The Response of Plants toward N-Acyl Homoserine Lactones ......de Bruijn c73.tex V1 - 12/14/2012...

de Bruijn c73.tex V1 - 12/14/2012 12:14 A.M. Page 775

Chapter 73

The Response of Plants towardN -Acyl Homoserine Lactones ofQuorum-Sensing-Active Bacteria inthe Rhizosphere

Anton HartmannHelmholtz Zentrum Muenchen, German Research Center for Environmental HealthGmbH, Research Unit Microbe-Plant Interactions, Germany

Sebastian T. SchenkJustus-Liebig-University Giessen, Institute of Phytopathology and Applied Zoology,Centre for BioSystems, Land Use and Nutrition, Germany

Tina Riedel and Peter SchröderHelmholtz Zentrum Muenchen, German Research Center for Environmental HealthGmbH, Research Unit Microbe-Plant Interactions, Germany

Adam SchikoraJustus-Liebig-University Giessen, Institute of Phytopathology and Applied Zoology,Centre for BioSystems, Land Use and Nutrition, Germany

73.1 INTRODUCTION

Plants have evolved together with the associated micro-biota ever since their evolution. Thus, plants were alsoconfronted with the elaborate communication systems ofmicrobes colonizing the surfaces of leaves and roots orliving inside plant tissues. From an evolutionary point ofview, it was a major advantage to be able to eavesdropand interfere with the bacterial communication system thatespecially many pathogens are using for organizing theirattack—in addition to the recognition of the microbe-associated molecular patterns (MAMP) (Boller and Felix,2009). Even more, cooperative interactions of plants withsymbiotic microbes were also made possible using thesediffusible bacterial signal molecules.

Molecular Microbial Ecology of the Rhizosphere, Volume 2, First Edition. Edited by Frans J. de Bruijn.© 2013 Wiley-Blackwell. Published 2013 by John Wiley & Sons, Inc.

73.1.1 Bacterial Quorum SensingIt is now known for more than 40 years that single-cellprokaryotic bacteria are able to develop social behavior.Nealson et al. (1970) demonstrated, for the first time,chemical communication among members of a bacte-rial population controlling a luminescent system. Thediffusion of autoinducer molecules as the basis of thecooperative behavior was further studied in detail inVibrio fischeri living in symbiosis with a marine squidand was suggested as general principle of communication(Kaplan and Greenberg, 1985). Low density bacterial pop-ulations produce only minute amount of an autoinducersignal molecule, while in more dense populations (whenthe “quorum” is reached) the signal production is greatly

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776 Chapter 73 The Response of Plants toward N-Acyl Homoserine Lactones of Quorum-Sensing-Active

enhanced (Fuqua et al., 1996; Taga and Bassler, 2003).In the case of V. fischeri, which was the first bacteriumto be known as QS-regulated, the signaling moleculeN-(3-oxo)-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL) was identified to control bioluminescence as aneasily measurable outcome of cooperative behavior. Themajor biosynthetic enzyme (acyl homoserine lactonesynthase–LuxI) uses S-adenosyl methionine and anacyl chain carrier protein to form the HSL moiety. Thespecificity of the HSL molecule is conferred by thelength of the acyl-side chain (between 4 and 18 C-atoms)and the substitution at the C-3 position of the side chain,which can be hydroxylated or oxidized to a keto-group;in rare cases, there could be double bonds in the carbonside chains of triple carbon rings. In addition, severalmimetic or antagonistic molecular structures of HSLsare known (Stevens et al., 2011). Secreted HSLs areperceived by membrane-bound LuxR-type receptors.The HSL–receptor complex acts as a transcriptionalregulator by binding to DNA promoter sequences andactivating the expression of the lux operon and also otheroperons (see below). The response to the HSL-signalingcompounds is an autoinducing process, which makes thistype of regulation extremely economic and sensitive (interms of energy needed for the biosynthesis). Since theinitial description of the luciferase operon in V. fischeri,genome sequence analyses revealed HSL-mediated QShomologues to LuxR/LuxI in many Gram-negativebacteria. QS was found in bacteria living in associationor symbiosis with higher organisms such as plantsand humans as well as in the so-called free-livingbacteria, where it has a central role in biofilm formation.QS-regulated operons are very frequently also found inplant or human pathogenic bacteria, which harbor theirvirulence gene clusters under QS control (Eberl, 1999).

It has been argued that “quorum sensing” faces evo-lutionary problems with non- or overproducing cheaters,which the nonco-operative diffusion sensing does not have(Hense et al., 2007). Signaling in a complex environmentwith many interacting biota and a complex physical struc-ture of the microenvironment always has these interfer-ence problems. However, considering that the majority ofbacteria live in a homogenous environment, such as withina microcolony with signaling among identical progenies,these evolutionary problems do not exist. Small diffusibleautoinducing signal molecules exploring the environmen-tal space in addition to scoring the own population densityand the density of neighbors speaking the same languageimprove the value of such a signaling tool, because itintegrates information about the overall habitat quality.On the basis of this information, the expression of thegenetic potential can be optimized and therefore the effi-ciency of metabolic reactions improved. In evolutionaryterms, such regulation is of utmost relevance and thus

the term “efficiency sensing,” which implies a direct pos-itive impact on evolutionary selection, was suggested as analternative term for “quorum sensing” (Hense et al., 2007).Concerning the role of QS autoinducer molecules in cross-kingdom interactions, it has to be considered, whetherthey are simply used for recognition (chemical smell) ofa nonself-organism or a real “intended” signaling charac-ter (Diggle et al., 2007), for example, QS-molecules ofpathogenic Gram-negative bacteria (such as 3-oxo-C12-HSL) and similar molecules could constitute a warningsignal for eukaryotes and should therefore be called “cue”compounds, according to Stacy et al. (2012).

The signal character of other small bacterialmolecules is known for quinolones (for review see Huseand Whiteley, 2011), diketopiperazines of Pseudomonads Q1(McKnight et al., 2000), and AI2 (alternative autoin-ducer, furanosyl borate ester) (Taga and Bassler, 2003). InGram-positive bacteria, a variety of cyclic peptides (Lyonand Novick, 2004), AI2 and butyro-lactones (Folcheret al., 2001), were identified. Some of these signalmolecules have, in addition, important roles in processesunrelated to signaling, such as nutrient scavenging, ultra-structure modification, and competition between bacteria(Schertzer et al., 2009). In particular iron siderophores,such as pyochelin and quinolone (see above), whichinterfere with cellular iron homeostasis, are small signalmolecules with central functions in the iron uptake. Smalldiffusible molecules, which diffuse out of the cell orcan be even actively transported, are, however, commonin all these systems. It has been recently suggestedthat transport processes across the bacterial membranemay even improve the specificity and response towarddifferent chemical forms of HSL compounds (Minagawaet al., 2012).

73.1.2 Specific, Highly ResolvingN-Acyl Homoserine LactoneAnalysis ToolsThe availability of specific, sensitive, and quantitativebioreporter and chemical tools to analyze quorum sensingautoinducer molecules is an essential prerequisite forinnovative and in depth studies of quorum sensing(Fekete et al., 2010b). Common liquid chromatographicmethods usually fail because of the required clean upand the lack of favorable functions on the moleculesthat would allow easy detection. However, the use ofmodern analytical tools such as UPLC (ultra performanceliquid chromatography) coupled to high resolving Fouriertransform ion cyclotron mass spectrometry (FTICR-MS)makes detection and identification of HSLs very accurate(Li et al., 2006). The method of choice combines solid-phase extraction (SPE), followed by direct transfer to aUPLC column; it allows the quantitative determination


73.3 Direct Evidence for HSL-Induced Responses in Plants 777

of different N-acyl homoserine lactones of several Gram-negative bacteria with UV detection, without sampleloss. Alternatively, additional information can be gainedby applying coupling with FTICR-MS, which allows thediscrimination of HSL molecule structures in extenso andidentification of homoserine compounds (after chemicalor enzymatic lactonase cleavage) (Frommberger et al.,2004). A completely different independent approach ofHSL detection is possible through the application ofspecific monoclonal antibodies against HSL molecules(Kaufmann et al., 2006; Chen et al., 2010a, b). Applyingthese techniques, a specific tracing of these QS moleculesin the environment and within eukaryotic hosts, colonizedby HSL-producing bacteria, was made feasible (seebelow).

73.2 DIVERSE EUKARYOTESRESPOND TO HSLs

73.2.1 HSLs and Responsein Invertebrates and MammalsLong as well as short-side-chain N-acyl-homoserinelactones modulate the host immune response and inflam-matory signaling pathways of invertebrates provokingsevere defense reactions by the host tissue in invertebrates(for review, see Cooley et al., 2008; Teplitski et al.,2011). In mammals, the effects of 3-oxo-C12-HSL, themajor autoinducer of the lung infection-causing bacteriumPseudomonas aeruginosa, were thoroughly investigated.Q2The P. aeruginosa HSL interacts with the host’s immuneresponse via a mechanism independent of the canoni-cal pathogen-associated molecular pattern recognitionreceptor pathway (Kravchenko et al., 2006). For theimmune-modulatory activity of HSL, a chain lengthlonger than 10 C-atoms, an intact homoserine lactonering, and oxo- or hydroxyl substitutes are impor-tant prerequisites. It seems that the primary immuneresponse triggered by microbial elicitors such as bacteriallipopolysaccharide was halted or neutralized by theinteraction with 3-oxo-C12-HSL.

73.2.2 HSL-Producing BacteriaInduce Plant ResponsesThe first indication that QS molecules of rhizospherebacteria influence plant defense responses came fromstudies on the interaction between Serratia liquefaciensMG1 and tomato plants (Schuhegger et al., 2006). S.liquefaciens MG1 produces C4- and C6-homoserinelactones when colonizing the root surface (Gantner et al.,2006; see also Chapter 74). Colonization of the rootsurface with S. liquefaciens induced systemic resistance

against the leaf pathogenic fungus Alternaria alternatain tomato, whereas the HSL-negative S. liquefaciensmutant MG44 was not able to induce such resistance(Schuhegger et al., 2006). The authors also observed anincreased salicylic acid (SA) level in the leaves and highinduction of SA- and ethylene-dependent defense-relatedgenes (pathogenesis-related 1a [PR1a] and basic chiti-nase) in tomato plants colonized by the HSL-producingS. liquefaciens MG1. In a similar manner, colonizationwith the HSL-producing Serratia plymuthica wild-typestrain HRO-C48 protected cucumber plants (Cucumissativus) from the damping-off disease caused by thePythium aphanidermatum oomycete, as well as tomato(Solanum lycopersicum) and bean plants (Phaseolusvulgaris) from infection with the gray mold-causingfungus Botrytis cinerea (Pang et al., 2009). Similar to thestudy with S. liquefaciens MG44 and tomato, the authorsdemonstrated that the splI– mutant of S. plymuthica,impaired in the production of HSLs, could not provideprotection against P. aphanidermatum and B. cinerea.These results provided evidence that HSLs play animportant role in the induction of plant defense. How-ever, contrasting results were reported for the interactionbetween Arabidopsis thaliana and S. liquefaciens MG1and its HSL-negative mutant MG44 (von Rad et al.,2008). Because the resistance against the pathogenicbacterium Pseudomonas syringae on A. thaliana leaveswas not differentially induced by the S. liquefaciens wildtype and its HSL-negative mutant, authors suggested anHSL-independent resistance increasing effect against P.syringae caused by root colonization with S. liquefaciens.This effect is probably based on the resistance inducedby MAMPs, the so-called MAMP-triggered immunity(MTI) (von Rad et al., 2008). Thus, bacteria–plantinteraction experiments with living microbial cells haveto be interpreted very carefully, as different bacteriacan induce different plant responses independent of theQS autoinducer system. A helpful means to circumventthis disturbing overlap effect is the use of pure HSLcompounds.

73.3 DIRECT EVIDENCE FORHSL-INDUCED RESPONSESIN PLANTS

73.3.1 HSL-Induced Resistancein PlantsThe application of many different HSL molecules at aconcentration range of 1–10 μM to roots in an axenicsystem was shown to induce resistance in diverse plants.In tomato plants, treatment with C4- or C6-HSL leads toa systemic induction of genes involved in defenses (PR1a



and PR1) as well as the 26 kDa acidic chitinase, putativeascorbate peroxidase, and the proteinase inhibitor CEVI57(Schuhegger et al., 2006). Different long-side-chain HSLsinduced resistance against biotrophic and hemibiotrophicpathogens in A. thaliana plants (Schikora et al., 2011).For example, the N-3-oxo-tetradecanoyl-l-homoserinelactone (oxo-C14-HSL) significantly enhanced the resis-tance against P. syringae pv. tomato DC3000 (Pst).Similar observations were made in the case of the fungalpathogens Golovinomyces orontii, the causal agent ofpowdery mildew on A. thaliana. In barley (Hordeumvulgare), oxo-C14-HSL also induced systemic resistanceagainst Blumeria graminis, the causal agent of powderymildew on barley (Fig. 73.1) (Schikora et al., 2011).Likewise, OH-C14-HSL and oxo-C12-HSL have alsoresistance-inducing potential, although weaker thanC14-HSL derivatives.

In contrast to those findings, treatment with the short-chain C6-HSL did not induce resistance in the A. thalianaplants, as has been found by von Rad et al. (2008). Theinduction of genes related to systemic pathogen response,such as chitinase and PR1, in Arabidopsis treated withC4- or C6-HSLs was not elevated, implying no effect onresistance by these compounds. Accordingly, no effect ofC6-HSL treatment was found on the resistance against Pstbacteria (von Rad et al., 2008).

73.3.2 Up-Regulation of DefenseMechanisms on Treatment withSpecific HSLsPersistent induction of defense mechanisms can lead tosevere deregulation of plant metabolism and inhibitionof plant growth. In order to avoid the negative effects ofhigh concentrations of SA and reactive oxygen species(ROS), which are the consequence of induced defensemechanisms, plants tightly regulated their defenseresponses. Preferentially, such responses are induced onlyshort time after recognition of pathogens. During thestudy of HSL-induced resistance in Arabidopsis plants,a deregulation of mitogen-activated protein kinase 3 and6 (MPK3 and MPK6) was discovered (Schikora et al.,2011). Both MPK3 and MPK6 are known to be involvedin plant defense (Asai et al., 2002). In Arabidopsis,as in other plants, treatment with the bacterial MAMPflg22 triggers transient activation of MPK3 and MPK6;this activity normally decreases shortly after treatment.However, in plants treated with 3-oxo-C14-HSL, theactivation of MPK3 and MPK6 was enhanced (Schikoraet al., 2011). In a normal case, flg22-induced activationof MAPKs is followed by the induced expression ofspecific transcription factors and PR-genes, for example,WRKY22, WRKY29, and PR1 (for review see Colcombetand Hirt, 2008; Pitzschke et al., 2009). In the case of

(a)

(b) (c) (d)

Figure 73.1 Oxo-C14-HSL induced resistance in barley plants.Infection with Blumeria graminis f.sp. hordei , the causative agent ofpowdery mildew on barley (Hordeum vulgare) plants, causes diversedefense responses; ranging from no response and fungal proliferationevidenced by elongated secondary hyphae (ESH), the formation ofpapillae, which stops the fungal penetration attempt to hypersensiti-vity response (HR) abolishing the further growth of this biotrophicfungus. (a) Percentage of interactions on 5 days old barley plantspretreated for 3 days with 6 μM of oxo-C14-HSL, acetone or mock,48 h after inoculation with B. graminis f.sp. hordei conidia, (b)Elongated secondary hyphae from B. graminis on barley leaf, (c)Papillae formation on site of infection. (d) Cell death (HR) as aconsequence of response to infection.

HSL-treated plants, the prolonged activation of MAPKsleads to a stronger induction of several defense-relatedgenes (WRKY22 and WRKY29 as well as PR1). The obser-vation that MAPK activation through MPK6 is indeedthe molecular base of HSL-induced resistance was furthersupported by the fact that in the mpk6 mutant, the HSLeffects on transcriptional activation of WRKY22 andWRKY29, as well as the induced resistance against Pstwere abolished (Schikora et al., 2011).

73.3.3 HSLs Modulate PlantDevelopmentA detailed assessment of HSL effects on plant devel-opment revealed that C4- and C6-HSL promote rootelongation (von Rad et al., 2008). Transcriptome analysesindicated that many cell wall and cell-growth-related


73.4 Bi-Functionality Of HSLs as A Group of Molecules 779

Synopsis* of HSL-effects on Arabidopsis thaliana

C6-HSL C8-HSL C10-HSL C12-HSL C14-HSL

____________________________________________________________________

Growth - -

Resistance

(systemic - - -

and local)

*Data from literature included:

von Rad et al. 2008 Planta 229: 73–85

Pang et al. 2009 Eur J Plant Pathol 124: 261–268

Schikora et al. 2011 Plant Physiol 157: 1407–1418

Schenk et al. 2012 Plant Signal Behav 7: 178–181Figure 73.2 Synopsis of HSL-effects on Arabidopsisthaliana .

genes are differentially expressed on treatment with theseshort-chain HSLs. This was confirmed by Liu et al.(2012), who studied two accession lines of Arabidopsisthaliana. The authors found that oxo-C6-HSL and oxo-C8-HSL promoted root elongation at concentration rangeof 1–10 μM. C10-HSL alters root architecture similarto auxin, causing shortage and thickening of the primaryroot; however, at rather high concentrations (100 μM),further experiments indicated that those changes areauxin independent (Ortiz-Castro et al., 2008). The samegroup has shown that C12-HSL strongly induces roothair formation (Ortiz-Castro et al., 2008). Very recentlyoxo-C10-HSL, but not its unmodified homolog C10-HSL,was shown to induce the formation of adventitious rootin mung bean plants (Vigna radiata) (Bai et al., 2012).The authors suggest that oxo-C10-HSL accelerates thebasipetal auxin transport and that the auxin-dependentformation of adventitious root relies on H2O2- andNO-dependent cGMP signaling in these plants (Bai et al.,Q32012). The G-protein-coupled receptor GCR1 and thesole canonical Gα subunit GPA1 were very recentlyreported to be required for the HSL-mediated growthpromotion in Arabidopsis plant (Liu et al., 2012). Theroot elongation promoted by the short-chain oxo-C6-HSLand oxo-C8-HSL was lost in the gcr1 and gpa1 mutants,whereas plants overexpressing GPA1 reacted strongerto HSLs then the wild-type plants (Liu et al., 2012),

implicating an active perception mechanism of HSLs inplants.

73.4 BI-FUNCTIONALITY OF HSLsAS A GROUP OF MOLECULES

N-acyl homoserine lactones vary in the length of thelipid side chain and the substitution on the C3 carbon(O– or OH– group). Especially, the length of the lipidside chain seems to be important for the reaction ofplants. As indicated above, different HSLs trigger diverseplant reactions. C4-HSL, C6-HSL, oxo-C6-HSL, andoxo-C8-HSL promoted growth of Arabidopsis (von Radet al., 2008; Liu et al., 2012; Fig. 73.2). Oxo-C10-HSLinduced the formation of adventitious roots in mungbeans (Bai et al., 2012). On the other hand, oxo-C14-HSLand to lesser extent OH-C14-HSL induced resistancein Arabidopsis and barley plants toward biotrophicand hemibiotrophic pathogens (Schikora et al., 2011).Likewise, oxo-C12-HSL has also resistance-inducingpotential, although weaker than C14-HSL derivatives(Schenk et al., 2012). Comparison of five different HSLsdiffering in the length of their lipid moieties rangingfrom 6 to 14 carbons on plants growth revealed cleardifferences in plant reactions (Fig. 73.2). The increase inshoot’s biomass caused by short-chained HSL was gradu-ally smaller in plants treated with HSL possessing longer



lipid moiety and lost when the long-chained HSLs wereadded to the medium (Schenk et al., 2012). Regardingroot’s biomass, the situation was even more evident; onlyshort-chained HSLs were able to significantly increasethe accumulation of Arabidopsis root’s biomass, whiletreatment with other HSLs provoked no changes (vonRad et al., 2008; Liu et al., 2012; Schenk et al., 2012).In contrast to the growth promoting effect, moleculeswith longer lipid chains induce resistance in Arabidopsisplants (Fig. 73.2). The apparent different reactions tolong- and short-chained HSLs may suggest that differentreceptors or at least different signaling pathways areinvolved in these responses. Mathesius et al. (2003)showed that on treatment with different HSLs, one-thirdof the differentially accumulated proteins are specific forthe respective HSL (Mathesius et al., 2003). However, thequestion how plants perceive the HSLs and distinguishbetween those molecules remains open.

73.4.1 Hormonal Effectsin HSL-Treated PlantsThe various effects on plants raise the question whetherAHLs modulate the plant’s hormonal balance. Analysis ofroots and leaves of plants treated with C6-HSL revealed analtered expression of auxin-regulated genes and an alteredratio between the free IAA (auxin) and cytokinins, whencompared to nontreated plants (von Rad et al., 2008).Proteomic analysis performed on AHL-treated Medicagotruncatula plants suggested deregulation in auxin levelson AHL treatment (Mathesius et al., 2003). The authorsreported temporary changes in the auxin-induced proteinTC51487 and increased accumulation of indol-3-acetate-β-glucosyltransferase, an auxin-degrading enzyme, whichimplies changes in auxin levels. Because of the impor-tance of auxin in virtually all aspects of plant develop-ment, a deregulation in its level could cause the observedeffects. However, the relationship between altered auxinconcentration and HSL was not verified in the study ofaux1-7, axr2, and doc1 mutants in Arabidopsis (Ortiz-Castro et al., 2008), leaving an open question on the exactmechanism of HSL action. However, the concentrationdependency of HSL effects was not clearly taken intoaccount in these studies. Not only auxin but also SA levelsare altered after exposure to HSLs. SA is the most promi-nent hormone involved in induced-systemic resistance andcould control the HSL-induced defense. The SA levelsare elevated in S. liquefaciens MG1-treated tomato plants.However, they are not elevated in plants treated with theHSL-negative S. liquefaciens MG44 strain. Furthermore,an application of C6-HSL, the major HSL species pro-duced by S. liquefaciens, leads to a local accumulationof SA in roots. The enhanced expression of salicylic-acid-dependent PR1 and the ethylene-dependent 30 kDa

chitinase in HSL-treated tomato plants further supportsthe theory of hormone association in this defense mech-anism (Schuhegger et al., 2006; Schikora et al., 2011).How HSLs influence the levels of the different hormones,either on the level of genetic regulation or on the post-transcriptional level, is currently unknown. Many ques-tions on the underlying mechanism as well as on possiblefunctional associations between particular HSLs and spe-cific hormone(s) and other physiological responses remainstill unanswered.

73.4.2 Different HSL-Uptakeof Plants and New Hints of DirectHSL Effects on Plant PhysiologyA very important question in understanding the impactof HSLs on plants is whether HSLs are taken up byplants. The transport of HSLs within plants has been stud-ied initially in barley and Arabidopsis, which lack HSLlactonases (Götz et al., 2007; von Rad et al., 2008).

The use of radioactive-labeled HSLs (all-tritiatedcompounds, 3H-HSL) enabled the detection of HSLseven in picomolar concentrations. Götz et al. (2007) andRiedel et al. (manuscript in preparation) were able tofollow the uptake of 3H-HSLs in roots of barley in ahighly resolved manner in the first 24 h after application.First signals of C8-HSL were detected already after 2 hin the shoots; C10-HSL was transported at a significantlylower rate. Since a high amount of HSL is bound tothe root surface, the transport of HSL through the roothad to be scrutinized as real translocation into a thirdseparated compartment in excised roots using a Pitmanchamber to minimize the contact between the root surfaceand the radioactive-labeled compound (Pitman, 1971).On addition of ortho-vanadate (VO4

3−), an inhibitorof energy-dependent ABC transporters, the 3H-HSL-transport was abolished. In addition, the HSL transportwas also blocked in the presence of KCl, indicatingsymplastic transport through the roots. Furthermore, theuptake of C8-HSL was enhanced in transpiring plants,which demonstrates that apoplastic transport of HSLalso contributes to the observed transport. Interestingly,Joseph and Phillips (2003) detected a 30% enhanced tran-spiration rate after application of HSLs, which indicatedthat HSLs might influence the water transport throughroots. Using autoradiography, Riedel et al. (manuscript inpreparation) could show in addition to the high amountsof 3H-HSL bound to roots surface, a high concentrationof 3H-C8-HSL in the central cylinder, suggesting thatHSLs are taken up into the central cylinder and aretransported along the root. Since the radioactive detectioncould not discriminate between the original substanceand any metabolic alterations of the HSL within theplant, additional experiments, for example, using UPLC


73.6 Summary and Conclusions 781

and FTICR-MS are necessary. Götz et al. (2007) couldverify the C8-HSL compound in shoots of barley usingFTICR-MS. This was supported by the analysis of C8-and C10-HSLs using the highly specific HSL-biosensorassay in sap obtained from leaves of Arabidopsis andbarley, respectively (Schikora et al., 2011; Riedel et al.,manuscript in preparation). Using specific monoclonalantibodies against C10-HSL and the ELISA-technique(Chen et al., 2010a, 2010b), the identity of functionalHSLs in the stem sap was confirmed. The concentrationof C10-HSL in the stem sap was calculated to be 0.7μM, when measured 4 h after the application of 10 μMC10-HSL to the roots of 17 day old barley seedlings(Riedel et al., manuscript in preparation).

Most interestingly, using the DAF-2DA method, theproduction of NO in C8-HSL-treated roots was detectedonly 50 min after treatment (Riedel et al., manuscript inpreparation). Accordingly, NO could be the transmitterof HSL-effects to other physiological processes inroots and shoots. For example, a dramatic decrease ofcatalase and ascorbate-peroxidase activities in roots andan increase in the shoots was observed (Riedel et al.,manuscript in preparation). Glutathione S-transferaseisoenzymes responded in a differential way in shoots androots to HSL treatments. Finally, using in vitro enzymeassays, C8-HSL could be demonstrated to be a possiblesubstrate for an apoplastic peroxidase and cytochromeP450 monooxygenase in barley. This could be an initialstep for metabolism and finally degradation of HSLs inplanta.

73.5 DISTURBANCE OF BACTERIALQUORUM SENSING SIGNALING BYLACTONASES AND HSL MIMICS

QS molecules play a major role in the virulence of plantpathogens. Therefore, a good strategy to prevent patho-genesis is the interruption of the bacterial communicationsystem (Dong et al., 2001; Gonzalez and Keshavan, 2006;Zhang et al., 2007; Haudecoeur et al., 2009a; Haude-coeur et al., 2009b). Some plants and algae produce mimicsubstances for N-acyl homoserine lactones (Bauer andQ4Mathesius, 2004). These compounds specifically blockthe quorum sensing system of bacteria and are efficientlyinterfering, for example, with biofilm formation or viru-lence acquisition of these bacteria. Interestingly, it wasrecently discovered that certain root-associated bacteriaare able to convert plant compounds, such as coumaricacid, into homoserine lactones (Schaefer et al., 2008).However, the physiological impact of this activity forbacteria–plant interaction is not understood yet.Q5

As mentioned above, plants are able to perceiveQS molecules and respond in a specific manner. A very

interesting feedback mechanism, based on a fine tunedregulation of the QS communication, was describedin the interaction between the tumor-inducing bac-terium Agrobacterium tumefaciens and its host plant(Haudecoeur and Faure, 2010). In the case of A. tume-faciens, the virulence is dependent on the production ofoxo-C8-HSL, which stimulates the replication of the Tiplasmid. On the other hand, an unsuitable accumulationof the HSL and therefore excessive action of bacteriacan be prevented by two HSL-degrading lactonases:AttM and AiiB expressed by Agrobacterium itself(Haudecoeur et al., 2009a). This negative regulation ofHSL accumulation seems to be exploited by variousplants, as different plant-originated compounds mayregulate the expression of attM and aiiB genes. Theaccumulation of γ-aminobutyrate (GABA) and SA inA. tumefaciens-infected plants was shown to inhibitthe expression of the attJ repressor, which inhibits thetranscription of attM (Haudecoeur et al., 2009b). Anotherexample of a lactonase-like activity in HSL-producingrhizobacteria was described in the plant-growth pro-moting rhizosphere bacterium Pseudomonas putida IsoF(Fekete et al., 2010a). In this bacterium, the hydrolysisof the lactone ring starts shortly after their productionand release into the medium. Possibly, this inactivationof freshly excreted HSLs could increase the signalingcharacter and would avoid high signal background noise,reducing also the risk of provoking defense responses bythe plant.

Lactonases, produced by the host plant, are widelydistributed means to effectively interfere with the bacte-rial QS system. Transgenic tobacco (Nicotiana tabacum)and potato (Solanum tuberosum) plants expressing the aiiAlactonase from Bacillus spp. show increased resistanceagainst the soft rot-causing pathogen Erwinia carotovora(Dong et al., 2001). Moreover, the Tr5 strain of the insec-ticidal pesticide Bacillus thuringiensis, containing the slh-aiiA fusion gene, also inhibits soft rot disease caused byE. carotovora (Zhang et al., 2007). These findings openthe possibility to use the interactions between plant andsoil-borne bacteria in a crop protection approach. On theother hand, transgenic tomato plants, producing differenttypes of HSL compounds themselves, altered the activ-ity of plant growth promoting bacteria associated with itsroots, which resulted in quenching or enhancement of theirplant growth supportive activities (Barriuso et al., 2008).

73.6 SUMMARY ANDCONCLUSIONS

Different plants such as Arabidopsis, barley, or tomatoare able to respond to bacterial QS molecules of theN-acyl homoserine lactone (HSL) type. Plants change



their pattern of gene expression, protein profile, andmodify their development when HSLs are present inthe rhizosphere. A very interesting phenomenon is thatapparently two different response patterns (defense orgrowth stimulation) are induced by long-side-chain orshort-side-chain HSLs, respectively (Fig. 73.2). MPK6was identified as an essential kinase in the oxo-C14-HSL/C12-HSL signaling, whereas the GCR1 G-proteinand the canonical Gα subunit GAP1 are required forC6/C8/C10-HSL signaling in Arabidopsis. The lengthof the acyl moiety substantially decreased the HSLmotility within the plant. The short- and medium-lengthside-chain HSLs (C4–C10), but not the oxo-C14-HSL,were detected in shoot sap after treatment of roots.Nevertheless, oxo-C14-HSL induced resistance in leavesagainst biotrophic leaf pathogens, suggesting systemicallyinduced resistance. Therefore, a long-distance signal waspostulated to induce the systemic effect of hydrophobicHSLs (Schikora et al., 2011). Further investigations onthe detailed receptor and perception mechanism involvedin these two HSL-induced signal response patterns ofplants will be important for further understanding ofhow bacterial HSLs influence host plants. Very strikingis the fact that the perception of HSLs seems to haveopposite effects on plant and animal immunity. Whiletheir presence abolishes some of the immune responsesin animals, it seems to induce defense mechanisms inplants.

Studies of HSL effects on plants clearly show thatthe response of plants to microbes cannot be restrictedto the classical MAMP recognition. Consequently, plantsshould be considered as an organized “superorganism”of a eukaryote interacting in many ways with its spe-cific microbiome (Eberl, 2012). Apart from the MAMPsand QS compounds, auxins, diverse volatiles, and manymore yet unknown bacterial metabolites also have sig-naling characters for the host, originating from the plantenvironment including the rhizosphere. It can be assumedthat as a result of these multiple interacting signals, akind of “immunity homoeostasis” is reached in a well-performing, healthy plant “superorganism,” also called theholobiont (Zilber-Rosenberg and Rosenberg, 2008). Thisnew vision of real-world eukaryotes as holobiontic sys-tems may eventually provide new ideas of stabilizing orretaining a healthy state of eukaryotes (plants and ani-mals/human alike).

REFERENCES

Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, et al. MAP kinase signalling cascade in Arabidopsis innateimmunity. Nature 2002;415:977–983.

Bai X, Todd CD, Desikan R, Yang Y, Hu X. N-3-oxo-decanoyl-L-homoserine-lactone activates auxin-induced adventitious root forma-tion via hydrogen peroxide- and nitric oxide-dependent cyclic GMPsignalling in Mung bean. Plant Physiol 2012;158:725–736.

Barriuso J, Solano BR, Fray RG, Cámara M, Hartmann A,Gutierrez-Manero FJ. Transgenic tomato plants alter quorumsensing in plant-growth-promoting rhizobacteria. Plant Biotechnol2008;6:442–452.

Boller T, Felix G. A renaissance of elicitors: perceptionof microbe-associated molecular patterns and danger signals bypattern-recognition receptors. Annu Rev Plant Biol 2009;60:379–406.

Chen X, Buddrus-Schiemann K, Rothballer M, Krämer P, Hart-mann A. Detection of quorum sensing molecules in Burkholde-ria cepacia culture supernatants with enzyme-linked immunosorbentassays. Anal Bioanal Chem 2010a;398:2669–2676.

Chen X, Kremmer E, Gouzy MF, Clausen E, Starke M,Wöllner K, et al. Development and characterization of rat mon-oclonal antibodies for N-acylated homoserine lactones. Anal BioanalChem 2010b;398:2655–2667.

Diggle SP, Gardner A, West SA, Griffin AS. Evolutionary theory ofbacterial quorum sensing: when is a signal not a signal? Philos TransR Soc London Ser B 2007;362:1241–1249.

Colcombet J, Hirt H. Arabidopsis MAPKs: a complex signallingnetwork involved in multiple biological processes. Biochem J2008;413:217–226.

Cooley M, Chhabra SR, Williams P. N-acyl homoserine lactone-mediated quorum sensing: A twist in the tail and a blow for hostimmunity. Chem Biol 2008;15:1141–1147.

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;411:813–817.

Eberl G. A new vision of immunity: homoeostasis of the superorgan-ism. Mucosal Immunol 2012;3:450–460.

Eberl L. N-acyl-homoserine lactone-mediated gene regulationin Gram-negative bacteria. Syst Appl Microbiol 1999;22:493–506.

Fekete A, Kuttler C, Rothballer M, Fischer D, Buddrus K,Hense BA, et al. Dynamic regulation of AHL-production anddegradation in Pseudomonas putida IsoF. FEMS Microbiol Ecol2010a;72:22–34.

Fekete A, Rothballer M, Hartmann A, Schmitt-Kopplin P.Identification of bacterial autoinducers. In: Kraemer R, Jung K, edi-tors. Bacterial Signalling . Weinheim, Germany: Wiley Publishers;2010b. p 95–111.

Folcher M, Gaillard H, Nguyen LT, Nguyen KT, Lacroix P,Bamas-Jacques N, et al. Pleiotropic functions of a Streptomycespristinaespiralis autoregulator receptor in development, antibioticbiosynthesis, and expression of a superoxide dismutase. J Biol Chem2001;276:44297–44306.

Frommberger M, Schmitt-Kopplin P, Ping G, Frisch H, SchmidM, Zhang Y, et al. A simple and robust set-up for on-columnsample preconcentration - nano-liquid chromatography - electrosprayionization mass spectrometry for the analysis of N-homoserinelactone. Anal Bioanal Chem 2004;378:1014–1020.

Fuqua C, Winans SC, Greenberg EP. Census and consen-sus in bacterial ecosystems: the luxR-luxI family of quorumsensing transcriptional regulators. Annu Rev Microbiol 1996;50:727–751.

Gantner S, Schmid M, Dürr C, Schuhegger R, Steidle A,Hutzler P, et al. In situ quantitation of the spatial scale of call-ing distances and population density-independent N-acylhomoserinelactone-mediated communication by rhizobacteria colonized on plantroots. FEMS Microbiol Ecol 2006;56:188–194.


References 783

Götz C, Fekete A, Gebefügi I, Forczek ST, Fuksova K,Li X, et al. Uptake, degradation and chiral discrimination ofN-Acyl-D/L-homoserine lactones by barley (Hordeum vulgare)and yam bean (Pachyrhizus erosus) plants. Anal Bioanal Chem2007;389:1447–1457.

Gonzalez JE, Keshavan ND. Messing with bacterial quorum sensing.Microb Mol Biol Rev 2006;70:859–875.

Haudecoeur E, Faure D. A fine control of quorum-sensing com-munication in Agrobacterium tumefaciens. Commun Integr Biol2010;3:84–88.

Haudecoeur E, Planamente S, Cirou A, Tannieres M, Shelp BJ,Morera S, Faure D. Proline antagonizes GABA-induced quenchingof quorum-sensing in Agrobacterium tumefaciens. Proc Nat Acad SciUSA 2009a;106:14587–14592.

Haudecoeur E, Tannieres M, Cirou A, Raffoux A, Dessaux Y,Faure D. Different regulation and roles of lactonases AiiB andAttM in Agrobacterium tumefaciens C58. Mol Plant Microbe Interact2009b;22:529–537.

Hense BA, Kuttler C, Müller J, Rothballer M, Hartmann A,Kreft JU. Does efficiency sensing unify diffusion and quorum sens-ing? Nature Rev Microbiol 2007;5:230–239.

Huse H, Whiteley M. 4-quinolones: smart phones of the microbialworld. Chem Rev 2011;111:152–159.

Joseph CM, Phillips DA. Metabolites from soil bacteria affect plantwater relations. Plant Physiol Biochem 2003;41:189–192.

Kaplan HB, Greenberg EP. Diffusion of autoinducer is involved inregulation of the Vibrio fischeri luminescence system. J Bacteriol1985;163:1210–1214.

Kaufmann GF, Sartorio R, Lee SH, Mee JM, Altobell LJ,Kujawa DP, et al. Antibody interference with N-acyl homoser-ine lactone-mediated bacterial quorum sensing. J Am Chem Soc2006;128:2802–2803.

Kravchenko VV, Kaufmann GF, Mathison JC, Scott DA, KatzAZ, Wood MR, et al. N-(3-oxo-acyl)homoserine lactones signal cellactivation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways. J BiolChem 2006;281:28822–28830.

Li X, Fekete A, Englmann M, Götz C, Rothballer M,Buddrus K, et al. Development of a solid phase extraction - ultrapressure liquid chromatography method for the determination of N-acyl homoserine lactones from bacterial supernatants. J ChromatogrA 2006;1134:186–193.

Liu F, Bian Z, Jia Z, Zhao Q, Song S. The GCR1 and GPA1 participatein promotion of Arabidopsis primary root elongation induced by N-acyl-homoserine lactones, the bacterial quorum-sensing signals. MolPlant Microbe Interact 2012;25:677–683.

Lyon GJ, Novick C. Peptide signalling in Staphylococcus aureus andother Gram-positive bacteria. Peptides 2004;25:1389–1403.

Mathesius U, Mulders S, Gao M, Teplitski M, Caetano-Anolles G, Rolfe BG, Bauer WD. Extensive and specific responsesof a eukaryote to bacterial quorum-sensing signals. Proc Nat Acad SciUSA 2003;100:1444–1449.

McKnight SL, Iglewski BH, Pesci EC. The Pseudomonas quinolonesignal regulates rhl quorum sensing in Pseudomonas aeruginosa. JBacteriol 2000;182:2702–2708.

Minagawa S, Inami H, Kato T, Sawada S, Yasuki T, Miyairi S,Horikawa M, Okuda J, Gotoh N. 2012. RND type efflux pump

system MexAB-OprM of Pseudomonas aeruginosa selects bacteriallanguages, 3-oxo-acyl homoserine lactones, for cell-to-cell communi-cation. BMC Microbiol 12: 70 (doi: 10.1186/1471-2180-12-70)

Nealson KH, Platt T, Hastings JW. Cellular control of the syn-thesis and activity of the bacterial luminescent system. J Bacteriol1970;104:313–322.

Ortiz-Castro R, Martinez-Trujillo M, Lopez-Bucio J. N-acyl-L-homoserine lactones: a class of bacterial quorum-sensing signals alterpost-embryonic root development in Arabidopsis thaliana. Plant CellEnviron 2008;31:1497–1509.

Pang Y, Liu X, Ma Y, Chernin L, Berg G, Gao K. Induction ofsystemic resistance, root colonization and biocontrol activities of therhizospheric strain of Serratia plymuthica are dependent on N-acylhomoserine lactones. Eur J Plant Pathol 2009;124:261–268.

Pitman MG. Uptake and transport of ions in barley roots. Aust J BiolSci 1971;24:407–421.

Pitzschke A, Schikora A, Hirt H. MAPK cascade signalling networksin plant defence. Curr Opin Plant Biol 2009;12:421–426.

Schaefer AL, Greenberg EP, Oliver CM, Oda Y, Huang JJ,Bittan-Banin G, et al. A new class of homoserine lactone quorum-sensing signals. Nature 2008;454:595–599.

Schenk ST, Stein E, Kogel KH, Schikora A. Arabidopsis growthand defence are modulated by bacterial quorum sensing molecules.Plant Signal Behav 2012;7:178–181.

Schikora A, Schenk ST, Stein E, Molitor A, Zuccaro A,Kogel KH. N-acyl-homoserine lactone confers resistance towardsbiotrophic and hemibiotrophic pathogens via altered activation ofAtMPK6. Plant Physiol 2011;157:1407–1418.

Schertzer JW, Boulette ML, Whiteley M. More than a signal: non-signalling properties of quorum sensing molecules. Trends Microbiol2009;17:189–195.

Schuhegger R, Ihring A, Gantner S, Bahnweg G, Knappe C,Vogg G, et al. Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria. Plant CellEnviron 2006;29:909–918.

Stacy AR, Diggle SP, Whiteley M. Rules of engagement:defining bacterial communication. Curr Opin Microbiol 2012;15:155–161.

Stevens AM, Queneau Y, Soulère L, Beck von Bodman S,Doutheau A. Mechanisms and synthetic modulators of AHL-dependent gene regulation. Chem Rev 2011;111:4–27.

Taga ME, Bassler BL. Chemical communication among bacteria. ProcNat Acad Sci USA 2003;100:1449–1454.

Teplitski M, Mathesius U, Rumbaugh KP. Perception and degrada-tion of N-acyl homoserine lactone quorum sensing signals by mam-malian and plant cells. Chem Rev 2011;111:100–116.

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 inthe rhizosphere. Planta 2008;229:73–85.

Zhang L, Ruan L, Hu C, Wu H, Chen S, Yu Z, Sun M. Fusion of thegenes for AHL-lactonase and S-layer protein in Bacillus thuringiensisincreases its ability to inhibit soft rot caused by Erwinia carotovora.Appl Microbiol Biotechnol 2007;74:667–675.

Zilber-Rosenberg I, Rosenberg E. Role of microorganisms in theevolution of animals and plants: the hologenome theory of evolution.FEMS Microbiol Rev 2008;32:723–735.

de Bruijn c73.tex V1 - 12/14/2012 12:14 A.M.

Queries in Chapter 73

Q1. The citation “Huse and Whiley, 2011” (original) has been changed to “Huse and Whiteley, 2011”. Please check ifappropriate.

Q2. The citation “Teplitzki et al., 2011” (original) has been changed to “Teplitski et al., 2011”. Please check if appro-priate.

Q3. Please provide expansion for cGMP, AHL.

Q4. Reference Bauer and Mathesius (2004) has not been included in the Reference List, please supply full publicationdetails.

Q5. The citation “Schaefer et al., 2010” (original) has been changed to “Schaefer et al., 2008”. Please check if appro-priate.

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