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Oryza sativa rice plants contain molecules that activate different quorum-sensing N-acyl homoserine lactone biosensors and are sensitive to the specific AiiA lactonase

Giuliano Degrassi, Giulia Devescovi, Renando Solis, Laura Steindler, Vittorio Venturi
DOI: http://dx.doi.org/10.1111/j.1574-6968.2006.00624.x 213-220 First published online: 1 April 2007

Abstract

Gram-negative bacteria most often use N-acyl homoserine lactones (AHLs) as intercellular quorum-sensing signal molecules. In this study, it was demonstrated that rice plants contain AHL mimic molecules that are very sensitive to the highly specific AiiA lactonase enzyme and can activate three different AHL bacterial biosensors, indicating that the compounds have a homoserine lactone structure and could be AHLs. The possible source and biological significance of this finding are discussed.

Keywords
  • quorum sensing
  • N-acyl homoserine lactones
  • plants
  • AiiA lactonase
  • rice

Introduction

Bacteria use small signal molecules as intercellular communicators to coordinate gene expression within a population in a process called quorum sensing (QS) (Fuqua et al., 1994; Waters & Bassler, 2005). The concentration of the signal molecules increases alongside the bacterial population density and, when it reaches a critical level, bacteria respond and modulate target gene expression. In natural ecosystems, bacteria are aiming at establishing communities as QS provides significant advantages such as improving access to environmental niches, enhancing defense capabilities against other microorganisms or eukaryotic host-defense mechanisms and facilitating the adaptation to changing environmental conditions. A typical QS system in gram-negative bacteria involves the production and response to an acylated homoserine lactone (N-acyl homoserine lactones; AHL) signal molecule. An AHL-QS system is usually mediated by two proteins belonging to the LuxI–LuxR protein families; LuxI-type proteins are responsible for synthesizing AHLs, which then interact directly at quorum concentration with the cognate LuxR-type protein, and this protein–AHL complex can then bind to target promoters affecting expression of QS target genes. Many plant associated bacteria, including Agrobacterium tumefaciens, Erwinia carotovora, Pseudomonas syringae and Pseudomonas putida, use AHL QS to regulate important traits such as Ti plasmid transfer, biofilm formation, production of hydrolytic degradative enzymes and epiphytic fitness (Loh et al., 2002; Von Bodman et al., 2003).

Several recent studies have demonstrated that eukaryotes interfere and respond to bacteria AHL QS signals, indicating interkingdom communication via AHLs (Bauer & Mathesius, 2004; Shiner et al., 2005). AHLs modulate gene expression in animal and plant cells as for example observed with one of the AHLs (3-oxo-C12-AHL) produced by Pseudomonas aeruginosa, which modulates the expression of certain genes in human lung fibroblasts and leukocytes (Telford et al., 1998; Smith et al., 2002; Shiner et al., 2005). Similarly, in plant cells, a proteomic study in the model legume Medicago truncatula resulted in the alteration of abundance of 150 proteins after root exposure to AHLs (Mathesius et al., 2003; Shiner et al., 2005); a similar experiment with Arabidopsis thaliana involving microarray analysis, however, resulted in AHLs not affecting endogenous gene expression (You et al., 2006). In addition, AHLs produced by biocontrol rhizosphere bacteria have been recently reported to induce systemic resistance to fungal pathogens in tomato plants (Schuhegger et al., 2006). Alongside these responses, eukaryotes appear to produce chemical compounds that act as agonists or antagonists to bacterial AHL QS systems and are often called AHL mimics (Bauer & Mathesius, 2004). The first AHL mimics identified and best characterized are the furanones from the red alga Delisea pulchra (Givskov et al., 1996; Gram et al., 1996; Hentzer et al., 2003), which inhibit the AHL QS response by interfering with AHL–LuxR interactions (Manefield et al., 2002). Several other plant species secrete AHL mimics that can either stimulate or inhibit bacterial AHL QS systems (Teplitski et al., 2000; Gao et al., 2003). The precise source, structure and biological significance of these AHL mimics from plants are currently unknown.

In this study evidence is provided that from the leaves and stem of rice plants (Oryza sativa) two different compounds could repeatedly be detected, which activate three different AHL biosensors, both of which were sensitive to the specific AiiA lactonase. In these experiments, no other compound that could mimic AHLs was observed. It is significant that these compounds are found in healthy rice plants grown in a greenhouse under sterile conditions, implying that bacteria infecting rice in the wild will most probably encounter these molecules.

Materials and methods

Bacterial strains, culture conditions and recombinant DNA techniques

Bacterial strains used were grown in either M9 medium with the possible addition of 0.2% w/v casamino acids and glucose or in Luria–Bertani (LB) medium (Sambrook et al., 1989). Escherichia coli JM109 (pSB401; Winson et al., 1998) was grown in LB medium plus tetracycline 10 µg mL−1 at 37°C. Plasmid pSB401 contains the following genetic arrangement: luxR gene, the promoter of luxI fused to a promoterless luxCDABE. Providing exogenous AHL inducer molecules to E. coli (pSB401) results in the induction of bioluminescence. The biosensor strain E. coli pSB536 (an AhyR-based sensor with luxCDABE as a reporter system) was grown in LB medium plus ampicillin 100 µg mL−1 at 37°C (Swift et al., 1997). Escherichia coli DH5α (pSCR1) was grown in LB medium plus ampicillin 100 µg mL−1 at 37°C. Plasmid pSCR1 contains the following genetic arrangement: the Burkholderia cepacia cepR gene, the promoter of cepI fused to a promoterless lacZ (Aguilar et al., 2003). Providing exogenous AHL inducer molecules to E. coli (pSCR1) results in the induction of β-galactosidase production. Chromobacterium violaceum CV026 was grown in LB medium at 30°C and is a double mini-Tn5 mutant derived from ATCC 31 532; this mutant is nonpigmented and production of the purple pigment can be induced by providing exogenous AHL inducer molecules (McClean et al., 1997). Pseudomonas putida F117 (pAS-C8) was grown in LB medium plus 25 µg mL−1 gentamycine at 30°C and is an AHL biosensor based on components of the Burkholderia cenocepacia CepI/R system, and the plasmid contains the cepI promoter translationally fused to the gfp reporter gene together with, the cepR gene placed under control of Plac in opposite direction (Riedel et al., 2001).

Digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, ligation with T4 DNA ligase, transformation of E. coli and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described (Sambrook et al., 1989). Analytical amounts of plasmids were isolated as described (Birnboim, 1983), whereas preparative amounts were purified with Qiagen columns. β-Galactosidase activity was determined as previously described (Miller, 1972).

Purification and characterization of AHL-like molecules from rice

Seeds of rice cv. IR24 (obtained from PhilRice, Science City of Muñoz, the Philippines) or Italian variety cv. Selenio (obtained from the Italian Rice Research Institute, Pavia, Italy) were sterilized in a 5% active chlorine hypochlorite solution for 1 h, and repeatedly washed with sterile water. Rice plants were grown in a sterilized peat substrate (Klassmann substrate 4, Geeste, D) under full-containment greenhouse conditions (28±2°C, 70% humidity) under a 14 : 10 light–dark photoperiod. Rice plants were taken for extraction after 1.5 (vegetative phase) and 3 months (reproductive phase), the roots were then disposed and the aerial parts of the plants were repeatedly washed with sterile water. Twenty-five grams of fresh plant material were then frozen with liquid nitrogen and ground by a pestle and mortar, reduced into a fine powder, suspended into warm water at 50°C and mixed for 1 h. The water solution was centrifuged to remove the plant residues, and the supernatant was extracted with the same volume of ethyl acetate after acidification with 0.1% acetic acid. The extract was dried at room temperature and resuspended in 1 mL methanol/water (50 : 50 vol/vol). This extract was fractionated by HPLC with a semi-preparative C18 reverse-phase column (1 × 25 cm), using a methanol:water gradient 20 : 80–80 : 20 eluent composition for 35 min at a flow rate of 2.5 mL min−1. Fractions of 5 mL each were collected and dried and were analyzed by (1) C18-reverse-phase thin layer chromatography (TLC) plates as described previously (Shaw et al., 1997) and finally overlaid with AHL biosensors E. coli (pSB401), E. coli (pSB536) or C. violaceum CV026 and (2) determining β-galactosidase activities when exposed to E. coli (pSCR1).

Purification and use of the AiiA lactonase enzyme

The aiiA gene was amplified from pME6860 (Reimmann et al., 2002) using primers 5′-cgggatccacagtaaagaagct-3′ and 5′-aactgcagctatatatattcaggga-3′ and cloned as a 765 bp BamHI–PstI fragment in the corresponding sites in pQE30 (Qiagen, Hilden, D) yielding pQAIIA. Expression and purification of His6-tagged AiiA were carried out in E. coli M15 (pREP-4) according to the instructions of the supplier (Qiagen, Hilden, D). Enzyme reactions were performed in 0.1 M phosphate buffer (pH 7.4) with 0.5 µg of purified AiiA enzyme incubated at 30°C for 4 h and then used for AHL detection.

Isolation of endophytic bacteria

Endophytic bacteria were isolated from rice cv. IR24 grown as described above following the method reported previously (Stoltzfus et al., 1997). All plant tissues were surface sterilized as follows: 10 g of tissue was shaken for 30 min in 500 mL Erlenmeyer flasks containing 250 mL sterile deionized water and 25 g of glass beads. The tissue was transferred aseptically to a sterile beaker, washed two times with sterile distilled water, and sterilized for 1 min using 0.2% HgCl2. The tissue was then washed six times with sterile distilled water cut into small pieces and homogenized in a Warring blender containing 90 mL sterile distilled water. Serial dilutions were prepared and spread on plates containing bacterial growth media.

Results

Detection of AHL-like QS signals from rice extracts

In order to determine whether rice contained AHLs and/or AHL mimics, ethyl acetate extracts of rice leaves and stems were prepared as described in ‘Materials and methods’ and tested for activity towards two AHL bacterial biosensors. As depicted in Fig. 1 (a) and (c), AHL purification from vegetative phase grown rice stimulated two AHL biosensors, resulting in two spots of bioluminescence in the TLC plate overlaid with E. coli pSB401 and in β-galactosidase activity in the corresponding HPLC fractions (HPLC fractions 5–7) when these were exposed to E. coli (pSCR1). Similarly, AHL purification from reproductive phase rice (Fig. 1 sections b and d), resulted in the stimulation of both biosensors, however, from different HPLC fractions and migrating differently on TLC; interestingly, rice in the vegetative phase showed two spots when analyzed with E. coli pSB401, and the upper one also seems to be present in the reproductive phase, although at a lower concentration. These results provide clear indications that in healthy rice there are compounds that respond to AHL biosensors; the two biosensors used respond to a wide range of AHLs. Escherichia coli pSB401 based on LuxR displays a dynamic range of AHL response, whereas the CepR-based pSCR1 best responds to C8-AHLs, C10-AHLs and C12-AHLs. C8-AHL was used as control for both the reporters at concentrations ranging from 0.1 to 0.5 µM, respectively. This experiment was repeated threefold and provided consistent reproducible results. In addition, the AHL biosensor strain C. violaceum CV026 (McClean et al., 1997) positively responded only to HPLC fraction 16 of reproductive phase rice when the same amount of rice sample was analyzed via TLC (data not shown). The biosensor strain E. coli pSB536, which is sensitive to short-chain AHLs, on the other hand, was not sensitive to any of the extracts from the same amount of rice of either growth phases (data not shown).

1

AHL purification and detection from 25 g of rice plant extract. (a) AHL molecules were purified by organic extraction with ethyl acetate, followed by HPLC from an extract derived from 25 grams of rice plants in the vegetative phase. Shown here is a TLC analysis of the HPLC fractions (for each fraction, one-half of the total volume was loaded), which was then overlaid with AHL-biosensor Escherichia coli (pSB401). The black spots represent production of bioluminescence as a result of the presence of molecules that are able to induce the AHL biosensor and visualized using an autoradiographic film. (c) β-Galactosidase activities of each HPLC fraction as used for the experiment presented in part a (the remaining half of total amount of each fraction was used) using CepR-based AHL biosensor E. coli (pSCR1) as previously described (Aguilar et al., 2003). (b) AHL molecules were purified by organic extraction with ethyl acetate, followed by HPLC from an extract derived from 25 g of rice plants in the reproductive phase. Shown here is a TLC analysis of the HPLC fractions (for each fraction, one-half of the total volume was loaded), which was then overlaid with AHL biosensor E. coli (pSB401). The black spots represent production of bioluminescence as a result of the presence of molecules that are able to induce the AHL biosensor and visualized using an autoradiographic film. (d) β-Galactosidase activities of each HPLC fraction as used for the experiment presented in part c (the remaining half of the total amount of each fraction was used) using CepR-based AHL biosensor E. coli (pSCR1) as previously described (Aguilar et al., 2003).

The compounds detected in rice are sensitive to the AiiA lactonase enzyme

In order to gain an insight into the type of molecules that activate the AHL biosensors, it was decided to test whether these compounds were substrates for the AiiA AHL-lactonase, a quorum quenching enzyme from Bacillus sp. that inactivates AHLs (Dong et al., 2001; Wang et al., 2004). This enzyme has remarkable substrate specificity and is very potent towards different AHLs regardless of length and substitution of the acyl chain and showed very residual activity against nonacyl homoserine lactones (Wang et al., 2004). In order to test the rice AHL mimic compounds for AiiA susceptibility and thus indicate whether or not they are AHL-related, we purified the AiiA enzyme was purified and then it was tested whether these compounds retained their ability to activate AHL biosensors once exposed to AiiA. The Bacillus aiiA gene encoding the AiiA protein was cloned, expressed and purified to homogeneity as a His6-tagged protein. The purity of the AiiA protein was analyzed and confirmed by 12% SDS-PAGE (data not shown).

Twenty-five grams of fresh rice plant material of 1.5 (vegetative phase) and 3 months (reproductive phase) were grown and used for AHL extraction as in ‘Materials and methods’. The final HPLC fractions were then divided into two: (1) one half was resuspended in 40 µL of 0.1 M phosphate buffer (pH 7.4) and used directly for AHL detection in TLC overlaid with E. coli pSB401 or for AHL detection using E. coli (pSCR1) and (2) the other half resuspended in 40 µL of 0.1 M phosphate buffer (pH 7.4) with the addition of 10 µL containing 0.5 µg of purified AiiA enzyme incubated at 30°C for 4 h and then used for AHL detection (Fig. 2). As can be seen from Fig. 2, the TLC spot observed in the vegetative phase-grown rice as well as the β-galactosidase peak of activities in HPLC fractions from reproductive phase rice clearly disappeared when AiiA lactonase was incubated with these compounds (Fig. 3). Figure 2a is slightly different from Fig. 1a, with one spot only instead of two, probably due to minor differences in batches of rice extract and quantities used. From this figure, it is also evident that the AHL-like molecule is not C8-AHL-like. In Fig. 3, fractions 16 and 17 after the treatment with the AiiA enzyme are lower than the other fractions, probably due to a minimal background to which the reporter is slightly sensitive. It is also believed that the residual activity of the lactonase-treated C8-AHL is due to high sensitivity of the reporter to residual C8-AHL. This experiment provides a significant indication that the compounds detected in rice could be AHLs and have a lactone structure thus are not compounds of a distant chemical structure that interact with and activate the LuxR family proteins. As a positive control, C8-AHL incubated with AiiA lactonase clearly inactivated the AHL response by E. coli (pSB401) and E. coli (pSCR1) (Fig. 3). As all experiments thus far were performed using rice of an indica variety cv. IR24 obtained from The Philippines, AHLs were also extracted as described above from 25 g of leaves and stems of rice of Italian variety cv. Selenio. The extract obtained was fractionated by HPLC, fractions were dried, resuspended in ethyl acetate and pooled. Precipitated material was eliminated by centrifugation, and the supernatant was dried and resuspended again in ethyl acetate. Half of the material was then loaded directly in the TLC plate and the other half was first treated with the AiiA-purified lactonase enzyme. The TLC was then overlaid with AHL biosensor C. violaceum CV026 and as can be seen in Fig. 4, compounds mimicking AHLs with Rf higher than C8-AHL were clearly detected, which completely disappeared when the extract was treated with AiiA-lactonase. This result cannot be directly compared with the above-reported results as the rice variety and the procedure used are different. However, it further demonstrates that healthy rice of European origin also contains AHL-like or closely related compounds.

2

TLC analysis of HPLC fractions from extracts derived from 25 g rice (vegetative phase) extract before (a) and after (b) AHL lactonase treatment. One half of each HPLC fraction was loaded directly in the TLC plate, and the other half was treated with the purified AiiA lactonase. Both TLCs were overlaid with AHL biosensor Escherichia coli (pSB401). In TLC A, the black spot present in fraction 4 represents production of bioluminescence as a result of the presence of molecules that are able to induce the AHL-biosensor and visualized using an autoradiographic film. This spot disappeared in TLC B following treatment with lactonase. In each TLC synthetic C8-AHL was also loaded. As control, in TLC B the same amount of C8-AHL was also treated with lactonase and the ability to induce the AHL biosensor almost disappeared (lane C8+L). See text for all details.

3

β-Galactosidase activities (y-axis) of AHL CepR-based AHL-biosensor Escherichia coli (pSCR1) grown in the presence of fractions (x-axis) from HPLC fractionation of extracts derived from 25 g of rice extract from the reproductive phase. One reaction was also performed using purified C8-AHL as indicated. SD of three independent experiments is reported. On set of HPLC fractions 16 and 17, and C8-AHL (lane C8+L) were treated with purified lactonase and β-galactosidase-induced activity disappeared as shown by black-filled bars. See text for all details.

4

Purification of AHL-like molecules from Italian rice variety Selenio (vegetative phase). TLC overlaid with AHL biosensor Chromobacterium violaceum CV026. Lane 1, C8-AHL; 2, C8-AHL after lactonase treatment; 3, half of the sample obtained from a purification of 25 g of rice extract; 4, other half of the sample obtained form 25 g of rice extract after treatment with purified lactonase. See text for all details.

Rice endophytic bacteria

In order to evaluate the role of rice-associated bacteria in determining the presence of AHL-like molecules in rice extracts, endophytes were isolated from the IR24 rice variety. Epiphytic bacteria were not considered because the rice used for extraction of AHL-like molecules was extensively washed with sterile water in order to eliminate epiphytic AHL contaminants. The maximum number of culturable bacteria obtained was 4.5 × 103 CFU in 10 g of rice plant (stems and leaves only). All colonies isolated from 200 µL of rice suspension were tested on petridishes (as a ‘T’-streak) for the ability to induce a response to several AHL-reporter strains covering most of the common AHL structural molecules. None of the tested colonies was able to induce the reporter strains.

Do the AHL-like compounds present in rice activate AHL biosensors in vivo?

In order to test whether in rice the presence of AHL-like compounds could activate AHL biosensors in vivo, the CepR-green fluorescent protein (GFP)-based AHL sensor P. putida F117 (pAS-C8) (this sensor is similar to pSCR1, a CepR-lacZ AHL sensor, which was used in the detection of AHLs in rice, see above) was introduced into rice leaves (Riedel et al., 2001). The bacterial suspension [P. putida F117 (pAS-C8)-108 CFU mL−1)] was injected with a needle-less syringe into the leaves; after 24 h, sections of leaves that included the inoculation site were placed in eppendorf tubes for 8 h in M9 minimal medium that either contained or did not contain added synthetic C8-AHL. Then, the sections were mounted on slides and observed under an inverted Zeiss LSM 510 Laser Scanning Microscope with a Plan–Neofluar × 40, N.A. 0.75 lens. GFP fluorescence was excited with a 488 nm Ar-laser, and confocal sections were collected using a BP 505–530 nm (GFP) and LP 585 (chlorophyll autofluorescence) filter emission settings. GFP was observed only in the samples that had been treated with C8-AHL, indicating that the bacteria were present in the leaf but the natural/biological concentration of AHL-like compounds inside the leaves was not sufficient to induce the GFP expression of the plasmid sensor pAS-C8 (data not shown).

Discussion

The understanding of how plants interfere and/or respond to bacterial AHL QS signals is at a very early stage and this will be an important research topic to address in the future. Several plant pathogens use AHL QS to regulate expression of virulence–associated factors and beneficial bacteria to modulate plant–bacteria interactions (Loh et al., 2002; Von Bodman et al., 2003; Schuhegger et al., 2006). Hence, plants could have evolved strategies to interfere with this signaling system, which may include production of signal-degrading enzymes, signal blockers or signal mimics. A few years ago, it has been reported that root exudates from several plant secrete compounds that mimic AHLs (Teplitski et al., 2000; Gao et al., 2003; Mathesius et al., 2003) as these can activate bacterial AHL biosensors; however, their chemical structure and precise biological source are currently unknown.

In this study, it was determined that stems and leaves of rice contained AHL mimic compounds that could activate several bacterial AHL biosensors. In addition through the use of the specific AHL AiiA-lactonase, it was observed that these compounds are very sensitive to the enzyme and therefore are most likely AHLs or have a very related lactone structure. The AiiA lactonase has only residual activity to nonacyl lactones and the differences in length and composition of the acyl chain do not affect enzyme activity (Wang et al., 2004). From these results, it cannot be stated that the compounds reported here are unequivocally AHLs but it can, however, be said that they are most likely AHLs or have a very closely related structure. In all the studies, no compound that was resistant to the AiiA lactonase and activated the bacterial AHL biosensors was purified from rice. This result raises two important questions: firstly, are the AHL-like compounds found in rice of bacterial and/or plant origin and, secondly, does bacteria infecting the rice plant encounter these compounds at a biologically significant concentration? With respect to the first question, it is unlikely that they are produced by epiphytic bacteria as the seeds were sterilized, plants were grown under sterile conditions in a full containment greenhouse and extensively washed with sterile water before AHL extraction was performed. It is also unlikely that they are due to rhizosphere bacteria for similar reasons and also because no root material was used for AHL extraction. It is possible, however, that the AHLs detected in rice are produced by endophytic bacteria. There is scarce information regarding bacterial endophytic colonization of rice as well as on the response of rice to these bacteria. Several gram-negative endophytic rice bacteria have been reported (Mukhopadhyay et al., 1996; Adhikari et al., 2001; Tan et al., 2001; Singh et al., 2006), many of which are regarded as beneficial bacteria and future study needs to establish whether the AHLs detected in this study originate from endophytic bacteria. It appears that densities of endophytic bacteria, if rice colonization-infection experiments are performed, are usually low, lower than 107 cells g−1 (James et al., 2002; You et al., 2005). Very low concentrations of natural endophytic bacteria have been detected here, all of which did not appear to produce AHLs under the conditions that were tested.

It is noteworthy to mention that two predominant AHL-like compounds were repeatedly detected from two different rice cultivars and thus it cannot be excluded that the plant could be synthesizing them as several different endophytic species could be present and at very low CFU per gram of plant material. Unfortunately, the amount of molecules obtained was too low to perform structural studies; however, their sensitivity to the very specific AiiA-lactonase and ability to activate three different AHL biosensors is important evidence that these molecules are most likely AHLs. The presence of these molecules in rice could have important biological significance as rice pathogens such as Burkholderia glumae and Burkholderia plantarii utilize AHL QS for expressing virulence-associated factors (Kim et al., 2004; Solis et al., 2006). At present, it is not known whether the compounds detected here are found in rice at a biologically relevant concentration (it was determined that an AHL biosensor based on a GFP reporter system could not be activated in vivo in rice leaves; this detection method, however, does not detect the possible presence of agonist molecules in vacuoles): in addition, the purification procedure used here does not guarantee that most AHL-like compounds present in the stem and roots of rice have been extracted.

In summary, this study has demonstrated the presence of AHL-like compounds inside the rice plant, thus extending the few previous studies of AHL mimic compounds present in root exudates. The important result from this study is that AHL-like molecules are present in rice and future study will be directed towards understanding their source and possible biological significance.

Acknowledgements

R.S. and research activities at the ICGEB Outstation in Ca' Tron, TV, Italy, were supported by the Fondazione Cassamarca, Treviso, Italy.

Footnotes

  • Editor: Yaacov Okon

References

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