studied family of QS 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 becoming better understood to have an influence on other species, families and kingdoms of organisms occupying the rhizosphere [6–9]. 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 spp. through the lactonase enzyme AiiA [13] which cleaves the lactone ring. QQ has been shown to inhibit both the virulence of plant pathogens [14–16] 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 implemented from the outset. 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 Escheri 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 [23–28].

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) [29–31]. Biosensors are also used to identify specific AHLs produced in PGPR [32] and to screen root isolates for QQ activity [33,34]. A recent protocol from Begum et al. (2019) presents a more streamlined approach to screening by placing detached rice roots from the field placed directly on agar containing C. violaceum CV026 and A. tumefaciens NTL1 AHL biosensors [35].

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 plant roots. Recent work by Rosier et al. (2020) 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 suitable for visually assessing the presence of quorum sensing bacteria, as well as the effects of QQ on Medicago truncatula A17 plant roots. This technique represents a potentially valuable tool for research for QS beneficial and pathogenic bacteria observable dynamics in the rhizosphere.

Preparation of seeds and seedling growth

Seeds of M. truncatula A17 ‘Jemalong’ were scarified in sulfuric acid for 6 minutes and sterilized in 70% ethanol for 1 minute and 3% bleach for 10 minutes, rinsing thoroughly between each. Seeds were resuspended in sterile Nanopure^TM water and placed on shaker at room temperature for 4 hours, rinsing and replacing water every hour. After final rinse seeds were resuspended and placed in 4 °C for 48 hours. Seeds were again rinsed and placed on sterile 120 mm² plates, sealed with parafilm and germinated vertically in dark conditions for 24 hours. Seedlings were placed onto Lullien media [36] agar 120 mm² plates + 0.5 AVG (2- aminoethoxyvinyl glycine, ethylene inhibitor, Sigma) 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 hours. 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. The plasmid PLS1 harboring P_exoY-mTFP [37] was introduced into S. meliloti strain Rm8530 (intact QS system) through tri-parental mating. The Rm8530 P_exoY-mTFP fusion (‘Rm8530-tfp’) confers spectinomycin resistance along with the teal fluorescence gene. Rm8530-tfp was grown on TYC agar with 250 µg/ml^-1 streptomycin and 50 µg ml^-1 spectinomycin for four days at 28 °C. Colonies were re-suspended in 5 mL TYC + Ab to OD₆₀₀ ~ 0.5 and grown shaking for 3 hours. Cells were washed once, resuspended in sterile Nanopure^TM and diluted to OD₆₀₀ = 0.2 in preparation to inoculate onto plant roots.

Table 1. Bacterial strains used in this study.

Strain	Description	Reference
Rm8530-tfp	S. meliloti Rm8530 with PLS1 (P_exoY^-mTFP)	This work, [37]
UD1022 ycbU	B. subtilis UD1022 ycbU-spc-ImrB (intergenic resistance)	Gift, Dr. Pascale Beauregard
KYC55	A. tumefaciens KYC55 (pJZ372)(pJZ384) (pJZ410)	[39]

QQ strain

Spectinomycin resistance was introduced into the PGPR B. subtilis UD1022 (‘UD1022 ycbU’). UD1022 ycbU was streaked onto LB agar plates with 100 µg ml^-1 spectinomycin and grown 24 hours at 37 °C. Colonies were re-suspended in 5 ml LB + 100 µg ml^-1 spectinomycin and grown shaking for 3 hours. Cells were washed once, resuspended in sterile Nanopure^TM and diluted to OD₆₀₀ = 1.0 in preparation to inoculate onto plant roots.

Biosensor strain

A. tumefaciens ‘KYC55’ pre-induced cells [38] were inoculated into minimal glutamate mannitol (MGM) broth and grown shaking at 28 °C for 24 hours. KYC55 cells were used to make X-gal soft agar following the protocol of Joelsson and Zhu (2006) with the following modifications. Addition of 0.5 µM AVG final concentration and 50 µg ml^-1 spectinomycin to MGM-based agar media. Twenty-five ml KYC55 soft agar was aliquoted per 120 mm² plate and allowed to dry 30 minutes.

Assembly of the biosensor growth plates

Three-day old M. truncatula seedlings were transferred from Lullien media plates to freshly poured KYC55 + 50 µg ml^-1 spectinomycin soft agar plates (5 seedlings per plate). 10 µl drops of UD1022 ycbU were pipetted 1 cm below the root tips (‘UD1022 + Rm8530’ treatments) and allowed to dry. 10 µl drops of Rm8530-tfp were pipetted onto appropriate treatments (‘Rm8530’ and ‘UD1022 + 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 PBS to 100 µg ml^-1. Aliquots were split with one set subjected to ‘heat treatment’ at 100 °C for 15 minutes to inactivate the protein activity. 10 µl 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 resuspended in methanol and diluted in PBS to 10 µM and 10 µl drops pipetted as previous. All treatments included one plate without plants. Three replicate plates were included per treatment and experiments were repeated three times.

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 fused with antibiotic resistance for spectinomycin, gentamycin and tetracycline [39]. Spectinomycin is also simple to introduce to a range of different types of bacteria genomes. Though spectinomycin has been reported to bleach M. sativa cultured cells [40], in this protocol, plants were initially grown without the antibiotic to ensure vigor. We observed no deleterious effects of 50 µg ml^-1 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 (Supplementary Figure 1).

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 (Figure 1). Further, this techniques allowed for the visualization of YtnP lactonase activity in the presence of live M. truncatula roots (Figure 2). Recent work by Veliz-Vallejos et al. (2020) 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 [41]. The potential QQ of seed-borne bacteria in their system could be visualized through replicating such experiments on KYC55 as described in this protocol.

Our protocol would be easily adapted to elucidate the QS and QQ interactions of rhizosphere bacteria on plant roots and for evaluating direct plant/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. 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/roots to QS bacteria/AHLs and to observe potential plant influences on the same. 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 [42], and work by Liu et al. (2012). Addition of long-chain AHLs significantly increased the number of symbiotic root nodules formed by the symbiotic mutualist Sinorhizobium meliloti on its legume host Medicago truncatula [43]. 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 [8,44,45]. 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 [46]. Calatrava-Morales et al. (2018) reported positive responses of suite of AHL bioreporters (not including KYC55) to pea root extracts, possibly detecting substances which could competitively inhibit AHLs [47]. Substances such as L-canavanine found in alfalfa and legume root exudates and rosmarinic acid are also reported as AHL mimics inhibiting QS [48,49]. Many other plant natural products and phytochemicals, including phytohormones have also been reported to interfere with bacterial QS [19,50–56]. In this system, we observed no detectable plant derived quorum signals (Figure 1a) or quenching of S. meliloti AHL signal (Supplementary Figure 2). Adaptation of various other AHL biosensors or use of other AHLs such as those from the afore-mentioned studies could be incorporated into this assay; KYC55 and long-chain AHLs may not reflect the types of plant-bacteria interactions described previously. This method could also be suitable for evaluating transgenic plants expressing QQ enzymes. [14,57,58], a technique proposed for control of QS plant pathogens.

Although the use of various mass spectroscopy and HPLC techniques [59–61] for AHL detection and identification is the gold standard, but the relatively simple visual assays using AHL biosensors are most useful for screening and validation [34]. Here we demonstrate an effective protocol which expands on the foundations of AHL biosensor assays and could be applied toward multiple different lines of research. This application of the broad range AHL biosensor KYC55 combined with the advantages of the low requirement for technical equipment and the ability to comprehensively observe interspecies interactions makes this protocol extremely useful for the active field of QS research in the rhizosphere.

Authors’ contributions

All authors contributed equally to the work presented.

Acknowledgements

Authors would like to acknowledge Dr. Sharon R. Long, Department of Biology, Stanford University, USA for providing the plasmid PLS1 carrying P_exoY-mTFP. We would also like to extend our appreciation to 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.

Competing interests

The authors declare no competing interests.

Availability of data and material

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

Funding

H.P.B. acknowledges the support from Multistate Hatch grant from USDA. A.R. acknowledges support from USDA SARE.

Ethics approval and consent to participate (kindly mention the name of the Ethics Committee and the Ethical Approval Number): Not applicable

Consent for publication

All authors approve the submission of the manuscript.

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No competing interests reported.

RosierMethodsSuppFig1.jpg
Spectinomycin resistant bacteria tests of the KYC55 biosensor (a) Rm8530-tfp (b) Rm8530-tfp + UD1022 ycbU (c) Rm8530-tfp + 100 µg ml^-1 YtnP (d) Rm8530-tfp + 100 µg ml^-1 heat killed YtnP.
RosierMethodsSuppFig2.jpg
M. truncatula does not QQ oxo-C16 AHL (a) above bar: Rm8530 alone, below bar: 10 µM oxo-C16 AHL alone (d) M. truncatula with 10 µM oxo-C16 AHL.

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Editorial decision: Major revision
01 Jul, 2022
Reviews received at journal
22 Jun, 2022
Reviewers agreed at journal
11 Jun, 2022
Reviewers invited by journal
11 Jun, 2022
Submission checks completed at journal
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First submitted to journal
09 Jun, 2022

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Visualizing bacterial quorum sensing and quorum quenching interaction on Medicago roots

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