Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial and fungal infection

Arabidopsis histidine kinase 5 regulates salt sensitivity andresistance against bacterial and fungal infection

Jasmine Pham, Jasmine Liu, Mark H. Bennett, John W. Mansfield and Radhika Desikan

Department of Life Sciences, Imperial College London, London SW7 2AZ, UK

Author for correspondence:Radhika Desikan

Tel: +44 20 7594 3895Email: [email protected]

Received: 28 November 2011

Accepted: 1 December 2011

New Phytologist (2012) 194: 168–180doi: 10.1111/j.1469-8137.2011.04033.x

Key words: Arabidopsis histidine kinase 5,Botrytis cinerea, histidine kinase,Pseudomonas syringae, redox, salt stress.

Summary

• The ability of plants to adapt to multiple stresses imposed by the natural environment

requires cross-talk and fine-tuning of stress signalling pathways. The hybrid histidine kinase

Arabidopsis histidine kinase 5 (AHK5) is known to mediate stomatal responses to exogenous

and endogenous signals in Arabidopsis thaliana. The purpose of this study was to determine

whether the function of AHK5 in stress signalling extends beyond stomatal responses.

• Plant growth responses to abiotic stresses, tissue susceptibility to bacterial and fungal

pathogens, and hormone production and metabolism of reactive oxygen species were moni-

tored in a T-DNA insertion mutant of AHK5.

• The findings of this study indicate that AHK5 positively regulates salt sensitivity and contrib-

utes to resistance to the bacterium Pseudomonas syringae pv. tomato DC3000 and the fungal

pathogen Botrytis cinerea.

• This is the first report of a role for AHK5 in the regulation of survival following challenge by

a hemi-biotrophic bacterium and a necrotrophic fungus, as well as in the growth response to

salt stress. The function of AHK5 in regulating the production of hormones and redox homeo-

stasis is discussed.

Introduction

Plants are challenged by many different organisms and, becauseof their sessile lifestyle, must defend themselves not only againstparasites, pathogens and herbivores (biotic factors) but alsoagainst stresses imposed by the environment (abiotic factors).These stresses and stimuli can act in succession or in combina-tion. As a result, plants have developed complex signallingnetworks with common components acting to integrate responsesto multiple stimuli.

Cross-talk and overlap between signalling pathways allowplants to mediate rapid responses, either when multiple stimuliare likely to occur together or where different stresses require asimilar physiological response. By integration of signalling path-ways based on the physiological effects of the stress, plants areable to coordinate and fine-tune responses without the need forseparate signalling pathways for every stimulus or combination ofstimuli that they may encounter.

An important mechanism used for intracellular signalling isthe phosphorylation of proteins mediated by protein kinases. Ofrecent interest are the plant histidine kinases (HKs) of the two-component systems which have also been identified in yeast,bacteria, amoeba and plants (Urao et al., 2000; Hwang et al.,2002; Grefen & Harter, 2004). In Arabidopsis, 11 HKs havebeen identified, of which nine are of the hybrid type, the generalstructure of which consists of an input domain, an HK domain

and a receiver domain, whereas the archetypal nonhybrid HKslack the receiver domain. Signal transduction occurs via aphosphorelay between the HK, a histidine-containing phospho-transfer protein (AHP) and a response regulator (ARR), leadingto changes in target gene expression or protein activity (Uraoet al., 2001; Hwang et al., 2002; Grefen & Harter, 2004).

The nine hybrid HKs of Arabidopsis are known to function inethylene signalling (ETR1 ETHYLENE RESPONSE1, ETR2ETHYLENE RESPONSE2 and ERS1 ETHYLENE RESPONSESENSOR1) (Chang et al., 1993; Schaller et al., 2008), as osmo-sensors (Arabidopsis histidine kinase 1 (AHK1) ⁄ AtHK1, AHK2,AHK3, AHK4 ⁄ CRE1 ⁄ WOL CYTOKININ RESPONSE1 ⁄WOODEN LEG and ETR1) (Urao et al., 1999; Zhao & Schaller,2004; Tran et al., 2007), as cytokinin receptors (AHK2, AHK3and AHK4 ⁄ CRE1 ⁄ WOL) (Higuchi et al., 2004), in megagame-tophyte development (CKI1 CYTOKININ INDEPENDENT1)(Hejatko et al., 2003), in temperature perception (AHK2 andAHK3) (Jeon et al., 2010) and more recently as integrators ofenvironmental and endogenous signals (AHK5 ⁄ CKI2) (Desikanet al., 2008).

Of these kinases, the least is known about AHK5, the onlyhybrid HK initially predicted to have a cytoplasmic location andsubsequently shown to be localized in both the cytoplasm andthe plasma membrane (Desikan et al., 2008). AHK5 was firstshown to function as a negative regulator of root growth inhibi-tion mediated by ABA ⁄ ethylene (Iwama et al., 2007).

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Subsequently, we have shown that AHK5 integrates abiotic andbiotic stimuli in stomatal guard cells through regulation of H2O2

homeostasis (Desikan et al., 2008).Here, we demonstrate that the function of AHK5 in abiotic

and biotic stress signalling is not restricted to stomata. Using theahk5-1 T-DNA insertion line (Desikan et al., 2008) and ahk5-1complemented with full-length AHK5, we show that AHK5function is required for full immunity to the bacterial pathogenPseudomonas syringae pv. tomato DC3000 (PstDC3000) and thenecrotrophic fungus Botrytis cinerea. In addition, loss of functionof AHK5 also confers tolerance to high salinity, suggesting thatAHK5 acts to integrate multiple stress responses. Data are alsopresented to suggest a regulatory function for AHK5 in redoxand hormone balance.

Materials and Methods

Plant lines and growth conditions

All plants were sown on Levington’s F2 compost + sand (Scotts,Ipswich, UK) and grown under short-day conditions of 10 hlight : 14 h dark cycles with a light intensity of 120–150 lmolm)2 s)1 at 23�C and 55–65% relative humidity. Plants used forexperiments were 5–6 wk old, unless otherwise stated.

The ahk5-1 mutant containing a T-DNA insertion in thereceiver domain of the AHK5 histidine kinase was originallyobtained from Syngenta (Minnetonka, Minnesota, USA) (SAIL50_H11). Two complemented lines, PAHK5-AHK5 ⁄ ahk5-1-1and PAHK5-AHK5 ⁄ ahk5-1-4, were generated by complementa-tion of the ahk5-1 mutant with full-length AHK5 including 3205bases upstream of the ATG start codon. The plasmid pMKC111containing this construct was generated as described by Desikanet al. (2008) and used for complementation of ahk5-1 mutantplants. pMKC111 was transformed into Agrobacterium tumefac-iens strain GV3101 and subsequently transformed into ahk5-1plants by vacuum transformation. T3 lines were isolated byselection on 50 lg ml)1 hygromycin B (Duchefa, Haarlem, theNetherlands). Confirmation of expression of AHK5 in themutant background was obtained by RT-PCR (SupportingInformation Fig. S1).

Salinity treatment of seedlings in tissue culture

For seedling root growth assays, seeds were surface-sterilized andplated onto half-strength Murashige and Skoog medium(Duchefa), pH 5.7, with 1.5% agar and supplemented with 0,25, 50, 100 and 150 mM NaCl. Plated seeds were stratified at4�C for 2 d before transfer to a growth chamber and maintainedvertically under a 16-h photoperiod with a light intensity of120–150 lmol m)2 s)1 at 23�C. After 7 d of growth, rootlength was measured with a ruler to an accuracy of ± 0.5 mm.In addition, the number of germinated seeds was also recordedfor determination of percentage germination in response tosalinity.

For the seedling survival assay, seeds were surface-sterilized,stratified and grown on half-strength Murashige and Skoog

medium as above. After 7 d of growth, seedlings were transferredto plates containing half-strength Murashige and Skoog mediumwhich contained 200 mM NaCl and the number of bleachedseedlings three, five, seven, nine and 11 days after transfer weremonitored.

Salinity stress in mature plants

Three-wk-old plants were watered with 250 mM NaCl solutionor water as controls. At the start of the experiment, plants werewatered with 30 ml of salt solution directly into each pot. Plantswere then watered in the same fashion three times a week for2 wk, resulting in a total of six watering events. The aerial por-tion of the plant was then harvested on the third day after the lastwatering dose for determination of fresh shoot weight.

Bacterial growth and surface inoculation of plants

Pseudomonas syringae pv. tomato DC3000 (PstDC3000) used inthis study was provided by M. Grant (University of Exeter,Exeter, UK). Bacteria were maintained on solid PseudomonasAgar F Base medium (Merck, Darmstadt, Germany) grown at25�C supplemented with 50 lg ml)1 rifampicin.

For inocula, PstDC3000 was grown overnight in liquid LuriaBertani (LB) broth supplemented with 50 lg ml)1 rifampicin at25�C. Bacteria were pelleted, re-suspended in 10 mM MgCl2 anddiluted to the appropriate density by estimating absorbance at600 nm (A600). Silwet L-77 (Lehle Seeds, Round Rock, Texas,USA) was added to a concentration of 0.04% (v ⁄ v) and the inocu-lum gently coated onto both sides of the leaf. Plants were main-tained in a growth chamber for the duration of the experimentand covered with a transparent propagator lid to increase humi-dity. To determine in planta population counts of bacteria aftersurface inoculation, leaves were weighed and surface-sterilized asdescribed by Katagiri et al. (2002) and ground in quarter-strengthRinger’s solution (Merck, Darmstadt, Germany), and the numberof colony-forming units (cfu) was counted after 2 d and expressedas cfu g)1 FW of leaves.

Estimation of chlorophyll content

Leaves inoculated as described in the previous section wereexcised and chlorophyll was extracted from leaves in 100% meth-anol at 50�C for 1 h in a heated block. A665 and A652 of themethanolic extracts were measured for calculation of chlorophyllcontent. Concentrations of chlorophylls a and b in the methanolicextracts were calculated as described by Porra et al. (1989).

Inoculation with Botrytis cinerea and assessment of diseaseprogression

Botrytis cinerea (obtained from K. Denby, Horticulture ResearchInternational, Warwick, UK) was cultured on Potato GlucoseAgar (PGA) (Sigma-Aldrich, UK) supplemented with 500 lgml)1 spectinomycin and incubated at 18–20�C under MiniBlack Light Blue fluorescent lamps emitting long-wave UV light

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(Phillips, Guilford, UK) for 7–10 d. Spores were harvested andadjusted to a concentration of 2 · 105 spores ml)1 in one-eighthstrength Potato Dextrose Broth (PDB) (Sigma-Aldrich, UK).

Symptom development was assessed on detached leaves kept inclear plastic boxes lined with moist tissue with the petioles wrappedin tissue paper. The abaxial side was inoculated with 10-ll dropletsof the spore suspension. Boxes were kept at 23�C under short-dayconditions with a 10-h photoperiod at a light intensity of 25 lmolm)2 s)1. For assessment of the effect of diphenyleneiodiniumchloride (DPI) (Sigma-Aldridge) and 2-(4-carboxyphenyl)-4, 4, 4,5-tetramethylimidazoline-oxyl-3-oxide (cPTIO) (Sigma-Aldrich)treatment on lesion development, leaves were vacuum-infiltratedwith 1 lM DPI or 50 lM cPTIO for 5 min and floated in thesame solution for 2 h before inoculation with the B. cinerea sporesuspension as described earlier in this section.

The severity of disease was assessed by visual inspection andbased on the spread of the lesions. Symptoms were scored on ascale of 0–4, with each score denoting the severity of the lesionspresent as follows: 0, no lesions; 0.5, multiple small lesions withinthe inoculum droplet; 1, single large lesion within the inoculumdroplet; 2, single large lesion spreading beyond the inoculumdroplet; 3, tissue collapse; 4, collapse of tissue and sporulation.

For measurement of lesion area, images of individual lesionswere captured using a stereomicroscope (Leica MZ16F; Leica,Wetzlar, Germany) with an attached Leica DFC300FX cameraand lesion size was measured using IMAGE J software (Abramoff,2004).

Histological observations

Fungal hyphae on leaves were visualized by trypan blue stainingas described by Xiao et al. (2003). Leaves were decolourized in an8 : 1 : 1 : 1 mixture of ethanol : phenol : lactic acid : glycerol for24 h, during which the solution was changed twice. Leaves werethen stained for 30 min in 0.025% (w ⁄ v) trypan blue (Sigma-Aldrich) in a 1 : 1 : 1 mixture of lactic acid : glycerol : water,cleared in saturated chloral hydrate and mounted in 60%glycerol.

The presence of reactive oxygen species (ROS) was visualizedby staining with 3,3-diaminobenzidine (DAB) (Sigma-Aldrich)at a concentration of 1 mg ml)1 in water (pH 3.8). Excisedleaves were vacuum-infiltrated with DAB solution for 5 min.Leaves were then incubated in DAB in the dark overnight,destained in 100% ethanol and mounted in 60% glycerol.

To assess the effect of DPI on germination of B. cinerea sporesin vitro, 10-ll droplets of a 2 · 105 spores ml)1 suspension inone-eighth PDB supplemented with 1 lM DPI or 50 lMcPTIO were deposited onto glass slides which were placed inclear plastic boxes as described in the previous section. Beforemicroscopic observation, the hyphae were stained by adding adrop of lactophenol cotton blue solution (Pro-Lab Diagnostics,Cheshire UK) to the inoculum droplet; a coverslip was thenapplied and sealed with clear nail varnish.

Stained material was examined either with a stereomicroscope(Leica MZ16F), with images being captured with an attachedLeica DFC300FX camera, or with a Zeiss AxioSkop2 Plus

Microscope, with images being captured with an attached ZeissAxioCam with AxioVision 3.1 software (Zeiss). Hyphal lengthand area of cellular DAB staining was quantified using IMAGE Jsoftware.

Extraction and analysis of camalexin

Individual lesions were excised with a razor blade and freeze-dried, and camalexin was extracted from each individual lesion in200 ll of 30% methanol with a steel ball using a Tissue Lyser(Qiagen) at 30 Hz for 1 min and left to soak for 15 min. Theextraction procedure was performed three times on each sampleand supernatants from the successive extractions were collectedand pooled for analysis.

Analysis of samples was by high-pressure liquid chromato-graphy (HPLC) using an Agilent 1200 HPLC system (AgilentTechnologies, Cheshire, UK). The compounds were separatedon a Phenomenex C-18 column (100 mm · 2 mm; 3 lm)(Phenomenex, Torrance, California, USA) using an isocraticsolvent system of H2O : CH3CN (7 : 3) with a flow rate of0.25 ml min)1 at a temperature of 35�C. Typically, 20-ll injec-tions were used. Camalexin was detected with a Shimadzu RF535Fluorescence detector (Shimadzu, Milton Keynes, UK) set tocamalexin’s characteristic fluorescent spectra (318 nm excitationand 385 nm emission). Identification and quantification werewith reference to an authenticated standard from B. A. Halkier(University of Copenhagen, Copenhagen, Denmark); camalexineluted with a retention time of 13.5 min. The data were acquiredand integrated using Agilent’s CHEMSTATION software.

Hormone and coronatine extraction and analysis

Leaves for hormone extraction were inoculated with PstDC3000or mock-inoculated with 10 mM MgCl2. For each biologicalsample, three leaves from three plants were pooled. Harvestedleaves were snap-frozen in liquid nitrogen, subsequently freeze-dried and milled with a steel ball at 25 Hz for 3 min using atissue lyser. Hormones were extracted from 10 mg of freeze-driedmaterial and analysed by LC-MS ⁄ MS using an Agilent 1100 LCcoupled to an Applied Biosystems Q-TRAP LC ⁄ MS ⁄ MS system(Applied Biosystems, California, USA) fitted with a Turbo IonSpray source operating in negative mode as described by Forcatet al. (2008). For coronatine, a calibration curve was constructedfrom data obtained by injection of known quantities of corona-tine (purchased from S. Abrams, NRC, Ontario, Canada) andthe amount was quantified by monitoring the coronatine masstransition pair 318 > 163.

Statistical analysis

To test for statistical significance, data were analysed withStudent’s t-test (for comparison of wild-type and ahk5-1 mutantresponses), one-way ANOVA with Tukey’s post hoc test (forcomparison of the responses of the wild type, the ahk5-1 mutantand the complemented lines) or Kruskal–Wallis one-way ANOVAwith Dunn’s multiple comparison test for nonparametric data

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(for comparison of the responses of the wild type, the ahk5-1mutant and the complemented lines).

Results

AHK5 contributes to salt sensitivity

To determine whether AHK5 is involved in modulating reactionsto abiotic stress, phenotypic responses of wild-type and mutantseedlings to heat, cold, salt and osmotic stress were tested. As

AHK5 was shown to be most highly expressed in the roots(Iwama et al., 2007; Desikan et al., 2008), we focused on theroot growth of seedlings under diverse abiotic stresses. Under theconditions tested, a markedly increased inhibition of seedlingroot growth was observed in response to salinity (Fig. 1a). Thisinhibitory effect of salinity was greater in wild-type plants, withthe root length of the ahk5-1 mutant being significantly greaterat all concentrations of NaCl tested, suggesting that AHK5 posi-tively regulates salt-induced root growth inhibition.

The root growth of seedlings of two independent comple-mented lines, PAHK5-AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ ahk5-1-4 (see Fig. S1 for genotyping of these lines), on media containing100 mM NaCl was similar to that of wild-type Columbia(Col-0) seedlings, confirming that the insensitivity of the ahk5-1mutant in response to salinity was caused by loss of AHK5 func-tion (Fig. 1b). At a higher concentration of NaCl (200 mM),survival of the ahk5-1 mutant was similar to that of the wild typeand the complemented lines, suggesting that AHK5 is involvedin tolerance to, rather than survival under, saline conditions(Fig. S2). Seeds of ahk5-1 also showed a significantly higher per-centage of germination on 150 mM NaCl, with 90% of mutantseeds germinating compared with 72% of Col-0 seeds (P < 0.05using Student’s t-test). In the absence of NaCl, all seeds sown forboth Col-0 and ahk5-1 germinated, suggesting that the differencein germination of wild-type and ahk5-1 seeds in the presence ofNaCl was caused by differential sensitivity of the seeds to salinity.

The investigation of salt sensitivity was subsequently extendedto mature soil-grown plants, for which plants were irrigated with250 mM NaCl for a period of 2 wk, after which the fresh

Fig. 1 The AHK5 mutant is more resistant to salt stress. (a) Root length ofwild-type (open bars) and ahk5-1 mutant (closed bars) seedlings after 7 dof germination and growth on half-strength Murashige and Skoogmedium supplemented with 0–150 mM NaCl. Measurements are themean ± SEM for c. 140 seedlings for each line from three independentexperiments. Asterisks denote statistically significant differences betweenwild-type and mutant responses as determined by Student’s t-test. Controlroot lengths were as follows: Columbia (Col-0), 14.83 ± 0.78 mm;ahk5-1, 16.19 ± 0.67 mm. (b) Root length of wild-type, ahk5-1 mutantand complemented lines PAHK5-AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ahk5-1-4 after germination and 7 d of growth on half-strength Murashigeand Skoog medium supplemented with 100 mM NaCl. Root length ofseedlings grown in the presence of 100 mM NaCl is presented as apercentage of control treatments (no NaCl) from an average of at least130 seedlings for each line from three independent experiments. Asterisksdenote statistically significant differences between mutant responsescompared with the wild type and the complemented lines as determinedby one-way ANOVA with Tukey’s post hoc test. Control root lengthswere as follows: Col-0, 17.54 ± 0.48 mm; ahk5-1, 16.19 ± 0.67 mm;PAHK5-AHK5 ⁄ ahk5-1-1, 18.93 ± 0.28 mm; PAHK5-AHK5 ⁄ ahk5-1-4,16.42 ± 0.27 mm. (c) Fresh weight of mature soil-grown plants wateredwith a 250 mM solution of NaCl over the course of 2 wk. Data are themean ± SEM for the fresh weights of at least six plants for each line tested.Fresh weights of untreated control plants were as follows: Col-0,3.62 ± 0.20 g; ahk5-1, 2.24 ± 0.13 g; PAHK5-AHK5 ⁄ ahk5-1-1,2.94 ± 0.13 g; PAHK5-AHK5 ⁄ ahk5-1-4, 2.88 ± 0.13 g. Asterisks denotestatistically significant differences between wild-type and mutantresponses as determined by one-way ANOVA with Tukey’s post hoc test.*, P < 0.05; **, P < 0.005; ***, P < 0.0001.






weights of the plants were measured. The relative shoot freshweight (as a percentage of the shoot fresh weight of the respectivecontrol-treated plants) was determined and found to be signifi-cantly greater in ahk5-1 mutant plants compared with wild-typeplants and the complemented lines PAHK5-AHK5 ⁄ ahk5-1-1 andPAHK5-AHK5 ⁄ ahk5-1-4 (Fig. 1c), demonstrating that AHK5positively contributes to salt sensitivity in both seedlings andmature plants.

Salt is known to cause ionic toxicity as well as osmotic stress;build-up of ions in the cytoplasm inhibits enzymatic activity,whereas build-up in the cell wall causes dehydration and osmoticstress (Munns, 2002; Munns & Tester, 2008). No difference wasseen between the responses of Col-0 and ahk5-1 seedlings toosmotic stress with either sorbitol or KCl as the osmoticum(Fig. S3), suggesting that the differences seen in root length andgermination may be attributable to the ionic component (i.e.Na+) of salt stress.

AHK5 is involved in defence against Pseudomonas syringaepv DC3000

Symptom development and bacterial growth It was previouslyshown in our laboratory that the ahk5-1 mutant is defective instomatal responses to PstDC3000 and to bacterial flagellin(Desikan et al., 2008). Here we set out to determine whether thedefect in stomatal responses to bacterial pathogens in the ahk5-1mutant correlated with an increased susceptibility to infection.

When PstDC3000 was inoculated onto the leaf surface, diseaseprogression in the ahk5-1 mutant was accelerated compared withwild-type plants, as shown by increased chlorosis of leaves(Fig. S4). No obvious senescence phenotype was seen in ahk5-1mutant plants in the absence of bacterial inoculation (Fig. S4).Quantification of the extent of chlorosis via chlorophyll measure-ments revealed a significantly lower amount of chlorophyll in theahk5-1 mutant at 6 d post-inoculation (dpi) compared withwild-type Col-0 and the complemented lines PAHK5-AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ ahk5-1-4 (Fig. 2a). In plantabacterial populations also showed a clear increase in PstDC3000growth in the ahk5-1 mutant compared with wild-type plantsand the complemented lines (Fig. 2b). The increased susceptibil-ity of ahk5-1 plants indicated by higher bacterial numbers wasmost significant at 6 dpi, correlating with the decrease in chloro-phyll content observed at this time-point. The delayed increase insusceptibility indicates that the effects of the ahk5-1 mutationextended well beyond the early stages of stomatal penetration.

Hormones and infection Resistance to PstDC3000 involveschanges in concentrations of the phytohormones salicylic acid(SA), abscisic acid (ABA) and jasmonic acid (JA) (de TorresZabala et al., 2009; Verhage et al., 2010). SA is required for basalresistance against biotrophic pathogens such as PstDC3000,whereas manipulation of ABA can promote virulence of the bac-terium through inhibition of SA biosynthesis and responses(Block et al., 2005; de Torres Zabala et al., 2007, 2009). How-ever, an intact ABA signalling pathway is required for stomatalclosure in response to bacteria on the leaf surface, and the ability

of PstDC3000 to manipulate ABA concentrations and to over-come stomatal defences requires the presence of the bacterial phy-totoxin coronatine (Brooks et al., 2004; Melotto et al., 2006; deTorres Zabala et al., 2007, 2009).

In order to determine whether changes in hormone concen-trations may account for the altered ahk5-1 disease phenotype,hormone concentrations following surface inoculation of leaveswere measured. No significant differences were observed in theconcentrations of hormones in mock-inoculated leaves of wild-type and ahk5-1 plants (SA: 1.14 lg g)1 DW in Col-0 and1.53 lg g)1 DW in ahk5-1; ABA: 62.20 ng g)1 DW in Col-0and 61.95 ng g)1 DW in ahk5-1; JA: 1.07 lg g)1 DW in Col-0and 0.88 lg g)1 DW in ahk5-1). Following inoculation, similarpatterns of increase in hormone concentrations were observed inboth genotypes, but concentrations were significantly lower inahk5-1 leaves (Fig. 3). Differences were most striking for SA andJA after 2 and 6 dpi, respectively, but were consistently higher in

(a)

(b)

Fig. 2 The AHK5 mutant is more susceptible to infection withPseudomonas syringae pv. tomato DC3000 (PstDC3000). Leaves weresurface-inoculated with 4 · 108 cfu ml)1 PstDC3000. (a) Chlorophyllcontent of infected leaves expressed as a percentage of that of mock-inoculated (10 mM MgCl2) leaves. Values are mean ± SEM ofmeasurements from six plants per genotype. Asterisks denote a statisticallysignificant difference between the ahk5-1 mutant response andthe responses of wild-type Columbia (Col-0) and complemented PAHK5-AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ ahk5-1-4, as determined by one-wayANOVA with Tukey’s post hoc test. (b) In planta growth of PstDC3000.Values are mean ± SEM obtained from six plants per genotype. Asterisksdenote a statistically significant difference between the ahk5-1 mutantresponse and the responses of wild-type Col-0 and complemented PAHK5-

AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ ahk5-1-4 plants, as determined byone-way ANOVA with Tukey’s post hoc test. *, P < 0.05; ***,P < 0.0001.

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wild-type plants during the course of infection (Fig. 3a,c). ABAconcentrations were also lower in the mutant ahk5-1 plants com-pared with wild-type plants, and this difference was statisticallysignificant at 6 dpi (Fig. 3b). Although no overlap in error barswas seen for ABA concentrations at 2 dpi in wild-type Col-0 andthe ahk5-1 mutant, non-overlap of error bars does not necessarilyindicate a significant difference (Cumming et al., 2007). Inagreement with the observations of de Torres Zabala et al.(2009), JA was seen to increase later than ABA and SA duringinfection (Fig. 3c). On the other side of the interaction, corona-tine accumulation was observed by 2 dpi (Fig. 3d); however, theconcentrations of the phytotoxin were fourfold lower at 2 dpiand twofold lower at 4 and 6 dpi in ahk5-1 compared with wild-type plants (Fig. 3d).

AHK5 is involved in defence against the necrotroph Botrytiscinerea

Symptom development and camalexin production Infectionwith PstDC3000 has been shown to increase susceptibility to fun-gal pathogens (Spoel et al., 2007), demonstrating a link betweenresistance pathways operating against bacterial and fungal patho-gens. Here, the response of the ahk5-1 mutant was tested toinfection with the necrotroph B. cinerea, which is able to directlypenetrate the host plant cuticle (Williamson et al., 2007).

Symptom development ⁄ severity of disease was assessed bymonitoring lesion spread and the amount of tissue collapse. Thisrevealed that the ahk5-1 mutant was markedly more susceptible

to infection by the necrotrophic pathogen; significantly higherlesion scores were observed at 3 and 5 dpi in the ahk5-1 mutantcompared with leaves of the wild type and the complementedlines PAHK5-AHK5 ⁄ ahk5-1-1 and PAHK5-AHK5 ⁄ ahk5-1-4, withhigher lesion scores representing more severe disease symptoms(Fig. 4a). Measurement of lesion size at 3 dpi confirmed thatlesion size was significantly greater in the ahk5-1 mutant, approx-imately double the size of lesions seen in leaves of wild-typeplants and the complemented lines (Fig. 4b).

The phytoalexin camalexin has been shown to contribute toresistance to a number of necrotrophic fungi, including B. cinerea(Ferrari et al., 2003; Denby et al., 2004; Lazniewska et al.,2010). Concentrations of camalexin in individual lesions fromwild-type and mutant plants infected with B. cinerea were mea-sured at 1, 2, 3 and 4 dpi. Camalexin concentrations were seento increase as lesion development progressed but were not signifi-cantly different between wild-type and mutant leaves (Fig. 4c).This shows that the increased susceptibility of the ahk5-1 mutantis not attributable to a defect in camalexin production.

Differences in reactive oxygen species production and fungalgrowth in planta ROS production during infection withB. cinerea is known to occur in both plant cells and fungal struc-tures, and has been shown to enhance fungal growth and symp-tom development (Govrin & Levine, 2000). To determinewhether ROS production was altered in the mutant plant or inthe fungus during the infection process, ROS accumulation wasmonitored using DAB. At 8, 12 and 24 h post-inoculation (hpi),

Fig. 3 Concentrations of (a) salicylic acid (SA), (b) abscisic acid (ABA), (c) jasmonic acid (JA) and (d) coronatine (COR) in leaf tissue surface inoculatedwith 4 · 108 cfu ml)1 Pseudomonas syringae pv. tomato DC3000. Measurements are an average of two independent experiments. Asterisks denotestatistically significant differences between wild-type (open bars) and mutant (closed bars) plant responses as determined by Student’s t-test. *, P < 0.05;**, P < 0.005; ***, P < 0.0005.






an apparent increase in DAB staining at the site of inoculationwas seen, with more DAB staining recorded in the ahk5-1mutant earlier than in wild-type plants (Fig. 5a). On closerexamination, it appeared that the DAB staining seen at 8 and12 hpi was associated with fungal structures and not with the leaf

cells (Fig. S5). However, it was unclear whether the increasedamount of DAB associated with the fungus on the ahk5-1 mutantwas caused by increased production of fungal ROS or an increasein the actual mass of fungus present. To determine whether thelatter was the case, fungal hyphae were stained with trypan blue

(a)

(b)

(c)

Fig. 4 The AHK5 mutant is more susceptible to infection with Botrytis

cinerea. Leaves were inoculated with 2 · 105 spores ml)1. (a) Lesionscores (mean ± SEM of 37–108 lesions) observed at 3 and 5 d post-inoculation (dpi); experiments were repeated three times. Asterisks denotestatistically significant differences as determined by the Kruskal–Wallis test.(b) Lesion area as measured at 3 dpi (mean ± SEM of 37–108 lesions).Asterisks denote statistically significant differences as determined byone-way ANOVA with Tukey’s post hoc test. (c) Mean concentrations ofcamalexin extracted from a total of 20 individual lesions caused byB. cinerea, on wild type (open bars) and ahk5-1 mutant (closed bars)plants per time-point. *, P < 0.05; **, P < 0.005; ***, P < 0.0001.

(a)

(b)

(c)

Fig. 5 Botrytis cinerea growth is greater on leaves of AHK5 mutant thanwild-type leaves. Leaves were inoculated with 2 · 105 spores ml)1 in1 ⁄ 8th potato dextrose broth (PDB). (a) 3,3-Diaminobenzidine (DAB)staining of plant cells and fungal hyphae on wild-type and ahk5-1 leaves.Representative images are shown for each time-point. Arrows indicate theposition of the inoculation droplet. Bar, 3 mm. (b) Length of fungal hyphaeon wild-type and ahk5-1 leaves measured at 8 h post-inoculation usingtrypan blue staining. Values are mean ± SEM of 237–271 measurements.Asterisks denote a statistically significant difference between Col-0 andahk5-1 as determined by Student’s t-test. (c) Hyphal length of B. cinerea

spores germinated in vitro in 1 ⁄ 8th PDB supplemented with water(control, open bars) or 1 lM diphenyleneiodinium chloride (DPI; closedbars) for various durations. Asterisks denote a significant differencebetween DPI-treated and control samples as determined by Student’s t-test.Values are mean ± SEM of at least 120 measurements for each treatmentand time-point. ***, P < 0.0001.

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and hyphal length was measured. A significant difference in thelength of fungal hyphae was seen as early as 8 hpi, with hyphallength an average of 45% greater on the ahk5-1 mutant leavesthan that measured on wild-type plants (Figs 5b, S6).

To further investigate the role of ROS in fungal growth, theeffect of the NADPH oxidase inhibitor DPI on hyphal growth ofB. cinerea was also investigated. Botrytis cinerea contains twoNADPH oxidases, BcnoxA and BcnoxB, which have been shownto contribute to the virulence and pathogenicity of the fungus(Segmuller et al., 2008). The hyphal length of spores germinatedin vitro in the presence of 1 lM DPI was measured at 4, 6 and8 hpi. At all three time-points, hyphal length was significantlyshorter in spores germinated in the presence of DPI comparedwith control spores germinated without DPI (Fig. 5c), suggestingthat fungal-derived ROS produced by NADPH oxidase(s) con-tributes to hyphal growth.

Whereas DAB staining in fungal hyphae on leaves wasobserved at all experimental time-points (Fig. 5a), plant-derivedROS in response to infection with B. cinerea was not seen until24 hpi and was noticeably less extensive in the ahk5-1 mutantcompared with wild-type leaves (Fig. 6a). Analysis of the propor-tion of DAB staining at the site of inoculation revealed thatahk5-1 displayed 28% less staining than wild type, suggestingthat the ahk5-1 mutant is attenuated in ROS production inresponse to infection with B. cinerea at 24 hpi (Fig. 6b). At later

time-points (up to 3 dpi), the extent of DAB staining corre-sponded to lesion size, that is, in the mutant there appeared to bemore ROS at the lesion sites as a result of the larger lesionsformed. Interestingly, pretreatment of wild-type Col-0 leaveswith DPI 2 h before inoculation with B. cinerea resulted in mea-surably smaller lesions at 3 dpi compared with mock-treated,inoculated leaves (Fig. S7).

Discussion

In response to the changing environment, plants have the abilityto sense and respond to single or multiple stimuli via activationof unique signalling cascades. The two-component systems inbacteria and yeast act as unique sensing and signalling systemsresponding to osmotic (Forst & Roberts, 1994; Posas et al.,1996; Paithoonrangsarid et al., 2004), temperature (Aguilaret al., 2001; Suzuki et al., 2005) and oxidative stimuli (Singh,2000; Kanesaki et al., 2007). More recently, some plant HKshave been shown to function in response to environmental stim-uli such as drought ⁄ osmotic (AtHK1, AHK2, AHK3 andAHK4) (Urao et al., 1999; Tran et al., 2007, 2010) and salinitystress (AHK2, AHK3 and ETR1) (Zhao & Schaller, 2004; Tranet al., 2007; Wang et al., 2008; Tran et al., 2010). Additionally,a role for AHK2 and AHK3 in modulating disease resistance inresponse to infection with PstDC3000 has been reported (Choiet al., 2010). Previously, our laboratory has shown that AHK5mediates stomatal responses to endogenous and environmentalcues (Desikan et al., 2008). Here we demonstrate the ability ofAHK5 to function in response to both abiotic and biotic stimulito affect the growth and survival of Arabidopsis.

AHK5 regulates tolerance to salinity

The present study shows that, of the various abiotic stressestested, AHK5 positively regulates growth inhibition to salinitystress. This is the first demonstration of a function for AHK5 insalinity-induced growth responses in Arabidopsis. ABA is knownto play a role in drought and salinity stress tolerance (Zhu,2002). However, the finding that AHK5 negatively regulatesABA-induced root growth inhibition (Iwama et al., 2007) is hardto reconcile with the finding that it positively regulates salt-induced root growth inhibition. It is possible that ABA concen-trations induced following salt stress are insufficient to enableAHK5-mediated signal transduction to act in an inhibitory man-ner. This possibility is supported by the promotion of rootgrowth seen with low concentrations of ABA in both wild-typeand ahk5-1 mutant plants (Ghassemian et al. (2000) andFig. S8). In addition, there is evidence suggesting that ABA mayfunction to maintain rather than to inhibit root growth underconditions of stress (Sharp & LeNoble, 2002).

Salinity stress imposes both osmotic and ionic stress on theplant (Munns, 2002; Munns & Tester, 2008). Here, we haveobtained evidence to suggest that the ahk5-1 mutant is less sensi-tive to the ionic component of salt stress. Under salt stress, theincreased uptake of Na+ disrupts homeostasis of ions and inhibi-tion of cellular processes, through toxicity to cellular enzymes

(a)

(b)

Fig. 6 The AHK5 mutant produces less reactive oxygen species (ROS) inresponse to infection with Botrytis cinerea. Leaves were inoculated with aspore suspension at a concentration of 2 · 105 spores ml)1 in 1 ⁄ 8thpotato dextrose broth. (a) Wild-type Columbia (Col-0) and ahk5-1 mutantleaves stained with 3,3-diaminobenzidine (DAB) at 24 h post-inoculation.Representative pictures at the site of inoculation are shown. Bar, 3 mm.(b) Quantification of the amount of DAB staining measured at the site ofinoculation 24 h post-inoculation. Values are an average obtained fromIMAGE J analysis of 20 lesions per plant. The asterisk denotes a statisticallysignificant difference as determined by Student’s t-test. *, P < 0.05.






and disruption of K+ uptake by the plant (Munns, 2002; Zhu,2002). Tolerance to salt stress in Arabidopsis is mediated in partthrough efflux of Na+ by the Na+ ⁄ H+ antiporter SOS1 (SALTOVERLY SENSITIVE1) at the plasma membrane, which is acti-vated by the myristolated calcium-binding protein SOS3 and theSer ⁄ Thr kinase SOS2 as part of the SOS pathway (Zhu, 2002,2003). The phenotype we observed in the ahk5-1 mutantappeared to be specific to the presence of Na+ (Figs 1a, S3),raising the possibility that AHK5 may interact with the SOSpathway. Whether AHK5 directly regulates uptake of Na+ ⁄ K+ isnot yet known. However, a link with the SOS pathway via ROSis plausible.

ROS are known to accumulate to varying concentrations inresponse to salinity treatment in different subcellular compart-ments (Miller et al., 2010) and the SOS1 protein has beenshown to interact with RCD1 (RADICAL-INDUCED CELLDEATH1), a regulator of oxidative stress responses, to regulateROS concentrations (Katiyar-Agarwal et al., 2006). Preliminaryobservations indicate that ahk5-1 seedlings also accumulatehigher concentrations of H2O2 following salinity stress than trea-ted wild-type Col-0 seedlings (2.3-fold and 1.4-fold increasesover untreated controls, respectively). Given the previously iden-tified function for AHK5 in regulating H2O2 concentrations(Desikan et al., 2008), it is possible that AHK5 acts to regulateredox balance following salinity stress challenge to mediategrowth responses.

Loss of AHK5 function increases susceptibility to thebacterial pathogen PstDC3000

Stomata form an active part of plant defences against bacterialpathogens (Melotto et al., 2006; Underwood et al., 2007; Zenget al., 2010) and previous work has shown that stomata of theahk5-1 mutant are unresponsive to the presence of PstDC3000on the leaf surface (Desikan et al., 2008). Here, we report thatthe ahk5-1 mutant is more susceptible to bacterial infection,when surface-inoculated with PstDC3000. These observationswere similar to those reported by Zipfel et al. (2004) with the fls2(flagellin sensitive2) mutant, which is defective in the perceptionof bacterial flagellin and does not close stomata in response toeither flg22 or PstDC3000 at the leaf surface (Melotto et al.,2006; Zeng & He, 2010). In addition to PstDC3000, the ahk5-1mutant is also defective in stomatal closure in response to flg22,ethylene, H2O2 and darkness (Desikan et al., 2008). AHK5 maytherefore act to integrate responses at the leaf surface to diverseexogenous stimuli.

Further analysis of metabolic changes following PstDC3000challenge revealed quantitative differences between wild-type andahk5-1 mutant plants in the hormones SA, ABA and JA. To ourknowledge, this is the first report of changes in plant hormonesfollowing surface inoculation of bacteria onto Arabidopsis leaves,with the timing of hormone changes correlating with the spreadof symptoms. Concentrations of all hormones were maintainedat low levels following infection of ahk5-1 leaves. It is clear fromrecent studies that the balance between the concentrations of thedifferent hormones influences the outcome of disease (Block

et al., 2005; de Torres Zabala et al., 2009). During the Arabid-opsis–PstDC3000 interaction, antagonism of SA defencesthrough manipulation of ABA production promotes virulence ofthe bacterium (de Torres Zabala et al., 2007, 2009). In thisstudy, AHK5 differentially affected the temporal accumulationof SA, JA and ABA in response to PstDC3000. In the absence ofa stomatal closure response to bacteria, as in ahk5-1, PstDC3000would enter at an accelerated rate into the apoplastic space andmultiply to higher numbers than that seen in wild-type plants.This is likely to suppress basal defence more efficiently, and haveprolonged effects on hormonal imbalance, as seen in ahk5-1,throughout the course of the infection.

Another obvious cause for increased virulence in the mutantcould be increased production of the bacterial phytotoxin corona-tine, also known to suppress basal defences and to manipulatehormone signalling to promote disease (Brooks et al., 2005;Uppalapati et al., 2007; Ishiga et al., 2010; Zeng et al., 2010).Surprisingly, however, PstDC3000 produced lower concen-trations of coronatine in ahk5-1 plants. This might result fromhost defences (e.g., hormones) already being suppressed to a largeextent, thereby not necessitating the production of coronatine byPstDC3000. In support of this, Block et al. (2005) suggest thatcoronatine concentrations do not necessarily correlate with thegrowth of bacteria. Rather, PstDC3000 might be reallocating itsresources to other virulence mechanisms, such as expression of thetype-three secretion system and effector proteins, and expressionof genes involved in nutrient assimilation or adaptation to theapoplastic environment (Boch et al., 2002; Rico & Preston,2008).

An alternative explanation for the reduced synthesis of corona-tine in ahk5-1 plants is that AHK5 indirectly regulates coronatinebiosynthesis. There have been some early reports in the literatureof plant-derived factors from the shikimate pathway regulatingcoronatine biosynthesis in PstDC3000 (Li et al., 1998). In thisstudy, we observed a metabolite unique to ahk5-1 mutant leaveswhich appeared to be independent of bacterial infection(Fig. S9). Preliminary mass spectrometry analysis is indicative ofthis metabolite belonging to the indolic class of compounds (partof the shikimate pathway). Is it possible that AHK5 normallysuppresses the synthesis of this unique metabolite, and lack ofAHK5 removes this suppression, leading to altered susceptibilityto PstDC3000? Further work to identify this compound willaddress this question.

AHK5 contributes to resistance to the necrotrophic fungalpathogen B. cinerea

In addition to increased susceptibility to the hemibiotrophPstDC3000, the ahk5-1 mutant was also strikingly susceptible tothe necrotrophic fungal pathogen B. cinerea. Although differ-ences in lesion formation between wild-type and mutant plantswere not apparent until 3 dpi in response to B. cinerea, closeinspection of infected leaves revealed an increase in fungal growthin ahk5-1 as early as 8 h after contact of the fungal spores withthe leaf surface. The cause of this early difference in fungalgrowth could be due to differences in the properties of the leaf

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surface structure and composition and ⁄ or compounds exudedonto the leaf surface affecting fungal growth (Rossall et al., 1977;Doss et al., 1993; Calo et al., 2006; Bessire et al., 2007; Chassotet al., 2008; Curvers et al., 2010). DAB staining of leaves at earlytime-points showed more fungal structures being stained, andfrom the images obtained it appeared that individual hyphae onthe mutant showed more DAB staining (Fig. S5). Although thereis an increase in fungal hyphal length in the mutant, the experi-ments here cannot reveal clearly whether or not the mutant leavessomehow enhance fungal production of ROS, thereby enhancingfungal growth. As our data also reveal a requirement for ROS inin vitro hyphal growth, this is a strong possibility.

In addition to the early increase in fungal growth observed onthe ahk5-1 mutant, in planta ROS production in ahk5-1 leaf cellswas significantly lower than that in wild-type tissue at 24 hpi atthe site of inoculation. Early ROS production in tomato (Solanumlycopersicum) and bean (Phaseolus vulgaris) has previously beenlinked to resistance to infection with B. cinerea (Unger et al.,2005; Asselbergh et al., 2007), and the early attenuation in ROSproduction by the ahk5-1 mutant may be linked to a dampenedimmune response resulting in more severe disease symptoms seenin this mutant. However, plant-derived ROS is also required forvirulence of the fungus, as ROS-triggered cell death, althougheffective against biotrophic pathogens in the form of the hyper-sensitive response (HR), enhances infection by Botrytis cinerea(Govrin & Levine, 2000). This is in agreement with the DPIinhibition of lesions seen here at 3 dpi (Fig. 5a). Moreover, after24 hpi, the presence of ROS correlated to the area covered by thelesions appearing later (i.e. there appeared to be more extensiveROS production in ahk5-1 leaves as a consequence of largerlesions). Clearly, the role of ROS in the outcome of necrotroph–plant interactions is complex and is determined by factors such asthe timing of induction, and the cellular location in the host andthe pathogen, as well as the source and concentration of ROSgenerated. In response to infection with B. cinerea, whereas earlyROS production regulated by AHK5 contributes to triggeringdefence mechanisms against B. cinerea infection, at the laterstages of infection, loss of AHK5 function results in increasedROS production associated with cell death, which further facili-tates disease progression.

Aside from ROS, nitric oxide (NO) is also known to contrib-ute to resistance to B. cinerea (Asai & Yoshioka, 2009). Addition-ally, a recent study found that NO produced in B. cinerea wasable to diffuse into the surrounding growth medium, raising thepossibility that fungal-derived NO may influence plant signalling(Turrion-Gomez & Benito, 2011). However, we found that pre-treatment of leaves with the NO scavenger cPTIO did not signifi-cantly affect the size of lesions caused by B. cinerea in wild-typeCol-0 (Fig. S7). Similarly, treatment of spores with cPTIO didnot affect hyphal elongation in vitro (Fig. S10), suggesting thatneither a defect in NO production in the ahk5-1 mutant noralteration in NO production in B. cinerea contributes to theincreased susceptibility of the ahk5-1 mutant.

Another factor that contributes to resistance to B. cinerea is thephytoalexin camalexin, which is known to accumulate at thesite of infection (Denby et al., 2004; Kliebenstein et al., 2005).

In this study, the increased susceptibility of the ahk5-1 mutantwas not attributed to a defect in camalexin production, indicatingthat factors other than camalexin determine host suscepti-bility or that a mutation in AHK5 results in the secretion ofother compounds that influence the sensitivity of B. cinerea tocamalexin.

In response to necrotrophic pathogens such as B. cinerea, theplant hormones ethylene (ET), SA and JA are known to posi-tively contribute to resistance (Thomma et al., 1998, 1999,2000; Zimmerli et al., 2001; Ferrari et al., 2003; van Baarlenet al., 2007; Vicedo et al., 2009). Although the endogenousconcentrations of the plant hormones measured were similar inahk5-1 and wild-type leaf tissue, it would be interesting to seehow concentrations of these hormones change over the course ofinfection with B. cinerea in the ahk5-1 mutant.

Interestingly, pretreatment of leaves with flg22 has been shownto increase resistance to infection with B. cinerea (Ferrari et al.,2007; Galletti et al., 2008), and the kinase Botrytis-induced kinase1 (BIK1) which is involved in resistance to B. cinerea infection(Veronese et al., 2006) has also been shown to interact withthe flagellin receptor FLS2 and BAK1 (BRI1 ASSOCIATEDRECEPTOR KINASE1) to mediate flagellin responses (Lu et al.,2010). Given the link with AHK5 and flagellin signalling (Desikanet al., 2008), it is possible that AHK5 acts to integrate basaldefence signalling pathways activated by diverse pathogens.

In conclusion, we have shown here that the hybrid histidinekinase AHK5 functions in salinity tolerance and resistance toPstDC3000 and B. cinerea. An underlying theme linking AHK5to the signalling pathways of the responses tested here is phyto-hormones and ROS. It is possible that, depending on the stimu-lus, AHK5 functions differentially to perturb hormoneaccumulation ⁄ signalling and redox balance, resulting in theahk5-1 mutant being more tolerant to salinity stress, yet less resis-tant to infection with bacterial and fungal pathogens. In previousstudies, AHK2 and AHK3 were found to positively contribute toresistance to PstDC3000 but to negatively regulate cold, salinityand drought stress (Tran et al., 2007, 2010; Choi et al., 2010;Jeon et al., 2010). The opposing functions of both AHK2 andAHK3 in biotic and abiotic stress responses are similar to thatfound for AHK5 in this study, whereby AHK5 is required for fullresistance to the plant pathogens PstDC3000 and B. cinerea butloss of function increases resistance to salinity stress. This studyprovides further evidence for the importance of hybrid HK func-tion in regulation of growth and survival responses to both abi-otic and biotic stresses. Further studies to identify AHK5interactors will be important for determining the mechanism ofAHK5 function. Other key genes which appear to integrate abi-otic and biotic stress responses via redox changes and hormonesinclude the DELLA proteins (Achard et al., 2008), UPS1(UNDERINDUCER AFTER PATHOGEN AND STRESS1)(Denby et al., 2005), BOS1 (BOTRYTIS SUSCEPTIBLE1)(Mengiste et al., 2003) and ATAF1 (ARABIDOPSIS NACDOMAIN CONTAINING PROTEIN2) (Wu et al., 2009).Thus, single genes controlling multiple stress responses do existand are undoubtedly important for the phenomenon of cross-tolerance and acclimation to stress. Our novel findings highlight






the importance of identifying key nodes in the signalling path-ways that mediate multiple stress responses in plants.

Acknowledgements

This research was funded by a Biotechnology and Biological Sci-ences Research Council (BBSRC) DTG to J.P. We thank EmmyMcGarry (BSPP summer student) for preliminary work pheno-typing plant responses to B. cinerea. We also thank YvonneStewart for her comments on the manuscript.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Confirmation of expression of Arabidopsis histidinekinase 5 (AHK5) in genetically complemented lines by semiquan-titative RT-PCR.

Fig. S2 Seedling survival assay in response to 200mM NaCl.

Fig. S3 Root growth of wild-type and Arabidopsis histidinekinase 5-1 mutant (ahk5-1) seedlings in response to sorbitol andKCl.

Fig. S4 Symptom development in wild-type Columbia (Col-0)and Arabidopsis histidine kinase 5-1 mutant (ahk5-1) leaves sur-face-inoculated with 4 · 108 cfu ml)1 Pseudomonas syringae pv.tomato DC3000 (PstDC3000) 7 d post-inoculation.

Fig. S5 3,3-Diaminobenzidine (DAB) staining of fungal hyphaegrowing on wild-type and Arabidopsis histidine kinase 5-1(ahk5-1) mutant leaves at 12 h post-inoculation.

Fig. S6 Trypan blue staining of fungal hyphae growing on wild-type and Arabidopsis histidine kinase 5-1 (ahk5-1) mutant leavesat 8 h post-inoculation.

Fig. S7 Lesion size in wild-type Columbia (Col-0) leaves 3 dpost-inoculation with pre-(vacuum) infiltration of diphenylenei-odinium chloride (DPI; 1 lM) or 2-(4-carboxyphenyl)-4, 4, 4,5-tetramethylimidazoline-oxyl-3-oxide (cPTIO; 50 lM) 2 hbefore inoculation of leaves with 2 · 105 spores ml)1 Botrytiscinerea in 1 ⁄ 8th potato dextrose broth.

Fig. S8 Dose-dependent response of seedling root growth toexogenous ABA.

Fig. S9 Unique metabolite detected in leaf tissue of the Arabid-opsis histidine kinase 5-1 (ahk5-1) mutant mock-inoculated with10mM MgCl2 (control) or surface-inoculated with 4 · 108 cfuml)1 Pseudomonas syringae pv. tomato DC3000 (PstDC3000) byLC-MS ⁄ MS.

Fig. S10 Hyphal length of Botrytis cinerea spores germinated in1 ⁄ 8th potato dextrose broth with or without the nitric oxide(NO) scavenger 2-(4-carboxyphenyl)-4, 4, 4, 5-tetramethylimi-dazoline-oxyl-3-oxide (cPTIO) (50 lM) for various durations.

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Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial and fungal infection