A Deletion in NRT2.1 Attenuates Pseudomonas syringae ... · A Deletion inNRT2.1 Attenuates Pseudomonas syringae-Induced Hormonal Perturbation, Resulting in Primed Plant Defenses1[C][W]

A Deletion in NRT2.1 Attenuates Pseudomonassyringae-Induced Hormonal Perturbation, Resultingin Primed Plant Defenses1[C][W]

Gemma Camanes, Victoria Pastor, Miguel Cerezo, Javier Garcıa-Andrade, Begonya Vicedo,Pilar Garcıa-Agustın, and Victor Flors*

Area de Fisiologıa Vegetal, Departamento de Ciencias Agrarias y del Medio Natural, Escuela Superiorde Tecnologıa y Ciencias Experimentales Universitat Jaume I, Castellon 12071, Spain (G.C., V.P., M.C., B.V.,P.G.-A., V.F.); and Instituto de Biologıa Molecular y Celular de Plantas, Universidad Politecnica de Valencia-Consejo Superior de Investigaciones Cientıficas, Ciudad Politecnica de la Innovacion, 46022 Valencia, Spain(J.G.-A.)

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce anincompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, wepresent an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plantdefenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient intwo genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environ-mental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the totallevel of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming ofsalicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection.Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due toreduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis(Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatinesynthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibilityof nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to thebacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistanceby down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.

In higher plants, nitrate uptake through the roots ismainly achieved by two gene families, NRT1 andNRT2. In Arabidopsis (Arabidopsis thaliana), there are53 NRT1 genes and seven NRT2 genes (Tsay et al.,2007). NRT1 codes for components of the nitrate low-affinity transport system (LATS), which regulatesnitrate uptake mainly when it is present at highconcentrations in the root environment. NRT1 cancombine its LATS function with a high-affinity trans-port system (HATS) function (Ho et al., 2009), and thisdual role is regulated by phosphorylation. Functionalcharacterization has shown that NRT2 genes belong tothe family of nitrate HATS (Gansel et al., 2001). The

NRT2.1 gene is activated when nitrate concentrationsare below 1mM, and it mediates systemic signals to theshoot in split-root systems depending on the nitrateconcentration (Cerezo et al., 2001; Gansel et al., 2001).The net contribution to total nitrate uptake of NRT2 islow compared withNRT1 (Little et al., 2005; Tsay et al.,2007). Interestingly, mutations in theNRT2.1 gene haveno influence on total nitrate or nitrogen content innormal fertilization conditions (Orsel et al., 2004). Inaddition, individual NRT2.1 mutations in Arabidopsisseem to be compensated for by NRT2.2; therefore, it isnecessary to have both genes blocked to induce asignificant reduction of nitrate uptake mediated byHATS (Li et al., 2007). Therefore, additional roles forthe NRT2 gene family have been proposed. Recently,the term “transceptor” has been applied to membraneproteins that fulfill dual-nutrient transport/signalingfunctions. Although it is now clear that NRT1.1 can beconsidered a transceptor, there is also evidence sug-gesting a signaling role for NRT2.1 (Gojon et al., 2011).NRT2 genes are related to environmental sensing andsignal transduction that integrates nitrogen metabo-lism with carbon metabolism (Little et al., 2005).NRT1.1 and NRT2.1 can both perceive small amountsof nitrate and transmit signals to the plant in order to

1 This work was supported by Generalitat Valenciana (grant no.GV/2007/099) and Plan Promocion Bancaja (grant nos. UJIP1.1A2007–07 and P1.1B2007–42).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Victor Flors ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.111.184424

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integrate growth with nutrient availability (Krouket al., 2010). Moreover, the 150-bp promoter region ofNRT2.1 is sufficient to mediate induction by nitrateand repression by nitrogen metabolites (Castaingset al., 2011).NAR2.1/NRT3.1 has been described as a transmem-

brane protein that is necessary for NRT2.1 function,but it has no transporter activity by itself (Okamotoet al., 2006; Tsay et al., 2007). This finding demonstratesthe complex regulation of the NRT2.1 gene, the role ofwhich as a nitrate transporter has been questioned andwhich has been proposed to be an environmentalsignal sensor that controls the development of the rootsystem and coordinates it with nutritional cues (Littleet al., 2005; Tsay et al., 2007).Nitrate uptake mediated by HATS has also been

demonstrated to be regulated by hormonal and met-abolic signals in wheat (Triticum aestivum; Cai et al.,2007). Abscisic acid (ABA) and Gln enhance NRT2.1and NAR2.1 gene expression when nitrate is notpresent in the nutrient solution, but they have noeffect when nitrate fertilization is appropriate. Thisphenomenon suggests the existence of feedback be-tween hormonal signaling and NRT2.1-mediated en-vironmental sensing (Cai et al., 2007).Nitrate transporter mutants have been shown to

display unusual phenotypes in response to abioticstress (Yao et al., 2008), but there is little or no evidencethat links nitrate HATS activity to resistance to bioticstresses. One of the few links between nitrogen trans-porters and biotic stress resistance is found in the geneNAR2/NRT3.1, also known as Wound Responsive3,which is induced in response to wounding througha jasmonic acid (JA)-independent pathway (www.arabidopsis.org).Several findings have indicated that the nutritional

status of a plant affects its defensive capacity againstthe bacterium Pseudomonas syringae (Modolo et al.,2006). When the main nitrogen source is ammonium,Arabidopsis is more sensitive to P. syringae infection.Feeding plants with ammonium as the sole nitrogensource prevents nitric oxide (NO) formation and in-hibits the hypersensitive reaction (HR), which corre-lates with altered susceptibility. By contrast, low levelsof nitrate favor NO formation through an enhance-ment of the NO synthase activity of nitrate reductase(NR; Modolo et al., 2005). Overfertilization leading toelevated internal nitrogen levels also contributes torice plant susceptibility (Long et al., 2000). This resultcould be explained by inhibition of the NO synthaseactivity of NR and stimulation of its reductase activity(Modolo et al., 2005). Although the reasons for thesealterations of basal plant resistance are not yet clear,net nitrate rates influence the dual activity of NR andalter the generation of reactive oxygen species andNO. This result may have a direct effect on plantdefense responses against pathogens. Lozano-Justeand Leon (2010) have shown that nia1nia2noa1 triplemutants (NIA and NOA genes involved in nitratereductase enzyme and NO synthesis, respectively)

display enhanced ABA sensitivity and drought toler-ance, due to the uncoupling of NO from ABA-triggeredresponses linked to defense responses. In addition tothese indications, alterations in amino acid transporters,which are tightly regulated by nitrogen status, havedirect effects on disease resistance. For example, the Lystransporter mutant lht1 (for Lys and His transport)displays constitutive activation of salicylic acid (SA)-dependent signaling and resistance to P. syringae (Liuet al., 2010).

Although the constitutive expression of defensegenes results in resistant plants, this property usuallyhas a negative impact on normal plant development(van Hulten et al., 2006). Therefore, among plantdefense strategies, priming has emerged as a notablyeffective one (Ton et al., 2005; Conrath et al., 2006;Conrath, 2011). Plants, upon appropriate stimulation,can activate faster and stronger defense signals bypotentiating their basal immune system. Interestingly,van Hulten et al. (2006) have demonstrated thatprimed responsiveness to SA in the edr1 mutant cor-relates with elevated levels of basal resistance againstPseudomonas syringae pv tomato DC3000 (Pst) with nomajor loss of plant growth or seed production. Al-though the mechanisms underlying priming are notfully known, the accumulation of dormant mitogen-activated protein kinases and chromatin remodelingare two processes likely to be involved in this phe-nomenon (Conrath, 2011). Although a link has not yetbeen established, the up-regulation of SA-dependentresponses may be under the control of such processes.

In this study, we demonstrate that alterations in theNRT2 gene family modify basal susceptibility to thebacterial pathogen Pst. The nrt2 mutant is less suscep-tible to Pst, and this reduced susceptibility correlateswith both SA pathway priming and reduced sensitiv-ity to pathogenic effectors. These results suggest anadditional role for membrane-located nitrate trans-porters in environmental stress perception and signaltransduction.

RESULTS

Disruption of NRT2.1 and NRT2.2 Results in Reduced

Susceptibility to Pst

Little et al. (2005) demonstrated that NRT2.1 actsas a nitrate sensor and signal transducer to coordi-nate the development of the root system with nutri-tional and abiotic cues. Given the similarities andclose link between biotic and abiotic sensing (Jakabet al., 2005; Ton et al., 2005), the levels of basalresistance against the hemibiotrophic pathogen Pstwere tested in the mutant nrt2. To this end, normallyfertilized Wassilewskija (Ws) and nrt2 plants wereinoculated by dipping at two different stages ofgrowth. Both adult plants and seedlings of nrt2showed reduced susceptibility to the bacteria, asevidenced by reduced symptom severity and re-duced bacterial proliferation (Fig. 1, A–C).

NRT2.1 Implications in Biotic Stress

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Figure 1. (Legend appears on following page.)

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Because total levels of nitrogen in a plant have astrong influence on pathogen colonization and prolif-eration (Hoffland et al., 1999; Ali et al., 2009), wedetermined whether alterations in NRT2 gene expres-sion or short periods of nitrate deprivation influencetotal nitrogen content, because it can induce the ex-pression of several NRT2 family members (derepres-sion; Lejay et al., 1999). Therefore, we transferredhydroponically growing plants to nitrate-free mediumbefore inoculation. Forty-eight hours of deprivationwas not sufficient to modify total nitrogen levels inplants that had been grown with 1 mM NH4NO3 for 6weeks (Supplemental Fig. S1A). We also confirmedthatNRT2.1 expression was enhanced not only in rootsbut also in leaves in both ecotypes tested (Ws andColumbia [Col-0]; Supplemental Fig. S1, B and C).Interestingly, after the induction of NRT2 gene expres-sion by nitrate depletion, Ws plants became moresensitive compared with normally fertilized Wsplants, whereas the level of resistance of nrt2 plantsdid not change (Fig. 1E). This finding might be attrib-utable to the plants’ lack of responsiveness to rootenvironmental sensing. Therefore, the derepressed (ac-tive transcription) versus repressed (nontranscribed)state of NRT2.1 affects the basal resistance level ofArabidopsis against Pst.It is noteworthy that nrt2 is a mutant that lacks the

function of both the NRT2.1 and NRT2.2 genes (Liet al., 2007). To clarify whether the reduced suscepti-bility of nrt2 is due to one or both genes, we testedbasal resistance in individual mutants, such as lin1(blocked in NRT2.1; Little et al., 2005), the knockoutmutant Salk_043543, and the double knockoutmutant Salk_035429, all in the Col-0 background. Thesingle NRT2.1 mutant lin1 and the double knockout(Salk_035429) also showed reduced susceptibility, butthe single mutant Salk_043543 did not, suggesting thatmutation of both genes may not be necessary to obtainsignificantly reduced bacterial growth (Fig. 1D). Thefact that Salk_035429 and lin1 are altered in their basalresistance reinforces the hypothesis that these genesplay additional roles in stress sensing and/or signaltransduction rather than being only involved in nitratetransport at low concentrations. In fact, NRT2.1 ex-pression after nitrate depletion was lower in Col-0than in Ws, and this finding correlates well with thenaturally greater resistance of Col-0. This natural

genetic variation has been recently reported to belinked to two quantitative trait loci, one of whichregulates SA-induced PR1 expression (Ahmad et al.,2011).

nrt2 Shows Primed SA-Dependent Responses upon

Pst Infection

Using chemical agents, such as b-aminobutyric acid(BABA), benzothiadiazole, or azelaic acid, to primeSA-dependent defenses results in enhanced resistanceagainst Pst (Conrath et al., 2006; Jung et al., 2009). Themutant nrt2 was found to display slightly increasedlevels of PR1 and PR5 compared with those of Ws inthe absence of infection (Fig. 2A). Interestingly, whenthe infection was present, nrt2 showed stronger andfaster induction of these SA marker genes (Fig. 2A),mainly at early time points. Accordingly, SA levelswere higher in nrt2 at 48 h after infection (Fig. 2B).

Salk_035429 and lin1 in the Col-0 backgroundshowed enhanced PR1 and SA responses concomitantwith their reduced susceptibility to the bacterium(Supplemental Fig. S2). To assess the relevance of SApriming in nrt2, we crossed lin1 and Salk_035429 withsid2.1 (isochorismate synthase deficient; Wildermuthet al., 2001). We tested two independent lines of thedouble and triple mutants lin1-sid2.1 and Salk_035429-sid2.1, and all of them displayed wild-type levels ofresistance, which demonstrates that enhancement ofthe SA pathway is a necessary component of thereduced susceptibility of nrt2 (Fig. 2C). The potentialhypersensitivity of nrt2 to SA was tested by treatingthe plants with BABA. BABAwas effective at protect-ing both wild-type and nrt2 plants to the same levels(Supplemental Fig. S3). Therefore, the mutant retainsits sensitivity to the SA priming induced by BABA.

nrt2 Has Reduced Sensitivity to P. syringae Effectors

Among the many impacts of bacterial effectors,manipulation of plant hormones is one of the maintargets. The role of ABA in Pst-Arabidopsis interac-tions is complex and not fully understood (Ton et al.,2009). It is known that Pst can take control of stomatalmovements during preinvasive and postinvasivestages by hijacking ABA signaling (Melotto et al.,2008; de Torres-Zabala et al., 2009; Ton et al., 2009).We checked for a contribution of ABA to nrt2-reduced

Figure 1. Disease rate and bacterial proliferation in wild-type plants and mutants lacking the NRT2 gene infected with Pst. Two-week-old (A and D) or 5-week-old (B, C, and E) plants were challenge inoculated with a bacterial suspension of Pst at 2.53 107

colony-forming units (cfu) mL21. Data are from a representative experiment that was repeated at least three times with similarresults. A and B, Bacterial growth in the leaves was determined over a 3-d time interval. The values presented are means6 SD ofthe log of the proliferation values. C, Disease symptomswere determined 3 d after inoculation and quantified as the proportion ofleaves with symptoms. The data presented are means of the percentage of diseased leaves per plant 6 SD. D, Bacterialproliferation in Col-0 wild-type plants and Salk_043543, Salk_035429, and lin1mutants infected with Pst. Two-week-old plantswere challenge inoculated with a bacterial suspension of Pst. The values presented are means6 SD of the log of the proliferationvalues. Asterisks indicate statistically significant differences (LSD test; P , 0.05, n = 15–25). E, Bacterial proliferation in Pst-infected plants fertilized normally (Ws and nrt2) and exposed to nitrate depletion 2 d before inoculation (Ws-N and nrt2-N). fw,Fresh weight.





susceptibility by studying the expression of severalABA marker genes and ABA levels. We monitoredthree different ABA-responsive genes, ABI1, RD22,and RAB18. The expression of ABI1 was up-regulatedby infection in nrt2, but it remained unchanged in Ws.By contrast, RD22 was down-regulated by the bacte-rium. Surprisingly, both ABA marker genes showedlower expression in nrt2 than in Ws in the absence ofchallenge. RAB18 showed the most dramatic change,as it was up-regulated by the bacterium in Ws butdisplayed lower expression in the mutant after infec-tion (Fig. 3A). This situation may lead to deficient ABAsignaling manipulation by the bacterium (Ton et al.,2009). Accordingly, the levels of ABA remained un-

changed during infection in the nrt2 mutant, whereasABA levels increased in wild-type plants (Fig. 3B).

To study the abnormal ABA signaling in nrt2, itsability to sense this hormone was verified by treatingWs and nrt2 plants with ABA 48 h before the infection(Fig. 3C). ABA treatment increased the disease rate inboth wild-type and mutant plants; however, bacterialgrowth only showed significant differences in ABA-treated nrt2 plants, probably due to the high inoculumused for all the experiments. The observation of thealtered behavior of ABA marker genes in nrt2 in theabsence of infection prompted us to test the response ofnrt2 plants to drought stress; however, the mutantshowed wild-type levels of water loss and tolerance

Figure 2. Effect of Pst infection on PR1and PR5 gene expression levels and SAaccumulation in 5-week-old Ws wild-type plants and nrt2 mutants. A, TotalRNA was isolated from infected leavesat 24 and 48 h after inoculation (hpi),converted to cDNA, and subjected toquantitative RT-PCR analysis. Theplants were mock or Pst inoculated,as described in Figure 1. The PR1 andPR5 transcript levels in the mock- andPst-infected plants were normalized tothe expression of At1g13320measuredin the same sample. The experimentwas repeated using b-tubulinwith sim-ilar results. The data show averages oftwo independent experiments ob-tained with a pool of 10 plants perpoint. The experiment was repeatedthree times with similar results. Aster-isks indicate statistical differencescompared with Ws for each time point(LSD test; P , 0.05, n = 3). B, RelativeSA accumulation in mock- or Pst-infected wild-type and nrt2 plants.Plant tissue was collected at varioustime points, and SA levels were deter-mined in freeze-dried material byHPLC-MS. The results are means 6 SD

(n = 5). The data are from a represen-tative experiment that was repeatedthree times with similar results. Theasterisk indicates a statistical differ-ence compared with Ws for that timepoint (LSD test; P , 0.05, n = 5). C,Four-week-old Col-0 plants, sid2.1,Salk_035429, and lin1 mutants, andtwo independent lines of the doubleand triple mutants lin1-sid2.1 andSalk_035429-sid2.1, all in the Col-0background, were challenged, as de-scribed in Figure 1. Bacterial growth inthe leaves was determined at 3-d timeintervals. The values presented aremeans 6 SD. Different letters indicatestatistically significant differences (LSDtest; P, 0.05, n = 15–25). cfu, Colony-forming units; dw, dry weight.

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(Supplemental Fig. S4). Therefore, ABA, which is impli-cated in abiotic stress signaling, seems to functionnormally in nrt2 plants. Subsequently, we studiedwhether coronatine sensitivity was altered in thismutant, because it has been reported that coronatinealters ABA functioning during infection (de Torres-Zabala et al., 2007; Melotto et al., 2008). To this end,several bioassays were performed. In the first exper-iment, the resistance of nrt2mutants against Pst COR2

was tested in both the Ws and Col-0 backgrounds.Interestingly, the absence of coronatine eliminated the

reduced susceptibility in all individual and doublemutants compared with their corresponding wild-typeplants (Fig. 4; Supplemental Fig. S5). We subsequentlycomplemented the Pst COR2 by treating both genotypeswith coronatine (Fig. 4B; Brooks et al., 2004). The resultconfirmed that the initial reduced susceptibility pheno-type of nrt2 was reestablished; therefore, it is clear thatthe presence of coronatine makes a contribution to thereduced susceptibility of this mutant.

This indication that nrt2 displays altered effectorsensitivity was further verified by treatingWs and nrt2

Figure 3. Analysis of the ABA-dependent signaling pathway in the mutant nrt2 versus Ws upon Pst infection. A, Total RNA wasisolated from infected leaves at 24 and 48 h after inoculation (hpi), converted to cDNA, and subjected to quantitative RT-PCRanalysis. Plants were mock or Pst inoculated, as described in Figure 1. The ABI1, RD22, and RAB18 transcript levels in wild-typeand nrt2 plants were normalized to the expression of At1g13320 measured in the same sample. The experiment was repeatedusing b-tubulin with similar results. The data show averages of two independent experiments obtained with a pool of 10 plantsper point. The experiment was repeated three times with similar results. Asterisks indicate statistical differences compared withWs for each time point (LSD test; P , 0.05, n = 3). B, Relative ABA accumulation in wild-type and nrt2 plants upon infection byPst. Plant tissue was collected at various time points, and ABA levels were determined in freeze-dried material by HPLC-MS. Theresults are means6 SD (n = 5). The data are from a representative experiment that was repeated with similar results. The asteriskindicates a statistical differences compared with Ws for that time point (LSD test; P , 0.05, n = 5). C, Four-week-old plants weresoil drenched with water or 80 mM ABA. At 2 d after the chemical treatment, the plants were challenged, as described in Figure 1.Pst infection was determined by quantifying both the disease rate and colony-forming units (cfu), as described previously.Different letters indicate statistically significant differences between wild-type and mutant plants (LSD test; P, 0.05, n = 15–25).dw, Dry weight; fw, fresh weight.





plants grown in Murashige and Skoog (MS) mediumwith the effector coronatine (Fig. 5). Treatment withcoronatine significantly induced hydrogen peroxide(H2O2; a marker of coronatine’s effect) in Ws, whereasnrt2 plants remained unaltered after treatment (Fig. 5,A and B). This finding suggests that H2O2 may be atarget of coronatine in a pathway requiring NRT2.1.An additional experiment confirmed the insensitivityof nrt2 plants to coronatine. Ws and nrt2 plants weresprayed with coronatine. After the treatment, de-tached leaves of Ws remained turgid, while nrt2 leavespresented clear wilting at 4 and 6 h post treatment, asdid water-treated leaves (Fig. 5C). This result might bedue to an induction of stomatal closure at 4 and 6 hpost treatment by coronatine in Ws, while in nrt2plants, the stoma would remain unaltered. We also

infected Arabidopsis by direct infiltration to circum-vent possible stomatal aperture defects in the mutant.Surprisingly, nrt2 remained resistant, suggesting thatcoronatine sensitivity may contribute to its enhancedresistance but is not essential for it (Supplemental Fig.S6). Finally, we performed the classical test of thesensitivity of germination to coronatine. In this case,nrt2 showed wild-type sensitivity upon coronatinetreatment, which was visible as purple leaves and adelay in germination (Fig. 5D).

Another major target of coronatine is the COI1 re-ceptor and subsequent JA pathway activation (Fonsecaet al., 2009). Accordingly, the JA pathway showedslower activation in nrt2 compared with Ws. The JAmarker genes VSP2 and MYC2, which are not reducedin the mutant in the absence of infection, showed a

Figure 4. Quantification of coronatine’s influ-ence on the basal resistance of Ws wild-typeand nrt2 plants against Pst. A, Four-week-oldplants were challenged, as described in Figure1, but using Pst COR2 instead. Disease wasassessed by quantifying both disease rate andcolony-forming units, as described previously.Different letters indicate statistically significantdifferences (LSD test; P , 0.05, n = 15–25). B,Four-week-old plants were sprayed with water or0.5 ng mL21 coronatine. At 2 d after the chemicaltreatment, the plants were challenged with PstCOR2, as described in Figure 1. Different lettersindicate statistically significant differences be-tween wild-type and mutant plants (LSD test; P ,0.05, n = 15–25). cfu, Colony-forming units; fw,fresh weight.

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lower level of induction at 24 h after inoculation in nrt2upon infection (Supplemental Fig. S7A). The fact thatthese genes are not altered without pathogen confirmsthat the negative influence of SA priming in the mutantis only present when the plant is challenged. In addi-

tion, infected mutants displayed reduced JA levelscompared with infected wild-type plants (Supplemen-tal Fig. S7B).

Therefore, the down-regulation of ABA and JAsignaling after Pst infection in nrt2 could be a conse-

Figure 5. Sensitivity to exogenously applied coronatine inWs wild-type and nrt2 plants. A and B, One-week-old plants grown in1 mL of MS medium were treated with coronatine at 0.5 ng mL21 final concentration. H2O2 accumulation was determined by3,3#-diaminobenzidine staining. The data show averages from visible microscopy of wild-type and mutant plants6 SD. Differentletters indicate statistically significant differences (LSD test; P, 0.05, n = 15–25). C, Four-week old plants were sprayedwith wateror 0.5 ng mL21 coronatine. After the treatment, leaves of the same size were detached and exposed towilting at the indicated timepoints. The data show averages of 25 leaves. Different letters indicate statistically significant differences (LSD test; P , 0.05,n = 25). The experiment was repeated twice with similar results. hpt, Hours post treatment. D, Four-day-old wild-type andmutantnrt2 seedlings grown in MS medium supplemented with water or coronatine at 0.5 ng mL21. [See online article for color versionof this figure.]





quence of a partial loss of bacterial effector sensitivityrather than a defect in those pathways.

Gene Expression Analysis Confirms the Reduced

Sensitivity of nrt2 to Coronatine

We tested whether nrt2 plants present abnormalresponses to direct coronatine treatment. We observedthat coronatine treatment up-regulated such genes asPDF1.2, CORI1, and CORI3; however, such inductionwas always lower in nrt2 compared with the wild type(Supplemental Fig. S7C). In contrast to Pst infection(Fig. 3A), RAB18 was down-regulated by direct treat-ment with coronatine in Ws, whereas in nrt2 itremained unaltered (Supplemental Fig. S7C). Thisresult reinforces the hypothesis that nrt2 is less sensi-tive to coronatine-induced alterations in the plant.

To characterize the molecular response of nrt2 to Pstfurther, whole-genome transcriptional profiles of Wsand nrt2 plants mock treated or infected with Pstwereobtained using Arabidopsis ATH1 arrays from Affy-metrix. An examination of the differentially expressedgenes in Ws and nrt2 upon infection compared withmock treatment revealed a group of 574 genes thatwere differentially expressed in the mutant but wereunchanged in Ws, whereas 550 genes were up- ordown-regulated in Ws as a consequence of infectionbut remained unaltered in nrt2 (Supplemental Fig. S8;Supplemental Table S2).

Following the line of evidence suggesting that nrt2has reduced sensitivity to bacterial effectors, we se-lected genes from the array that are induced bycoronatine. This set of genes was described by Tsaiet al. (2011) after a comparative full-genome analysis ofplants infected by a P. syringae COR2 strain or by astrain with functional coronatine. In addition, a secondcomparative analysis performed by Tsai et al. (2011)discriminated from among all of the coronatine-inducible genes a group of 31 genes that were down-regulated by the priming agent BABA. We looked atthe behavior of this set of genes in our study and,interestingly, 18 out of 31 genes repressed by BABAwere also less induced in infected nrt2 plants com-pared with wild-type infected plants (SupplementalFig. S8B; Supplemental Table S3). This finding sup-ports the idea that in the absence of NRT2, the plantdisplays behavior similar to that of BABA-primedplants.

DISCUSSION

In this work, we investigated additional roles for theNRT2.1 gene. Mutations affecting NRT2.1 reduce thesusceptibility of Arabidopsis to the virulent pathogenPst. nrt2 mutants display primed SA signaling uponinfection (Figs. 1 and 2). In addition, these plantspresent defects in sensitivity to the bacterial effectorcoronatine and down-regulation of the JA pathway,probably as a consequence of SA priming, reduced

coronatine sensitivity, or both (Supplemental Fig. S7).This result suggests that one or several components ofthe NRT2 gene family may function as environmentalsensors, coordinating not only root and nutritionalcues but also abiotic and biotic responses by enhancingabiotic responses and down-regulating plant defensesagainst biotic challenges. The term transceptor hasbeen applied to such genes as NRT1.1 that act astransporters but also coordinate other features of plantdevelopment and nitrogen metabolism (Gojon et al.,2011). In fact, mutants defective for NRT1 familymembers are impaired in NRT2.1 regulation, whichhighlights the tight regulatory link between nitrateperception by NRT1.1 and the function of other NRTfamily members. NRT2.1 and NRT2.2 have been pro-posed to play roles in the integration of sugar andamino acid metabolism. Other studies point to NRT2.1as a putative abiotic stress sensor that coordinates thedevelopment of the root system with the root envi-ronment and nutrition (Little et al., 2005; Tsay et al.,2007). It is worth mentioning that downstream nitrateuptake mutants, such as nia1nia2noa1, display hyper-sensitivity to ABA and faster stomatal closure upondrought stress, being subsequently more drought tol-erant (Lozano-Juste and Leon, 2010).

In our research, we were interested in studying thelinks between nitrate perception and biotic stressresponses. Several reports have described changes inplant susceptibility to Pst that depend on the nitrogen,nitrate, or ammonium content of the plant (Long et al.,2000; Modolo et al., 2005, 2006). To avoid this possibleinterference, we performed all experiments with nor-mally fertilized plants (unless otherwise mentioned).Arabidopsis fertilized with ammonium nitrate at 1 mM

has its LATS activated and NRT2.1, NRT2.2, and NIArepressed (Krouk et al., 2010). In this condition, weshow that mutants lacking either NRT2.1 or NRT2.2have reduced susceptibility (Fig. 1). This phenotype isdue to the lack of the nitrate HATS, because activation(derepression) versus down-regulation (repression) ofthese genes has clear consequences on wild-type Wssusceptibility to the bacterium.

One mechanism potentially able to explain the re-duced susceptibility of nrt2 is found in the SA signal-ing pathway. nrt2 mutants show priming of SAsignaling at early stages of infection, as confirmed bySA marker gene induction and hormone accumula-tion. SA priming is one of the main mechanisms ofinduced resistance against Pst (Zimmerli et al., 2000;Conrath et al., 2006). There are several priming in-ducers (such as benzothiadiazole, BABA, and azelaicacid) that clearly reduce bacterial disease symptomsand plant tissue colonization by stimulating the SApathway (Conrath et al., 2006; Jung et al., 2009).Interestingly, nrt2 mutants experience constitutivepriming, because basal levels of SA in the absence ofinfection are the same as in Ws, and this finding hasalso been reported for other mutants, such as enhanceddisease resistance1 (edr1; van Hulten et al., 2006). Mu-tants with direct activation of SA signaling, such as

Camanes et al.




cpr1, are highly resistant to biotrophic pathogens, butthis condition slows growth and reduces seed produc-tion (van Hulten et al., 2006). The nrt2mutant displaysno phenotype in the absence of the pathogen, and itshows enhanced defense responses upon infection,similar to edr1. Therefore, NRT2.1, bacterial percep-tion, and SA seem to be linked. Genevestigator stim-ulus analysis showed that both programmed celldeath (a possible mechanism of HR) and flg22 induceNRT2.1 expression strongly (data not shown). Re-cently, Ward et al. (2010) highlighted by metabolomicanalysis that nitrogen-containing compounds andamino acids are strongly altered upon Pst infection.Increasing evidence points to Gln reallocation in thecytoplasm as a checkpoint that clearly interacts withSA signaling. Wu et al. (2010) reported that Glnstrongly represses BABA priming against Pst bydown-regulating PR1. In addition, lht1 mutants showstrongly affected disease resistance (Liu et al., 2010).The LHT1 gene antagonizes SA-dependent signaling,and it is up-regulated by pathogen attack. lht1mutantsshow up-regulated PR1, NRT1.1, and AMT1.1, amongothers. In addition, the elevated resistance of lht1seems not to be directly linked to global nitrogenstatus, as it is with NRT2.1 (Liu et al., 2010). Thus, itseems clear that some of the genes regulating nitrogenand amino acid metabolism or transport have strongregulatory functions that play roles in plant-pathogeninteractions.Several studies suggest that hormones transfer

nitrate signals, which would explain the contextof nitrate’s influence on plants. INF1, an elicitin fromPhytophthora infestans, induces NRT2.1 expression intobacco (Nicotiana tabacum), and it stimulates manySA-dependent responses and HR. Thus, SA signalingand NRT2.1 seem to be coordinately up-regulated bythis pathogenic elicitor (Kawamura et al., 2009). It islikely that such oomycetes that combine necrotrophicand biotrophic lifestyles target certain plant genesrelated to nutrition in order to improve the plant’snutrient sources to their advantage. It is noteworthythat one-tenth of the genome is under the control ofnitrate (Gutierrez et al., 2003), and interestingly, anitrate-regulated biomodule of genes is also controlledby plant hormones (Krouk et al., 2010).Our experiments disproved the hypothesis that

irregular functioning of the ABA signaling pathwayupon infection is responsible for the reduced suscep-tibility of nrt2mutants (Fig. 3). The results indicate thatABA signaling functions normally in these mutants,because they show wild-type responses to droughtstress. In addition, external treatment with ABA in-creases the susceptibility of nrt2mutants to Pst, as waspreviously reported for wild-type plants (Mohr andCahill, 2003).Effector-triggered susceptibility (Jones and Dangl,

2006) is one of the main roles of the bacterial effectorcoronatine. Several studies have demonstrated thatcoronatine targets genes to stimulate the JA signalingpathway in order to repress SA signaling and take

advantage of plant colonization. In addition, coronatinealso hijacks ABA functions, such as stomatal closure inthe preinvasive stages by opening stomata and duringpostpenetration stages by closing stomata (de TorresZabala et al., 2007; Ton et al., 2009). In all cases,coronatine plays an active role in bacterial infection,but in our experiments, we found that nrt2 mutantswere partially unable to sense coronatine. This bacterialeffector failed to induce H2O2 in nrt2 mutants, and itwas also unable to reduce wilting in detached mutantleaves, probably due to a failure of stomatal closure(Figs. 4 and 5). In addition, nrt2 shows a wild-typedegree of basal resistance to Pst COR2, indicating thatthe presence of the effector is necessary to observe thereduced susceptibility of nrt2 mutants. Tsai et al. (2011)obtained similar results using the chemical primingagent BABA. These researchers determined that a func-tional coronatine response was needed to expressBABA priming and induce resistance against Pst. Theseobservations, together with the fact that many genesinduced by coronatine are down-regulated in nrt2,explain why the reduced susceptibility of nrt2 disap-

Figure 6. Model of the influence of theNRT2.1 gene on the SA, JA, andABA defense signaling pathways. In the absence of a functionalNRT2.1gene, the plant is less sensitive to coronatine; therefore, the manipu-lation of the plant’s defensive metabolism by the bacterium is lessefficient. In the mutant, JA- and ABA-dependent signaling displayirregular activation, and SA-dependent responses are primed in thepresence of the pathogen. Thus, the mutation affects the SA pathwaydirectly and also the negative cross talk between the ABA and JApathways. Only those signal transduction elements that are affected innrt2 are represented. Green indicates down-regulated processes, andred represents up-regulated processes. Black crosses indicate thealtered processes in the nrt2 mutant. [See online article for colorversion of this figure.]





pears when the bacterium lacks coronatine. To con-clude, the nrt2 mutant is less sensitive to coronatine-triggered susceptibility, and this property reduces theability of the bacterium to colonize the mutant. Anotherinteresting result of these experiments is that H2O2 is aputative target of coronatine, and this interaction isNRT2.1 dependent, as nrt2 is totally impaired in H2O2production upon coronatine treatment.

Tsai et al. (2011) reported that priming by BABAagainst Pst acts through the specific inhibition ofcoronatine-triggered responses, and this inhibition re-sembles nrt2 phenotypes that match partial coronatineinsensitivity with SA-dependent priming and reducedsusceptibility to the bacterium. Among all of the po-tential contributions to the reduced susceptibility ofnrt2mutants, SA priming seems to be the major player.In this study, we demonstrated the absence of theresistance phenotype in the triple and double mutantsSalk_035429-sid2.1 and lin-sid2.1 (Fig. 2). However, thereduced sensitivity to coronatine may also contribute tothe resistance; therefore, this phenotype could be acombination of both. The fact that most experimentsperformed with NRT2 mutants in the Col-0 and Wsbackgrounds have obtained almost identical resultsreinforces our main conclusions. However, the reducedSA signaling priming and the lower resistance of lin1and Salk_035429 mutants is justified because Col-0displays natural SA priming and thus enhanced resis-tance to Pst compared with Ws (Ahmad et al., 2011).Regarding the SA-JA cross talk, it is likely that the SApriming is also a consequence of the absence of repres-sion by JA signaling, which would be less stimulated,due to the reduced coronatine sensitivity. This inter-pretation is further confirmed by the delay in MYC2induction in nrt2 compared with the wild type, becauseMYC2 mediates the suppression of SA-dependent de-fenses by coronatine (Fig. 6). This cross talk has recentlybeen revised by Pieterse et al. (2009).

Indirectly, nrt2mutants show attenuated Pst-inducedhormonal perturbations that result in the priming ofplant defenses and reduced susceptibility.

In conclusion, although the full implications of thenitrate transporter NRT2 in biotic and abiotic stressesremain to be clarified, the implications may be linkedthough the enhancement of abiotic stress sensing andcoordinated biotic stress repression. At the same time,this transporter could be a direct or indirect targetused by bacterial pathogens to take advantage of plantcolonization. This research suggests that NRT2.1 is anovel transceptor of the nitrate transport family thatregulates plant disease resistance signaling.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Wild-type Arabidopsis (Arabidopsis thaliana) Ws and the Ws mutant nrt2.1-

2.2 (referred to as nrt2 herein; Filleur et al., 2001) were generously provided by

Alain Gojon (INRA-Montpelier), the knockout lines nrt2.1-2.2 (referred to as

Salk_035429 herein) and nrt2.2 (referred to as Salk_043543 herein) in the Col-0

background were obtained from the SALK Institute (Alonso et al., 2003), and

the ethyl methanesulfonate lin1 line was generously supplied by Jocelyn

Malamy (University of Chicago).

All plant genotypes were germinated in soil, and 2 weeks after germina-

tion, the seedlings were individually transferred to 33-mL pots containing

commercial potting soil (TKS1; Floragard; http://www.floragard.de) for adult

plants, or approximately 50 seedlings were germinated in 33-mL pots

containing commercial potting soil for seedling plants. The plants were

cultivated at 20�C day/18�C night with 8.5 h of light (105 mE m22 s21) per 24 h

and 60% relative humidity.

Growth Conditions for Starvation Experiments

Wild-type Ws and the mutant nrt2 were grown hydroponically, as de-

scribed by Lejay et al. (1999). The seeds were germinated directly on top of

modified Eppendorf tubes filled with prewetted sand. Plants were grown

until the age of 6 weeks on 1 mM NH4NO3 nutrient solution (repressed plants),

which prevented any growth difference between the two genotypes (Lejay

et al., 1999). Before inoculation experiments, the plants were transferred for 48 h

to nitrogen-free solution (derepressed plants).

Bacterial Strains and Bacterial Growth Assays

The bacterial strains were Pseudomonas syringae pv tomato DC3000 and P.

syringae pv tomato DC3000 cma (Brooks et al., 2004). Bacteria were grown

overnight at room temperature in King’s B solid medium with appropriate

antibiotics and diluted to the desired concentration with 10 mM MgSO4 for

plant inoculation. These bacteria were used to infect 5-week-old (or otherwise

mentioned) Arabidopsis plants by dipping in a suspension of 2.5 3 107

colony-forming units mL21 using 0.02% Silwet L-77 (Tornero and Dangl,

2001). After incubation at 28�C for 3 d, the number of rifampicin-resistant

colony-forming units per gram of infected leaf tissue was determined, and

bacterial proliferation over the 3-d time interval was calculated. Three days

after the challenge inoculation, the percentage of leaves with symptoms was

determined per plant (n = 20–25). Leaves showing necrotic or water-soaked

lesions surrounded by chlorosis were scored as diseased.

Double and Triple Mutant Generation and Selection ofHomozygous Lines

The sid2.1 mutant was crossed with Salk_035429 and lin1 (all in the Col-0

background), and the resulting F1 generation was self-crossed. Homozygous

F2 seedlings were selected by PCR, and F3 seeds were used for the experi-

ments. DNA was isolated from leaves of individual plants (Edwards et al.,

1991). To identify plants that were homozygous for the sid20.1 mutation, the

following primers were used: sid2.1-F (5#-GCTCTGCAGCTTCAATGC-3#)and sid2.1-R (5#-CGAAGAAATGAAGAGCTTGG-3#). A fragment of 243

nucleotides was obtained and subsequently digested by the enzyme TruI

(MBI Fermentas) and separated on a 2% agarose gel to evaluate the fragment

sizes of 154 and 89 nucleotides in the mutant. Next, homozygous sid2.1 plants

were selected to find the other mutations in homozygosity. In this case, the

primers used for Salk_035429 and lin1 were as follows: LP (5#-GCAAGC-

GACTATCATCACTCC-3#), RP (5#-GTTCTCCATGAGCTTCGTGAG-3#), andLB (5#-ATTTGCCGATTTCGGAAC-3#) following the SALK Institute instruc-

tions for Salk_035429 and lin-F (5#-ATCAAATCTCAAACTTGCAAAGAAC-3#)and lin-R (5#-GGGGACCAAAAGATCACACACGGATC-3#). To identify

plants homozygous for lin1, the amplification product from the wild-type

allele is cleaved with BamHI (MBI Fermentas), and the product from the lin1

mutant allele is uncut. These fragments were separated on a 2% agarose gel to

evaluate the fragment sizes.

Quantitative Real-Time Reverse Transcription-PCRAnalysis of Transcripts

Gene expression analysis by quantitative real-time reverse transcription

(RT)-PCR was performed using RNA samples extracted from leaf tissue using

the Total Quick RNA cells and tissues kit (Talent; http://www.spin.it/talent).

Arabidopsis leaf tissue samples for RNA isolation were collected at 24 and 48 h

after inoculation. Leaf tissue from five plants each of the mutant and wild-type

plants was collected. For quantitative real-time RT-PCR experiments, 1.5 mg of

total RNA was digested using 1 unit of RQ1 RNase-Free DNase (Promega;

Camanes et al.




http://www.promega.com) in 1 mL of DNase buffer and up to 10 mL of Milli-Q

water and was incubated for 30 min at 37�C. After the incubation, 1 mL of RQ1

DNase stop buffer was added, and the solution was incubated again at 65�C for

10 min to inactivate the DNase. Highly pure RNAwas used for the RT reaction.

The RT reaction was performed by adding 2 mL of RT buffer, 2 mL of 5 mM

deoxyribonucleotide triphosphate, 2 mL of 10 mM oligo(dT)15 primer (Promega),

1 mL of 10 units mL–1 Rnasin RNase inhibitor (Promega), and 1 mL of Omniscript

reverse transcriptase (Qiagen; http://www.qiagen.com). The reaction mixture

was incubated at 37�C for 60min. Less than 10% of the volume of the RTreaction

was used for the quantitative PCR. Forward and reverse primers (0.3 mM) were

added to 25 mL of QuantiTect SYBR Green PCR buffer (Qiagen), as were 2 mL of

cDNA and Milli-Q sterile water up to 50 mL total reaction volume. Quantitative

PCR was carried out using the Smart Cycler II sequence detector (Cepheid;

http://www.cepheid.com). PCR cycling conditions consisted of an initial

HotStarTaq (Qiagen; http://www.qiagen.com) polymerase activation step at

95�C for 15min followed by 45 cycles of 95�C for 15 s, 60�C for 30 s, and 72�C for

30 s Melting-curve analysis was performed at the end of the PCR to confirm

product purity. Differences in cycle numbers during the linear amplification

phase between samples containing cDNA from treated and untreated plants

were used to determine differential gene expression. The b-tubulin and

At1g13320 genes of Arabidopsis were used as housekeeping genes. Normalized

values of gene expression are presented on a log2 scale. A list of the primers used

in the quantitative RT-PCR is shown in Supplemental Table S1.

Determination of ABA, SA, and JA Levels

Fresh material was frozen in liquid nitrogen and lyophilized. Before

extraction, a mixture of internal standards containing 100 ng of [2H6]ABA, 100

ng of [2H4]SA, and 100 ng of dihydrojasmonic acid (Pinfield-Wells et al., 2005)

was added. Dry tissue (0.05 g) was immediately homogenized in 2.5 mL of

ultrapure water. After centrifugation (5,000g, 40 min), the supernatant was

recovered and adjusted to pH 2.8 with 6% acetic acid and subsequently

partitioned twice against an equal volume of diethyl ether. The aqueous phase

was discarded, the organic fraction was evaporated in a Speed Vacuum

Concentrator (Eppendorf; www.eppendorf.com) at room temperature, and

the solid residue was resuspended in 1 mL of a water:methanol (90:10)

solution and filtered through a 0.22-mm cellulose acetate filter. A 20-mL aliquot

of this solution was directly injected into the HPLC system. Analyses were

carried out using a Waters Alliance 2690 HPLC system with a nucleosil ODS

reverse-phase column (100 3 2 mm i.d., 5 mL; Scharlab; http://www.

scharlab.es). The chromatographic system was interfaced to a Quatro LC

(quadrupole-hexapole-quadrupole) mass spectrometer (Micromass; http://

www.micromass.co.uk). MASSLYNX NT software version 4.1 (Micromass)

was used to process the quantitative data from calibration standards and the

plant samples.

Microarray Analysis

Leaves of wild-type Arabidopsis Ws and the Ws mutant nrt2 infected with

Pst and not infected were taken for total RNA preparation using TRIZOL

(Invitrogen) reagent. Total RNA was purified with the RNeasy Mini Kit

(Qiagen). Double-stranded cDNA was synthesized with the one-cycle cDNA

Synthesis Kit (Affymetrix) and was purified with the GeneChip Sample

CleanupModule (Affymetrix). The purified cDNAwas used to prepare biotin-

labeled copy RNA using a GeneChip IVT Labeling Kit according to the

manufacturer’s instructions. The biotin-labeled copy RNAwas fragmented at

94�C for 35 min, which yielded the probes used for hybridization. The probes

were hybridized with the Affymetrix ATH1 array, and washing and scanning

were carried out according to the assay procedure. The hybridization image

was analyzed with Affymetrix Microarray Suite 5.0 software, and the data

were normalized. Three biological replicates from three different experiments

were used for transcriptomic comparisons. All basic operations, array hy-

bridization, image analysis, and statistical analysis were performed by

Progenika. Bioinformatic analysis was performed by using the PartekGeno-

mics Suite dChip (www.dchip.org) and the software Affy and affyPLM from

the consortium Bioconductor (www.bioconductor.org).

Chemical Treatments

Two-week-old seedlings were individually transferred to 33-mL pots. At the

age of 5 weeks, plants were soil drenched with water (control) or a solution of

BABA or ABA at a final concentration of 250 or 80 mM, respectively. Two days

after the chemical treatment, the plants were inoculated, as described above.

Growth Conditions and Coronatine Treatment in in

Vitro Assays

Approximately 15 sterilized Col-0 seeds were sown per well on sterile 12-

wells plates containing filter-sterilized MS medium amended with 1% Suc.

Seedlings were cultivated under standard growth conditions (15-h-day cycle;

20�C /17�C) with a light intensity of 105 mE m22 s21. After 7 d, the growth

medium was replaced with fresh MS medium. One day later, the plants were

challenged with coronatine (Sigma C8115) at a final concentration of 0.5 ng

mL21 in the growth medium. At day 9, the plants were stained with 3,3#-diaminobenzidine (Sigma, D-8001) to quantify H2O2 induction (Thordal-

Christensen et al., 1997). Samples were stained for 8 h and were subsequently

destained with 95% ethanol. Microscopic analysis was performed using a

microscope (Nikon Eclipse 11000) with a visible light filter. H2O2 was quan-

tified as the relative number of brown pixels in digital photographs using

GIMP 2.6 software.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AF019748 and AF019749.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Total nitrogen levels and NRT2 transcript levels.

Supplemental Figure S2. Effect of Pst infection on PR1 gene expression

levels and SA accumulation.

Supplemental Figure S3. Disease rate and bacterial proliferation.

Supplemental Figure S4. Water loss in Ws wild-type and nrt2 plants.

Supplemental Figure S5. Bacterial proliferation in Col-0 wild-type, lin1,

and Salk_035429

Supplemental Figure S6. Bacterial proliferation in Ws wild-type and nrt2

plants infected by infiltration.

Supplemental Figure S7. Effect of Pst infection on VSP2 and MYC2 gene

expression levels and JA accumulation.

Supplemental Figure S8. Analysis of differentially expressed genes in Ws

and nrt2 upon infection.

Supplemental Table S1. List of primers employed in quantitative RT-PCR

analysis.

Supplemental Table S2. List of genes and fold induction analysis

presented as a Venn diagram.

Supplemental Table S3. List of genes that are induced by coronatine and

down-regulated in nrt2-Pst/nrt2-mock versus Ws-Pst/Ws-mock.

ACKNOWLEDGMENTS

We thank the Servicios Centrales de Instrumentacion Cientıfica of the

Universitat Jaume I for its technical support. We also thank Brigitte Mauch-

Mani and Juan Antonio Lopez-Raez for proofreading and critically reviewing

the manuscript.

Received August 1, 2011; accepted December 9, 2011; published December 12,

2011.

LITERATURE CITED

Ahmad S, Van Hulten M, Martin J, Pieterse C, Van Wees S, Ton J (2011)

Genetic dissection of basal defence responsiveness in accessions of

Arabidopsis thaliana. Plant Cell Environ 34: 1191–1206

Ali FS, Zayed G, Saad OA, Abdul-Mohsen E (2009) Optimisation of

nitrogen fertiliser level for maximum colonisation of mycorrhizae on

roots of coriander plants. African Crop Science Conference Proceedings

9: 117–122

Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson





DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide inser-

tional mutagenesis of Arabidopsis thaliana. Science 301: 653–657

Brooks DM, Hernandez-Guzman G, Kloek AP, Alarcon-Chaidez F,

Sreedharan A, Rangaswamy V, Penaloza-Vazquez A, Bender CL,

Kunkel BN (2004) Identification and characterization of a well-defined

series of coronatine biosynthetic mutants of Pseudomonas syringae pv.

tomato DC3000. Mol Plant Microbe Interact 17: 162–174

Cai C, Zhao XQ, Zhu YG, Li B, Tong YP, Li ZS (2007) Regulation of the

high-affinity nitrate transport system in wheat roots by exogenous

abscisic acid and glutamine. J Integr Plant Biol 49: 1719–1725

Castaings L, Marchive C, Meyer C, Krapp A (2011) Nitrogen signalling in

Arabidopsis: how to obtain insights into a complex signalling network.

J Exp Bot 62: 1391–1397

Cerezo M, Tillard P, Filleur S, Munos S, Daniel-Vedele F, Gojon A (2001)

Major alterations of the regulation of root NO32 uptake are associated

with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis. Plant

Physiol 127: 262–271

Conrath U (2011) Molecular aspects of defence priming. Trends Plant Sci

16: 524–531

Conrath U, Beckers GJM, Flors V, Garcıa-Agustın P, Jakab G, Mauch F,

Newman MA, Pieterse CMJ, Poinssot B, Pozo MJ, et al (2006) Priming:

getting ready for battle. Mol Plant Microbe Interact 19: 1062–1071

de Torres Zabala M, Bennett MH, Truman WH, Grant MR (2009) Antag-

onism between salicylic and abscisic acid reflects early host-pathogen

conflict and moulds plant defence responses. Plant J 59: 375–386

de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW,

Rodriguez Egea P, Bogre L, Grant M (2007) Pseudomonas syringae pv.

tomato hijacks the Arabidopsis abscisic acid signalling pathway to

cause disease. EMBO J 26: 1434–1443

Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method

for the preparation of plant genomic DNA for PCR analysis. Nucleic

Acids Res 19: 1349

Filleur S, Dorbe MF, Cerezo M, Orsel M, Granier F, Gojon A, Daniel-

Vedele F (2001) An Arabidopsis T-DNAmutant affected in Nrt2 genes is

impaired in nitrate uptake. FEBS Lett 489: 220–224

Fonseca S, Chico JM, Solano R (2009) The jasmonate pathway: the ligand, the

receptor and the core signalling module. Curr Opin Plant Biol 12: 539–547

Gansel X, Munos S, Tillard P, Gojon A (2001) Differential regulation of the

NO32 and NH4

+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabi-

dopsis: relation with long-distance and local controls by N status of the

plant. Plant J 26: 143–155

Gojon A, Krouk G, Perrine-Walker F, Laugier E (2011) Nitrate transceptor

(s) in plants. J Exp Bot 62: 2299–2308

Gutierrez RA, Gifford ML, Poulthey C, Wang R, Shasha DE, Coruzzi GM,

Crawford NM (2003) Insights into the genomic nitrate response using

genetics and the Sungear Software system. J Exp Bot 58: 2359–2367

Ho CH, Lin SH, Hu HC, Tsay YF (2009) CHL1 functions as a nitrate sensor

in plants. Cell 138: 1184–1194

Hoffland E, van Beusichem ML, Jeger MJ (1999) Nitrogen availability and

susceptibility of tomato leaves to Botrytis cinerea. Plant Soil 210: 263–272

Jakab G, Ton J, Flors V, Zimmerli L, Metraux JP, Mauch-Mani B (2005)

Enhancing Arabidopsis salt and drought stress tolerance by chemical

priming for its abscisic acid responses. Plant Physiol 139: 267–274

Jones JDG, Dangl JL (2006) The plant immune system. Nature 444: 323–329

Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT (2009)

Priming in systemic plant immunity. Science 324: 89–91

Kawamura Y, Hase S, Takenaka S, Kanayama Y, Yoshikawa H, Takahashi H

(2009) INF1 elicitin activates jasmonic acid- and ethylene-mediated signaling

pathways and induces resistance to bacterial wilt disease in tomato.

J Phytopathol 157: 287–297

Krouk G, Crawford NM, Coruzzi GM, Tsay YF (2010) Nitrate signaling:

adaptation to fluctuating environments. Curr Opin Plant Biol 13: 266–273

Lejay L, Tillard P, Lepetit M, Olive FD, Filleur S, Daniel-Vedele F, Gojon

A (1999) Molecular and functional regulation of two NO32 uptake

systems by N- and C-status of Arabidopsis plants. Plant J 18: 509–519

Li W, Wang Y, Okamoto M, Crawford NM, Siddiqi MY, Glass AD (2007)

Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate

transporter gene cluster. Plant Physiol 143: 425–433

Little DY, Rao H, Oliva S, Daniel-Vedele F, Krapp A, Malamy JE (2005)

The putative high-affinity nitrate transporter NRT2.1 represses lateral

root initiation in response to nutritional cues. Proc Natl Acad Sci USA

102: 13693–13698

Liu G, Ji Y, Bhuiyan NH, Pilot G, Selvaraj G, Zou J, Wei Y (2010) Amino

acid homeostasis modulates salicylic acid-associated redox status and

defense responses in Arabidopsis. Plant Cell 22: 3845–3863

Long DH, Lee FN, TeBeest DO (2000) Effect of nitrogen fertilization on

disease progress of rice blast on susceptible and resistant cultivars. Plant

Dis 84: 403–409

Lozano-Juste J, Leon J (2010) Enhanced abscisic acid-mediated responses

in nia1nia2noa1-2 triple mutant impaired in NIA/NR- and AtNOA1-

dependent nitric oxide biosynthesis in Arabidopsis. Plant Physiol 152:

891–903

Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate

immunity and foliar bacterial diseases. Annu Rev Phytopathol 46:

101–122

Modolo LV, Augusto O, Almeida IMG, Magalhaes JR, Salgado I (2005)

Nitrite as the major source of nitric oxide production by Arabidopsis

thaliana in response to Pseudomonas syringae. FEBS Lett 579: 3814–3820

Modolo LV, Augusnt O, Almeida IMG, Pinto-Maglio CAF, Oliveira HC,

Seligman K, Salgado I (2006) Decreased arginine and nitrite levels in

nitrate reductase-deficient Arabidopsis thaliana plants impair nitric oxide

synthesis and the hypersensitive response to Pseudomonas syringae. Plant

Sci 171: 34–40

Mohr PG, Cahill DM (2003) Abscisic acid influences the susceptibility of

Arabidopsis thaliana to Pseudomonas syringae pv. tomato and Peronospora

parasitica. Funct Plant Biol 30: 461–469

Okamoto M, Kumar A, Li W, Wang Y, Siddiqi MY, Crawford NM, Glass

AD (2006) High-affinity nitrate transport in roots of Arabidopsis de-

pends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol

140: 1036–1046

Orsel M, Eulenburg K, Krapp AA, Daniel-Vedele F (2004) Disruption of

the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth

at low external nitrate concentration. Planta 219: 714–721

Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009)

Networking by small-molecule hormones in plant immunity. Nat Chem

Biol 5: 308–316

Pinfield-Wells H, Rylott EL, Gilday AD, Graham S, Job K, Larson TR,

Graham IA (2005) Sucrose rescues seedling establishment but not

germination of Arabidopsis mutants disrupted in peroxisomal fatty

acid catabolism. Plant J 43: 861–872

Thordal-Christensen H, Zhang Z, Wei YD, Collinge DB (1997) Subcellular

localization of H2O2 in plants: H2O2 accumulation in papillae and

hypersensitive response during the barley-powdery mildew interac-

tion. Plant J 11: 1187–1194

Ton J, Flors V, Mauch-Mani B (2009) The multifaceted role of ABA in

disease resistance. Trends Plant Sci 14: 310–317

Ton J, Jakab G, Toquin V, Flors V, Iavicoli A, Maeder MN, Metraux JP,

Mauch-Mani B (2005) Dissecting the b-aminobutyric acid-induced

priming phenomenon in Arabidopsis. Plant Cell 17: 987–999

Tornero P, Dangl JL (2001) A high-throughput method for quantifying

growth of phytopathogenic bacteria in Arabidopsis thaliana. Plant J 28:

475–481

Tsai CH, Singh P, Chen CW, Thomas J, Weber J, Mauch-Mani B, Zimmerli L

(2011) Priming for enhanced defence responses by specific inhibition of the

Arabidopsis response to coronatine. Plant J 65: 469–479

Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK (2007) Nitrate transporters and

peptide transporters. FEBS Lett 581: 2290–2300

van Hulten M, Pelser M, van Loon LC, Pieterse CMJ, Ton J (2006) Costs

and benefits of priming for defense in Arabidopsis. Proc Natl Acad Sci

USA 103: 5602–5607

Ward JL, Forcat S, Beckmann M, Bennett M, Miller SJ, Baker JM,

Hawkins ND, Vermeer CP, Lu C, Lin W, et al (2010) The metabolic

transition during disease following infection of Arabidopsis thaliana by

Pseudomonas syringae pv. tomato. Plant J 63: 443–457

Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate

synthase is required to synthesize salicylic acid for plant defence.

Nature 414: 562–565

Wu CC, Singh P, Chen MC, Zimmerli L (2010) L-Glutamine inhibits beta-

aminobutyric acid-induced stress resistance and priming in Arabidop-

sis. J Exp Bot 61: 995–1002

Yao J, Shi WM, Xu WF (2008) Effects of salt stress on expression of nitrate

transporter and assimilation-related genes in tomato roots. Russ J Plant

Physiol 55: 232–240

Zimmerli L, Jakab G, Metraux JP, Mauch-Mani B (2000) Potentiation of

pathogen-specific defense mechanisms in Arabidopsis by b-aminobutyric

acid. Proc Natl Acad Sci USA 97: 12920–12925

Camanes et al.




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