1 A deletion in NRT2.1 attenuates Pseudomonas syringae-induced

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A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting

in primed plant defenses

Víctor Flors Herrero

Área de Fisiología Vegetal, Departamento de Ciencias Agrarias y del Medio Natural, ESTCE

Universitat Jaume I, Campus de Riu Sec. 12071, Castellón, Spain

Tel: +34 (964) 729417

FAX: +34 (964) 728066

E-mail: [email protected]

Plant Physiology Preview. Published on December 12, 2011, as DOI:10.1104/pp.111.184424

Copyright 2011 by the American Society of Plant Biologists

www.plantphysiol.orgon March 24, 2018 - Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting

in primed plant defenses

Camañes Gemma1, Pastor Victoria1, Cerezo Miguel1, García-Andrade Javier2, Vicedo Begonya1,

García-Agustín Pilar1, Flors Victor1

1Área de Fisiología Vegetal. Departamento de Ciencias Agrarias y del Medio Natural, ESTCE

Universitat Jaume I, Campus de Riu Sec. 12071, Castellón, Spain 2Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-

Consejo Superior de Investigaciones Científicas, Ciudad Politécnica de la Innovación 46022,

Valencia, Spain



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Funding source:

Generalitat Valenciana GV/2007/099, Plan Promoción Bancaja-UJI P1.1A2007-07 and P1.1B2007-

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Corresponding author and e-mail address:

Víctor Flors; [email protected] (last position in order of appearance)



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Abstract

For an efficient defense response against pathogens, plants must coordinate rapid genetic

reprogramming to produce an incompatible interaction. NRT2 gene family members are sentinels of

nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance

against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen

Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes,

NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying

environmental conditions that stimulate the de-repression of the NRT2.1 gene influences resistance to

Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis

seemed to be affected in nrt2, which displays priming of salicylic acid (SA) signaling and concomitant

irregular functioning of the jasmonic acid (JA) and abscisic acid (ABA) pathways upon infection.

Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2,

probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and

metabolic targets of coronatine in Arabidopsis remain largely unstimulated in nrt2 mutants. In addition,

a Pseudomonas syringae strain defective in coronatine synthesis (Pst COR-) showed the same virulence

towards nrt2 as the coronatine-producing strain did. Taken together, the reduced susceptibility of nrt2

mutants seems to be a combination of priming of SA-dependent defenses and reduced sensitivity to the

bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence

plant disease resistance by downregulating biotic stress defense mechanisms and favoring abiotic stress

responses.



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Introduction

In higher plants, nitrate uptake through the roots is mainly achieved by two gene families, NRT1 and

NRT2. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes (Tsay et al., 2007). NRT1 codes for

components of the nitrate low-affinity transport system (LATS), which regulates nitrate uptake mainly

when it is present at high concentrations in the root environment. NRT1 can combine its LATS function

with a high-affinity transport system (HATS) function (Ho et al., 2009), and this dual role is regulated

by phosphorylation. Functional characterization has shown that NRT2 genes belong to the family of

nitrate high affinity transport systems (HATS) (Gansel et al., 2001). The NRT2.1 gene is activated when

nitrate concentrations are below 1 mM, and it mediates systemic signals to the shoot in split root

systems depending on the nitrate concentration (Cerezo et al., 2001; Gansel et al., 2001). The net

contribution to total nitrate uptake of NRT2 is low compared with NRT1 (Little et al., 2005; Tsay et al.,

2007). Interestingly, mutations in the NRT2.1 gene have no influence on total nitrate or nitrogen content

in normal fertilization conditions (Orsel et al., 2004). In addition, individual NRT2.1 mutations in

Arabidopsis seem to be compensated for by NRT2.2; therefore, it is necessary to have both genes

blocked to induce a significant reduction of nitrate uptake mediated by HATS (Li et al., 2007).

Therefore, additional roles for the NRT2 gene family have been proposed. Recently, the term

transceptor has been applied to membrane proteins that fulfill dual nutrient transport/signaling

functions . Although it is now clear that NRT1.1 can be considered a transceptor there is also evidence

suggesting a signaling role for NRT2.1 (Gojon et al., 2011). NRT2 genes are related to environmental

sensing and signal transduction that integrates the metabolism nitrogen with carbon metabolism (Little

et al., 2005). NRT1.1 and NRT2.1 can both perceive small amounts of nitrate and transmit signals to the

plant in order to integrate growth with nutrient availability (Krouk et al., 2010). Moreover, the 150-bp

promoter region of NRT2.1 is sufficient to mediate induction by nitrate and repression by nitrogen

metabolites (Castaings et al., 2010).

NAR2.1/NRT3.1 has been described as a trans-membrane protein that is necessary for NRT2.1

function, but it has no transporter activity by itself (Okamoto et al., 2006; Tsay et al., 2007). This

finding demonstrates complex regulation of the NRT2.1 gene, the role of which as a nitrate transporter

has been questioned, and which has been proposed to be an environmental signal sensor that controls

the development of the root system and coordinates it with nutritional cues (Little et al., 2005; Tsay et

al., 2007).



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Nitrate uptake mediated by HATS has also been demonstrated to be regulated by hormonal and

metabolic signals in wheat (Cai et al., 2007). ABA and Gln enhance NRT2.1 and NAR2.1 gene

expression when nitrate is not present in the nutrient solution, but they have no effect when nitrate

fertilization is appropriate. This phenomenon suggests the existence of feedback between hormonal

signaling and NRT2.1-mediated environmental sensing (Cai et al., 2007).

Nitrate transporter mutants have been shown to display unusual phenotypes in response to abiotic stress

(Yao et al., 2008), but there is little or no evidence that links nitrate HATS activity to resistance to

biotic stresses. One of the few links between nitrogen transporters and biotic stress resistance is found

in the gene NAR2/NRT3.1, also known as WR3 (wound responsive3), which is induced in response to

wounding through a JA-independent pathway (www.arabidopsis.org).

Several findings have indicated that the nutritional status of a plant affects its defensive capacity

against the bacterium Pseudomonas syringae (Modolo et al., 2006). When the main nitrogen source is

ammonium, Arabidopsis is more sensitive to Pseudomonas syringae infection. Feeding plants with

ammonium as the sole nitrogen source prevents nitric oxide (NO) formation and inhibits the

hypersensitive reaction (HR), which correlates with altered susceptibility. By contrast, low levels of

nitrate favor NO formation through enhancement of the NO-synthase activity of nitrate reductase (NR;

Modolo et al., 2005). Overfertilization leading to elevated internal nitrogen levels also contributes to

rice plant susceptibility (Long et al., 2000). This result could be explained by inhibition of the NO-

synthase activity of NR and stimulation of its reductase activity (Modolo et al., 2005). Although the

reasons for these alterations of basal plant resistance are not yet clear, net nitrate rates influence the

dual activity of NR and alter the generation of reactive oxygen species (ROS) and NO. This result may

have a direct effect on plant defense responses against pathogens. Lozano-Juste and León (2010) have

shown that nia1nia2noa1 triple mutants (genes involved in nitrate reductase enzyme and NO synthesis

respectively) display enhanced ABA sensitivity and drought tolerance, due to the uncoupling of NO

from ABA-triggered responses linked to defense responses. In addition to these indications, alterations

in amino-acid transporters, which are tightly regulated by nitrogen status, have direct effects on disease

resistance. For example, the lysine transporter mutant lht1 displays constitutive activation of SA-

dependent signaling and resistance to Pseudomonas syringae (Liu et al., 2010)

Although the constitutive expression of defense genes results in resistant plants, this property usually

has a negative impact on normal plant development (van Hulten et al., 2006). Therefore, among plant

defense strategies, priming has emerged as a notably effective one (Ton et al., 2005; Conrath et al.,



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2006; Conrath, 2011). Plants, upon appropriate stimulation, can activate faster and stronger defense

signals by potentiating their basal immune system. Interestingly, van Hulten and coworkers (2006)

have demonstrated that primed responsiveness to SA in the edr1 mutant correlates with elevated levels

of basal resistance against Pst with no major loss of plant growth or seed production. Although the

mechanisms underlying priming are not fully known, the accumulation of dormant mitogen-activated

protein kinases and chromatin remodeling are two processes likely to be involved in this phenomenon

(Conrath, 2011). Although a link has not yet been established, the upregulation of SA-dependent

responses may be under the control of such processes.

In this study, we demonstrate that alterations in the NRT2 gene family modify basal susceptibility to the

bacterial pathogen Pst. The nrt2 mutant is less susceptible to Pst, and this reduced susceptibility

correlates with both SA pathway priming and reduced sensitivity to pathogenic effectors. These results

suggest an additional role for membrane-located nitrate transporters in environmental stress perception

and signal transduction.

Results

Disruption of NRT2.1 and NRT2.2 results in reduced susceptibility to Pst

Little et al., (2005) demonstrated that NRT2.1 acts as a nitrate sensor and signal transducer to

coordinate the development of the root system with nutritional and abiotic cues. Given the similarities

and close link between biotic and abiotic sensing (Jakab et al., 2005; Ton et al., 2005), the levels of

basal resistance against the hemi-biotrophic pathogen Pst were tested in the mutant nrt2. To this end,

normally fertilized Ws and nrt2 plants were inoculated by dipping at two different stages of growth.

Both adult plants and seedlings of nrt2 showed reduced susceptibility to the bacteria, as evidenced by

reduced symptom severity and reduced bacterial proliferation (Fig 1A, B and C).

Because total levels of nitrogen in a plant have a strong influence on pathogen colonization and

proliferation ( Hoffland et al., 1999; Ali et al., 2009), we determined whether alterations in NRT2 gene

expression or short periods of nitrate deprivation influence total N content, because it can induce the

expression of several NRT2 family members (de-repression: Lejay et al., 1999). Therefore, we

transferred hydroponically growing plants to nitrate-free medium before inoculation. Forty-eight hours



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of deprivation was not sufficient to modify total N levels in plants that had been grown with 1 mM of

NH4NO3 for 6 weeks (Fig S1A). We also confirmed that NRT2.1 expression was enhanced not only in

roots but also in leaves in both ecotypes tested (Ws and Col-0; Fig S1B, C). Interestingly, after the

induction of NRT2 gene expression by nitrate depletion, Ws plants became more sensitive compared to

normally fertilized Ws plants, whereas the level of resistance of nrt2 plants did not change (Fig1E).

This finding might be attributable to the plants’ lack of responsiveness to root environmental sensing.

Therefore, the de-repressed (active transcription) versus repressed (non transcribed) state of NRT2.1

affects the basal resistance level of Arabidopsis against Pst.

It is noteworthy that nrt2 is a mutant that lacks the function of both the NRT2.1 and NRT2.2 genes (Li

et al., 2007). To clarify whether the reduced susceptibility of nrt2 is due to one or both genes, we tested

basal resistance in individual mutants, such as lin1 (blocked in NRT2.1; Little et al., 2005), the

knockout mutant Salk_043543 and the double knockout mutant Salk_035429, all in the Col-0

background. The single NRT2.1 mutant lin1 and the double knockout (Salk_035429) also showed

reduced susceptibility, but the single mutant Salk_043543 did not, suggesting that mutation of both

genes may not be necessary to obtain significantly reduced bacterial growth (Fig 1D). The fact that

Salk_035429 and lin1 are altered in their basal resistance reinforces the hypothesis that these genes

play additional roles in stress sensing and/or signal transduction rather than being only involved in

nitrate transport at low concentrations. In fact, NRT2.1 expression after nitrate depletion was lower on

Col-0 than in Ws, and this finding correlates well with the naturally greater resistance of Col-0. This

natural genetic variation has been recently reported to be linked to two QTLs, one of which regulates

SA-induced PR1 expression (Ahmad et al., 2011).

nrt2 shows primed SA-dependent responses upon Pst infection

Using chemical agents, such as β-aminobutyric acid (BABA), benzothiadiazole (BTH) or azelaic acid,

to prime SA-dependent defenses results in enhanced resistance against Pst (Conrath et al., 2006; Jung

et al., 2009). The mutant nrt2 was found to display slightly increased levels of PR1 and PR5 compared

to those of Ws in the absence of infection (Fig 2A). Interestingly, when the infection is present nrt2

showed stronger and faster induction of these SA marker genes (Fig 2A), mainly at early time-points.

Accordingly, SA levels were higher in nrt2 at 48 h after infection (Fig 2B).

Salk_035429 and lin1 in the Col-0 background showed enhanced PR1 and SA responses concomitant

with their reduced susceptibility to the bacterium (Fig S2). To assess the relevance of SA priming in



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nrt2, we crossed lin1 and Salk_035429 with sid2.1 (isochorismate synthase deficient, Wildermuth et

al., 2001). We tested two independent lines of the double and triple mutants lin1-sid2.1 and

Salk_035429 -sid2.1, and all of them displayed wild-type levels of resistance, which demonstrates that

enhancement of the SA pathway is necessary component of the reduced susceptibility of nrt2 (Fig 2C).

The potential hypersensitivity of nrt2 to SA was tested by treating the plants with β-aminobutyric acid.

BABA was effective at protecting both wild-type and nrt2 to the same levels (Fig S3). Therefore, the

mutant retains its sensitivity to the SA priming induced by BABA.

nrt2 has reduced sensitivity to Pseudomonas syringae effectors

Among the many impacts of bacterial effectors, manipulation of plant hormones is one of the main

targets. The role of ABA in Pst-Arabidopsis interactions is complex and not fully understood (Ton et a.,

2009). It is known that Pst can take control of stomatal movements during pre-invasive and post-

invasive stages by hijacking ABA signaling (Melotto et al., 2008; Ton et al., 2009; Torres-Zabala et al.,

2009). We checked for a contribution of ABA to nrt2-reduced susceptibility by studying the expression

of several ABA marker genes and ABA levels. We monitored three different ABA-responsive genes,

specifically, ABI1, RD22 and RAB18. The expression of ABI1 was upregulated by infection in nrt2, but

it remained unchanged in Ws. By contrast, RD22 was downregulated by the bacterium. Surprisingly,

both ABA marker genes showed lower expression in nrt2 than in Ws in the absence of challenge.

RAB18 showed the most dramatic change, as it was upregulated by the bacterium in Ws but displayed

lower expression in the mutant after infection (Fig 3A). This situation may lead to deficient ABA

signaling manipulation by the bacterium (Ton et al., 2009). Accordingly, the levels of ABA remained

unchanged during infection in the nrt2 mutant, whereas ABA levels increased in wild-type plants (Fig

3B).

To study the abnormal ABA signaling in nrt2, its ability to sense this hormone was verified by treating

Ws and nrt2 plants with ABA 48 h before the infection (Fig 3C). ABA treatment increased the disease

rate in both wild-type and mutant plants; however, bacterial growth only showed significative

differences in ABA-treated nrt2 plants, probably due to the high inoculum used for all the experiments.

The observation of the altered behavior of ABA marker genes in nrt2 in the absence of infection

prompted us to test the response of nrt2 plants to drought stress; however, the mutant showed wild-type

levels of water loss and tolerance (Fig S4). Therefore, ABA, which is implicated in abiotic stress

signaling, seems to function normally in nrt2 plants. Subsequently, we studied whether coronatine



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sensitivity was altered in this mutant because it has been reported that coronatine alters ABA

functioning during infection (Torres-Zabala et al., 2007; Melotto et al.,2008). To this end, several

bioassays were performed. In the first experiment, the resistance of nrt2 mutants against Pst COR- was

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

reduced susceptibility in all individual and double mutants compared with their corresponding wild-

type plants (Fig 4, Fig S5). We subsequently complemented the Pst COR- by treating both genotypes

with coronatine (Fig 4B; Brooks et al., 2004). The result confirmed that the initial reduced

susceptibility phenotype of nrt2 was reestablished; therefore, it is clear that the presence of coronatine

makes a contribution to the reduced susceptibility of this mutant.

This indication that nrt2 displays altered effector sensitivity was further verified by treating Ws and

nrt2 plants grown in MS medium with the effector coronatine (Fig 5). Treatment with coronatine

significantly induced H2O2 (a marker of coronatine's effect) in Ws, whereas nrt2 plants remained

unaltered after treatment (Fig 5A, B). This finding suggests that H2O2 may be a target of coronatine in a

pathway requiring NRT2.1. An additional experiment confirmed the insensitivity of nrt2 plants to

coronatine. Ws and nrt2 plants were sprayed with coronatine. After the treatment, detached leaves of

Ws remained turgid, while nrt2 leaves presented clear wilting at 4 and 6 hours post-treatment (hpt), as

did water-treated leaves (Fig 5C). This result might be due to an induction of stomatal closure at 4 and

6 hpt by coronatine in Ws, while in nrt2 plants the stoma would remain unaltered. We also infected

Arabidopsis by direct infiltration to circumvent possible stomatal aperture defects in the mutant.

Surprisingly, nrt2 remained resistant, suggesting that coronatine sensitivity may contribute to its

enhanced resistance but is not essential for it (Fig S6). Finally, we performed the classical test of

sensitivity of germination to coronatine. In this case, nrt2 showed wild-type sensitivity upon coronatine

treatment, which was visible as purple leaves and a delay in germination (Fig 5D).

Another major target of coronatine is the COI1 receptor and subsequent JA pathway activation

(Fonseca et al., 2009). Accordingly, the JA pathway showed slower activation in nrt2 compared with

Ws. The JA marker genes VSP2 and MYC2, which are not reduced in the mutant in the absence of

infection, showed a lower level of induction at 24 hpi in nrt2 upon infection (Fig S7A). The fact that

these genes are not altered without pathogen confirms that the negative influence of SA priming in the

mutant is only present when the plant is challenged. In addition, infected mutants displayed reduced JA

levels compared to infected wild-type plants (Fig S7B).

Therefore, the downregulation of ABA and JA signaling after Pst infection in nrt2 could be a



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consequence of a partial loss of bacterial effector sensitivity rather than a defect in those pathways.

Gene expression analysis confirms the reduced sensitivity of nrt2 to coronatine.

We tested whether nrt2 plants present abnormal responses to direct coronatine treatment. We observed

that coronatine treatment upregulated such genes as PDF1.2, CORI1 and CORI3, however such

induction was always lower in nrt2 compared with wild-type (Fig S7 C). In contrast to Pst infection

(Fig 3A), RAB18 was downregulated by direct treatment with coronatine in Ws, whereas in nrt2 it

remained unaltered (Fig S7 C). This result reinforces the hypothesis that nrt2 is less sensitive to

coronatine-induced alterations in the plant.

To characterize the molecular response of nrt2 to Pst further, whole-genome transcriptional profiles of

Ws and nrt2 plants mock-treated or infected with Pst were obtained using Arabidopsis ATH1 arrays

from Affymetrix. An examination of the differentially expressed genes in Ws and nrt2 upon infection

compared with mock treatment revealed a group of 574 genes that were differentially expressed in the

mutant but were unchanged in Ws, whereas 550 genes were up- or downregulated in Ws as a

consequence of infection but remained unaltered in nrt2 (Fig S8, Table S2).

Following the line of evidence suggesting that nrt2 has reduced sensitivity to bacterial effectors, we

selected genes from the array that are induced by coronatine. This set of genes was described by Tsai et

al. (2010) after a comparative full genome analysis of plants infected by a coronatineless Pseudomonas

syringae strain or by a strain with functional coronatine. In addition, a second comparative analysis

performed by Tsai and coworkers discriminated from among all of the coronatine-inducible genes a

group of 31 genes that were downregulated by the priming agent BABA. We looked at the behavior of

this set of genes in our study and, interestingly, 18 out of 31 genes repressed by BABA were also less

induced in infected nrt2 plants compared with wild-type-infected plants (Fig S8B, Table S3). This

finding supports the idea that in the absence of NRT2, the plant displays behavior similar to that of

BABA-primed plants.

Discussion

In the present work, we investigated additional roles for the NRT2.1 gene. Mutations affecting NRT2.1

reduce the susceptibility of Arabidopsis to the virulent pathogen Pst. nrt2 mutants displays primed SA

signaling upon infection (Fig 1 and 2). In addition, these plants present defects in sensitivity to the



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bacterial effector coronatine and downregulation of the JA pathway, probably as a consequence of SA

priming, reduced coronatine sensitivity or both (Fig S7). This result suggests that one or several

components of the NRT2 gene family may function as environmental sensors, coordinating not only

root and nutritional cues but also abiotic and biotic responses by enhancing abiotic responses and

downregulating plant defenses against biotic challenges. The term transceptor has been applied to such

genes as NRT1.1 that act as transporters but also coordinate other features of plant development and N

metabolism (Gojon et al., 2011). In fact, mutants defective for NRT1 family members are impaired in

NRT2.1 regulation, which highlights the tight regulatory link between nitrate perception by NRT1.1 and

the function of other NRT family members. NRT2.1 and NRT2.2 have been proposed to play roles in the

integration of sugar and amino-acid metabolism. Other studies point to NRT2.1 as a putative abiotic

stress sensor that coordinates the development of the root system with the root environment and

nutrition (Little et al., 2005; Tsay et al. 2007). It is worth mentioning that downstream nitrate uptake

mutants, such as nia1nia2noa1, display hypersensitivity to ABA and faster stomatal closure upon

drought stress, being subsequently more drought-tolerant (Lozano-Juste and León, 2010).

In our research, we were interested in studying links between nitrate perception and biotic stress

responses. Several reports have described changes in plant 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 possible interference, we performed all experiments with normally fertilized plants (unless

otherwise mentioned). Arabidopsis fertilized with ammonium nitrate at 1 mM has its LATS activated

and NRT2.1, NRT2.2 and NIA repressed (Krouk et al., 2010). In this condition, we show that mutants

lacking either NRT2.1 or NRT2.2 have reduced susceptibility (Fig 1). This phenotype is due to the lack

of the nitrate HATS because activation (de-repression) versus downregulation (repression) of these

genes has clear consequences on wild-type Ws susceptibility to the bacterium.

One mechanism potentially able to explain the reduced susceptibility of nrt2 is found in the SA

signaling pathway. nrt2 mutants show priming of SA signaling at early stages of infection, as

confirmed by SA marker gene induction and hormone accumulation. SA priming is one of the main

mechanisms of induced resistance against Pst (Zimmerli et al., 2000; Conrah et al., 2006). There are

several priming inducers (such as BTH, BABA and azelaic acid) that clearly reduce bacterial disease

symptoms and plant tissue colonization by stimulating the SA pathway (Conrath et al., 2006; Jung et

al., 2009). Interestingly, nrt2 mutants experience constitutive priming because basal levels of SA in the

absence of infection are the same as in Ws, and this finding has also been reported for other mutants,



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such as edr1 (enhanced disease resistance1) (van Hulten et al., 2006). Mutants with direct activation of

SA signaling, such as cpr1, are highly resistant to biotrophic pathogens, but this condition slows

growth and reduces seed production (van Hulten et al., 2006). The nrt2 mutant displays no phenotype

in the absence of the pathogen, and it shows enhanced defense responses upon infection similarly to

edr1. Therefore, NRT2.1, bacterial perception and SA seem to be linked. Genevestigator stimulus

analysis showed that both programmed cell death (PCD) (a possible mechanism of HR) and flg22

induce NRT2.1 expression strongly (data not shown). Recently, Ward et al. (2010) have highlighted by

metabolomic analysis that nitrogen containing compounds and amino acids are strongly altered upon

Pst infection. Increasing evidence points to Gln reallocation in the cytoplasm as a checkpoint that

clearly interacts with SA signaling. Wu et al. (2009) reported that Gln strongly represses BABA

priming against Pst by downregulating PR1. In addition, lysine and histidine transport (LHT1)

mutants show strongly affected disease resistance (Liu et al., 2010). The LHT1 gene antagonizes SA-

dependent signaling, and it is upregulated by pathogen attack. lht1 mutants show upregulated PR1,

NRT1.1 and AMT1.1, among others. In addition, the elevated resistance of lht1 seems not to be directly

linked to global N status as it is with NRT2.1 (Liu et al., 2010). Thus, it seems clear that some of the

genes regulating nitrogen and amino-acid metabolism or transport have strong regulatory functions that

play roles in plant-pathogen interactions.

Several studies suggest that hormones transfer nitrate signals, which would explain the context of

nitrate's influence on plants. INF1, an elicitin from Phytophthora infestans, induces NRT2.1 expression

in tobacco, and it stimulates many SA-dependent responses and HR. Thus, SA signaling and NRT2.1

seem to be coordinately upregulated by this pathogenic elicitor (Kawamura et al., 2009). It is likely that

such oomicetes that combine necrotrophyc and biotrophyc lifestyles target certain plant genes related to

nutrition in order to improve the plant's nutrient sources to their advantage. It is noteworthy that a tenth

of the genome is under the control of nitrate (Gutierrez et al., 2003), and interestingly, a nitrate-

regulated biomodule of genes is also controlled by plant hormones (Krouk et al., 2010).

Our experiments disproved the hypothesis that irregular functioning of the ABA signaling pathway

upon infection is responsible for the reduced susceptibility of nrt2 mutants (Fig 3). The results indicate

that ABA signaling functions normally in these mutants because they show wild-type responses to

drought stress. In addition, external treatment with ABA increases the susceptibility of nrt2 mutants to

Pst, as was previously reported for wild-type plants (Mohr and Cahill, 2003).

ETS (effector triggered susceptibility, Jones and Dangl, 2006) is one of the main roles of the bacterial



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effector coronatine. Several studies have demonstrated that coronatine targets genes to stimulate the JA

signaling pathway in order to repress SA signaling and take advantage of plant colonization. In

addition, coronatine also hijacks ABA functions, such as stomatal closure in the pre-invasive stages by

opening stomata and during post-penetration stages by closing stomata (Torres Zabala 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 mutants were partially unable to sense coronatine. This bacterial effector failed to

induce H2O2 in nrt2 mutants, and it was also unable to reduce wilting in detached mutant leaves,

probably due to a failure of stomatal closure (Fig 4 and 5). In addition, nrt2 shows a wild-type degree

of basal resistance to Pst COR-, indicating that the presence of the effector is necessary to observe the

reduced susceptibility of nrt2 mutants. Tsai et al. (2010) obtained similar results using the chemical

priming agent BABA. These researchers determined that a functional coronatine response was needed

to express BABA priming and induce resistance against Pst. These observations, together with the fact

that many genes induced by coronatine are downregulated in nrt2, explain why the reduced

susceptibility of nrt2 disappears when the bacterium lacks coronatine. To conclude, nrt2 mutant is less

sensitive to coronatine-triggered susceptibility, and this property reduces the ability of the bacterium to

colonize the mutant. Another interesting result of these experiments is that hydrogen peroxide is a

putative target of coronatine, and this interaction is NRT2.1–dependent, as nrt2 is totally impaired in

H2O2 production upon coronatine treatment.

Tsai et al. (2010) reported that priming by BABA against Pst acts through the specific inhibition of

coronatine-triggered responses, and this inhibition resembles nrt2 phenotypes that match partial

coronatine insensitivity with SA-dependent priming and reduced susceptibility to the bacterium.

Among all of the potential contributions to the reduced susceptibility of nrt2 mutants, SA priming

seems to be the major player. In this study, we demonstrated the absence of the resistance phenotype in

the triple and double mutants Salk_035429 -sid2.1 and lin1-sid2.1 (fig 2). However, the reduced

sensitivity to coronatine may also contribute to the resistance; therefore, the phenotype could be a

combination of both. The fact that most experiments performed with NRT2 mutants in the Col-0 and

Ws backgrounds have obtained almost identical results reinforces our main conclusions. However, the

reduced SA-signaling priming and the lower resistance of lin1 and Salk_035429 mutants is justified

because Col-0 displays natural SA-priming and thus enhanced resistance to Pst compared with Ws

(Ahmad et al., 2011). Regarding the SA-JA crosstalk, it is likely that the SA priming is also a

consequence of the absence of repression by JA signaling, which would be less stimulated, due to the



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reduced coronatine sensitivity. This interpretation is further confirmed by the delay in MYC2 induction

in nrt2 compared with wild-type because MYC2 mediates the suppression of SA-dependent defenses by

coronatine (Fig 6). This crosstalk has been recently revised by Pieterse et al. (2009).

Indirectly, nrt2 mutants show attenuated Pst-induced hormonal perturbations that result in the priming

of plant defenses and reduced susceptibility.

In conclusion, although the full implications of the nitrate transporter NRT2 in biotic and abiotic

stresses remain to be clarified, the implications may be linked though the enhancement of abiotic stress

sensing and coordinated biotic stress repression. At the same time, this transporter could be a direct or

indirect target used by bacterial pathogens to take advantage of plant colonization. This research

suggests that NRT2.1 is a novel transceptor of the nitrate transport family that regulates plant disease-

resistance signaling.

Material and Methods

Plant material and growth conditions

Wild-type Arabidopsis thaliana Wassilewskija (Ws) and the Ws mutant nrt2.1-2.2 (referred to as nrt2

in the text) (Filleur et al., 2001) were generously provided by Alain Gojon (INRA Montpelier France),

the knockout lines nrt2.1-2.2 (referred to as Salk_035429 in the text) and nrt2.2 (referred to as

Salk_043543 in the text) in the Col-0 background were obtained from the SALK institute (Alonso et

al., 2003) and the EMS lin1 line was generously supplied by Jocelyn Malamy (University of Chicago,

EEUU).

All plant genotypes were germinated in soil, and 2 weeks after germination, the seedlings were

individually transferred to 33-ml pots containing commercial potting soil (TKS1, Floragard GmbH;

http://www.floragard.de) (adult plants), or approximately 50 seedlings were germinated in 33-ml pots

containing commercial potting soil (seedling plants). The plants were cultivated at 20 ºC day/18 ºC

night with 8.5 h of light (105 μE m-2 s-1) per 24 h and 60% relative humidity.

Growth conditions for starvation experiments

Wild-type Arabidopsis thaliana (Ws) and the mutant nrt2 were grown hydroponically, as described in

Lejay et al. (1999). The seeds were germinated directly on top of modified Eppendorf tubes filled with



16

pre-wetted sand. Plants were grown until the age of 6 weeks on a 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

(de-repressed plants).

Bacterial strains and bacterial growth assays

The bacterial strains were Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae

pv tomato DC3000 cma (Pst COR-; 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 x 107 colony-forming

units (c.f.u)/ml using 0.02% Silwet L-77 (Tornero and Dangl, 2001). After incubation at 28 ºC for 3

days, the number of rifampicin-resistant colony-forming units per gram of infected leaf tissue was

determined, and bacterial proliferation over the 3-day 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 of homozygous 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 experiments. DNA was isolated from leaves of individual plants (Edwards et

al., 1991). To identify plants that were homozygous for the sid2.1 mutation, the following primers were

used: sid2.1-F 5’-gctctgcagcttcaatgc-3’ and sid2.1-R 5’-cgaagaaatgaagagcttgg. A fragment of 243 nt

was obtained and subsequently digested by the enzyme Tru1 (MBI Fermentas, Vilnius, Lithuania) and

separated on a 2 % agarose gel to evaluate the fragment sizes of 154 nt and 89 nt 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, respectively, LP 5'-gcaagcgactatcatcactcc-3', RP 5'-

gttctccatgagcttcgtgag-3' and LB 5'-atttgccgatttcggaac-3', following the SALK institute instructions 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

BamH1 (MBI Fermentas, Vilnius, Lithuania), and the product from the lin1 mutant allele is uncut.



17

These fragments were separated in a 2 % agarose gel to evaluate the fragment sizes.

Quantitative real-time RT-PCR analysis of transcripts

Gene expression analysis by quantitative real-time 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 μg of total RNA was digested using 1

unit of RQ1 RNase-Free DNase (Promega; http://www.promega.com) in 1 μl of DNase buffer and up to

10 μl of Milli-Q water and was incubated for 30 min at 37 ºC. After the incubation, 1 μl of RQ1 DNase

stop buffer was added, and the solution was incubated again at 65 ºC for 10 min to inactivate the

DNase. Highly pure RNA was used for the RT reaction. The RT reaction was performed by adding 2 μl

of RT buffer, 2 μl of 5 mM dNTP, 2 μl of 10 μM oligo(dT)15 primer (Promega), 1 μl of 10 U μl–1

Rnasin RNase inhibitor (Promega) and 1 μl of Omniscript reverse transcriptase (Qiagen,

http://www.qiagen.com/). The reaction mixture was incubated at 37 ºC for 60 min. Less than 10 % of

the volume of the RT reaction was used for the quantitative PCR. Forward and reverse primers (0.3

μM) were added to 25 μl of QuantiTectTM SYBR Green PCR reaction buffer (Qiagen), as were 2 μl of

cDNA and Milli-Q sterile water up to 50 μl 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 ºC for 15 min followed by 45 cycles of 95 ºC for 15 sec, 60ºC for 30 sec and 72 ºC for 30

sec. Melting-curve analysis was performed at the end of the PCR reaction to confirm the products'

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 β-

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 qRT-PCR is shown in

Table S1.

Determination of ABA, SA and JA levels

Fresh material was frozen in liquid nitrogen and lyophilized. Before extraction, a mixture of internal



18

standards containing 100 ng [2H6]- ABA, 100 ng [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 (5000 g, 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, and 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-μm cellulose

acetate filter. A 20-μl aliquot of this solution was directly injected into the HPLC system. Analyses

were carried out using a Waters Alliance 2690 HPLC system (Milford, MA, USA) with a nucleosil

ODS reversed-phase column (100 x 2 mm i.d.; 5 ml; Scharlab, Barcelona, Spain; 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 thaliana Wassilewskija 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 Cleanup Module

(Affymetrix). The purified cDNA was used to prepare biotin-labeled cRNA using a GeneChip IVT

Labeling Kit according to the manufacturer's instructions. The biotin-labeled cRNA was fragmented at

94 ºC 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 hybridization, image analysis and statistical

analysis were performed by Progenika Co® (Bilbao, Spain). Bioinformatic analysis was performed by

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

consortium BioConductor (www.bioconductor.org).



19

Chemical treatments

Two-week-old seedlings were individually transferred to 33-ml pots. At the age of five weeks, plants

were soil-drenched with water (control) or a solution of BABA or ABA at a final concentration of 250

μM or 80 μM, 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 in sterile 12-wells plates containing filter-

sterilized MS medium amended with 1% sucrose. Seedlings were cultivated under standard growth

conditions (15 h day cycle; 20ºC /17ºC) with a light intensity of 105 μE m-2 s-1. After 7 days, 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/μL in the growth medium. At day 9, the

plants were stained with 3,3'-diaminoenzidine (DAB; Sigma, D-8001) to quantify hydrogen peroxide

induction (Thordal-Christensen et al., 1997). Samples were stained for 8 h and were subsequently de-

stained with 95% ethanol. Microscopic analysis was performed using a microscope (Nikon Eclipse

11000) with a VIS filter. Hydrogen peroxide was quantified as the relative number of brown pixels in

digital photographs using GIMP 2.6 software.

Supplementary Material

Supplementary Figure 1. Total nitrogen levels and NRT2 transcript levels in leaves and roots of Ws and

Col-0 plants.

Supplementary Figure 2. Effect of Pst infection on PR1 gene expression levels and SA accumulation in

five-week-old Col-0 wild-type plants, Salk_035429 and lin1 mutants.

Supplementary Figure 3. Disease rate and bacterial proliferation in Ws wild-type plants and nrt2

mutants infected with Pst upon BABA treatment.

Supplementary Figure 4. Water loss in Ws wild-type and nrt2 plants.

Supplementary Figure 5. Bacterial proliferation in Col-0 wild-type, lin1 and Salk_035429 plants

infected with Pst and the coronatineless strain Pst COR-



20

Supplementary Figure 6. Bacterial proliferation in Ws wild-type and nrt2 plants infected by infiltration

with Pst.

Supplementary Figure 7. Effect of Pst infection on VSP2 and MYC2 gene expression levels and JA

accumulation in Ws wild-type and nrt2 plants.

Supplementary Figure 8. Analysis of differentially expressed genes in Ws and nrt2 upon infection.

Study of the reduced sensitivity of nrt2 to coronatine by array analysis and direct treatment with the

effector.

Supplementary Table 1. List of primers employed in qRT-PCR analysis.

Supplementary Table 2. List of genes and fold induction analysis present in the Venn diagram.

Supplementary Table3. List of genes that are induced by coronatine and down-regulated in nrt2-

Pst/nrt2-mock vs. Ws-Pst/Ws-mock. qRT-PCR confirmation of several genes present in the array.

ACKNOWLEDGMENTS

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

Mani and Juan Antonio López-Ráez for proofreading and critically reviewing the manuscript. We also

thank the funding provided by Generalitat Valenciana GV/2007/099, Plan Promoción Bancaja-UJI

P1.1A2007-07 and P1.1B2007-42.

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Figure 1. Disease rate and bacterial proliferation in wild-type plants and mutants lacking the NRT2

gene infected with Pst. Two- (A and D) or five- (B, C and E) week-old plants were challenge-

inoculated with a bacterial suspension of Pst at 2.5 x 107 c.f.u./ ml. Data are from a representative

experiment that was repeated at least three times with similar results.

(A, B) Bacterial growth in the leaves was determined over a 3-day time interval. The values presented

are means (±SD) of the log of the proliferation values.

(C) Disease symptoms were determined 3 days after inoculation and quantified as the proportion of

leaves with symptoms. The data presented are the means of the percentage of diseased leaves per plant

(±SD).

(D) Bacterial proliferation in Col-0 wild-type plants and Salk_043543, Salk_035429, and lin1 mutants

infected with Pst. Two-week-old plants were challenge-inoculated with a bacterial suspension of Pst.

The values presented are means (±SD) of the log of the proliferation values. Asterisk indicates

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 two days before inoculation (Ws-N and nrt2-N).

Figure 2. Effect of Pst infection on PR1 and PR5 gene expression levels and SA accumulation in five-

week-old Ws wild-type plants and nrt2 mutants.

(A) Total RNA was isolated from infected leaves at 24 and 48 hours after inoculation (hpi), converted

to cDNA, and subjected to quantitative RT-PCR analysis. The plants were mock- or Pst–inoculated, as

described in Figure 1. The PR1 and PR5 transcript levels in the mock- and Pst-infected plants were

normalized to the expression of At1g13320 measured in the same sample. The experiment was repeated

using β-tubulin with similar results. The data shows the average of two independent experiments

obtained with a pool of 10 plants per point. The experiment was repeated three times with similar

results. Asterisks mean statistical differences compared with Ws for each time-point (LSD test; P<

0.05, n=3)

(B) Relative SA accumulation in mock- or Pst-infected wild-type and nrt2 plants. Plant tissue was

collected at various time points, and SA levels were determined in freeze-dried material by HPLC-MS.

The results are means ±SD (n=5). The data are from a representative experiment that was repeated

three times with similar results. Asterisks mean statistical differences compared with Ws for each time-



27

point (LSD test; P< 0.05, n=5)

(C) Four-week-old Col-0 plants, sid2.1, Salk_035429 and lin1 mutants, and two independent lines of

the double and triple mutants lin1-sid2.1 and Salk_035429-sid2.1, all in the Col-0 background, were

challenged, as described in Figure 1. Bacterial growth in the leaves was determined at 3-day time

interval. The values presented are means (±SD). Different letters indicate statistically significant

differences (LSD test; P < 0.05, n = 15–25).

Figure 3. Analysis of the ABA-dependent signaling pathway in the mutant nrt2 vs. Ws upon Pst

infection.

(A) Total RNA was isolated from infected leaves at 24 and 48 hpi, converted to cDNA, and subjected

to quantitative RT-PCR analysis. Plants were mock- or Pst–inoculated, as described in Figure 1. The

ABI1, RD22 and RAB18 transcript levels in wild-type and nrt2 plants were normalized to the

expression of At1g13320 measured in the same sample. The experiment was repeated using β-tubulin

with similar results. The data show the average of two independent experiments obtained with a pool of

10 plants per point. The experiment was repeated three times with similar results. Asterisks mean

statistical differences compared with Ws for each time-point (LSD test; P< 0.05, n=3)

(B) Relative ABA accumulation in wild-type and nrt2 plants upon infection by Pst. Plant tissue was

collected at various time points, and ABA levels were determined in freeze-dried material by HPLC-

MS. The results are means ±SD (n = 5). The data are from a representative experiment that was

repeated with similar results. Asterisks mean statistical differences compared with Ws for each time-

point (LSD test; P< 0.05, n=5)

(C) Four-week-old plants were soil-drenched with water or 80 μM ABA. At 2 days 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, as described previously. Different letters

indicate statistically significant differences between wild-type and mutant plants (LSD test; P < 0.05,

n= 15–25).

Figure 4. Quantification of coronatine's influence on the basal resistance of Ws wild-type and nrt2

plants against Pst.

(A) Four-week-old plants were challenged as described in Figure 1 but using Pst COR- instead. Disease

was assessed by quantifying both disease rate and colony-forming units, as described previously.



28

Different letters indicate statistically significant differences (LSD test; P < 0.05, n = 15–25).

(B) Four-week-old plants were sprayed with water or 0.5 ng/μl coronatine. At 2 days after the chemical

treatment, the plants were challenged with Pst COR- , as described in Figure 1. Different letters

indicate statistically significant differences between wild-type and mutant plants (LSD test; P < 0.05,

n= 15–25).

Figure 5. Sensitivity to exogenously applied coronatine in Ws wild-type and nrt2 plants.

(A, B) One-week-old plants grown in 1 ml MS medium were treated with coronatine at 0.5 ng/μl final

concentration. Hydrogen peroxide accumulation was determined by DAB staining. The data show the

average from visible microscopy of wild-type and mutant plants ±SD. Different letters indicate

statistically significant differences (LSD test; P < 0.05, n = 15–25).

(C) Four-week old plants were sprayed with water or 0.5 ng/μl coronatine. After the treatment, leaves

of the same size were detached and exposed to wilting at the indicated time points. The data show the

average of 25 leaves. Different letters indicate statistically significant differences (LSD test; P < 0.05, n

=25). The experiment was repeated twice with similar results.

(D) Four-day-old wild-type and mutant nrt2 seedlings grown in MS medium supplemented with water

or coronatine at 0.5 ng/μl.

Figure 6. Model of the influence of the NRT2.1 gene on the SA, JA and ABA defense-signaling

pathways.

In the absence of a functional NRT2.1 gene, the plant is less sensitive to coronatine; therefore, the

manipulation of the plant's defensive metabolism by the bacterium is less efficient. In the mutant, JA-

and ABA-dependent signaling display irregular activation, and SA-dependent responses are primed in

the presence of the pathogen. Thus, the mutation affects the SA pathway directly and also the negative

crosstalk between the ABA and JA pathways. Only those signal transduction elements that are affected

in nrt2 are represented. Green color indicates downregulated processes and red represents upregulated

processes. Black crosses indicate the altered processes in the nrt2 mutant.



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Supplementary Figure 1.

(A) Total nitrogen levels in 6 week-old plants fertilized normally (Ws and nrt2) and plants subjected to

48 hours of nitrogen deprivation to induce NRT2.1 expression (Ws-N and nrt2-N).

(B, C) The NRT2.1 transcript levels in leaves and roots of Ws and Col-0 plants were normalized to the

expression of At1g13320 measured in the same sample. The experiment was repeated by using β-

tubulin with similar results. The data show the average of two independent experiments obtained with a

pool of 10 plants per point. The experiment was repeated three times with similar results. Asterisks

indicate statistically significant differences (LSD test; P < 0.05, n = 15–25).

Supplementary Figure 2. Effect of Pst infection on PR1 gene expression levels and SA accumulation in

five-week-old Col-0 wild-type plants, Salk_035429 and lin1 mutants.

(A) Total RNA was isolated from infected leaves at 24 and 48 hpi, converted to cDNA, and subjected

to quantitative RT-PCR analysis. Plants were mock- or Pst–inoculated, as described in Figure 1. The

PR1 transcript levels in mock- and Pst-infected plants were normalized to the expression of At1g13320

measured in the same sample. The experiment was repeated using β-tubulin with similar results. The

data show the average of two independent experiments obtained with a pool of 10 plants per point. The

experiment was repeated three times with similar results. Asterisks mean statistical differences

compared with wild-type (LSD test; P< 0.05, n=3)

(B) Relative SA accumulation in mock- or Pst-infected Col-0 wild-type, Salk_035429 and lin1

mutants. Plant tissue was collected at various time points, and SA levels were determined in freeze-

dried material by HPLC-MS. The results are means ±SD (n=5). The data are from a representative

experiment that was repeated three times with similar results. Asterisks mean statistical differences

compared with wild-type for each time-point (LSD test; P< 0.05, n=5)

Supplementary Figure 3. Disease rate and bacterial proliferation in Ws wild-type plants and nrt2

mutants infected with Pst upon BABA treatment and water loss in Ws wild-type and nrt2 plants.

Four week-old plants were soil-drenched with water or 250 μM BABA. After two days of chemical

treatment, the plants were inoculated with a bacterial suspension of Pst at 2.5 x 107 c.f.u./ ml. Pst

infection was determined by quantifying both the disease rate and colony-forming units. Different

letters indicate statistically significant differences (LSD test; P < 0.05, n = 15–25).



30

Supplementary Figure 4. Five-week-old plants were subjected to dehydration. The plants were flooded

and left to drain at day 0. Water loss was calculated by measuring the weight of the plants (n=25). The

values are means of water loss (g/plant; ±SD) after different numbers of days of dehydration. The

results shown are derived from a representative experiment that was repeated three times, yielding

comparable results.

Supplementary Figure 5. Bacterial proliferation in Col-0 wild-type plants, lin1 and Salk_035429

mutants infected with Pst or Pst COR-.

Four-week-old plants were challenge-inoculated with a bacterial suspension of Pst at 2.5 x 107 c.f.u./

ml. Disease was determined by quantifying both disease rate and colony-forming units as described

previously. Different letters indicate statistically significant differences (LSD test; P < 0.05, n = 15–25).

Supplementary Figure 6. Bacterial proliferation in Ws wild-type and nrt2 plants infected with Pst.

(A) Five-week-old plants were challenge-inoculated with a bacterial suspension of Pst at 105 c.f.u./ ml

by infiltration with a syringe without a needle. Bacterial growth in the leaves was determined over a 3-

day time interval. The values presented are means (±SD) of the log of the proliferation values.

Different letters indicate statistically significant differences (LSD test; P<0.05, n=15-25).

(B) The picture shows a representative sample of Pst-infected Ws and nrt2 plants three days after

inoculation.

Supplementary Figure 7. Effect of Pst infection on VSP2 and MYC2 gene expression levels and JA

accumulation in Ws wild-type and nrt2 plants.

(A) Total RNA was obtained, as described previously. The VSP2 and MYC2 transcript levels in wild-

type and nrt2 plants were normalized to the expression of At1g13320 measured in the same sample.

The experiment was repeated using β-tubulin with similar results. The data show the average of two

independent experiments obtained with a pool of 10 plants per point. The experiment was repeated

three times with similar results.

(B) Relative JA accumulation in wild-type and nrt2 plants upon infection by Pst. The plants were

inoculated, as described in Figure 1. Plant tissue was collected at various time points, and JA levels



31

were determined in freeze-dried material by HPLC-MS. The results are means ±SD (n = 5). The data

are from a representative experiment that was repeated with similar results.

(C) Transcript levels of several representative marker genes in Ws and nrt2 after coronatine treatment.

Plants were treated with either water or coronatine at 0.5 μM, and four hours after the treatment, leaf

material was harvested for analysis. Total RNA was obtained, and CORI1, CORI3, PDF1.2 and RAB18

transcript levels in wild-type and nrt2 plants were analyzed, as described previously. The data show the

average of two independent experiments obtained with a pool of 10 plants per point. The experiment

was repeated two times with similar results. Asterisks mean statistical differences compared with Wt

(LSD test; P< 0.05, n=3).

Supplementary Figure 8. Analysis of genes differentially expressed in wild-type and nrt2 plants after

inoculation with Pst or coronatine treatment.

(A) Venn diagram representing common up- or downregulated genes in wild-type and nrt2 plants upon

infection.

(B) Number of genes that are responsive to coronatine present in the Venn diagram for Ws-Pst

infected/Ws-mock and nrt2-Pst/nrt2-mock.















Documents

1 A deletion in NRT2.1 attenuates Pseudomonas syringae-induced