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. 2009 May 26;10(5):621–634. doi: 10.1111/j.1364-3703.2009.00557.x

Forward and reverse genetics to identify genes involved in the age‐related resistance response in Arabidopsis thaliana

JESSIE L CARVIEL 1, FADI AL‐DAOUD 1, MELODY NEUMANN 2, ASIF MOHAMMAD 1, NICHOLAS J PROVART 2,3, WOLFGANG MOEDER 2, KEIKO YOSHIOKA 2, ROBIN K CAMERON 1,✉
PMCID: PMC6640485  PMID: 19694953

SUMMARY

Age‐related resistance (ARR) occurs in numerous plant species, often resulting in increased disease resistance as plants mature. ARR in Arabidopsis to Pseudomonas syringae pv. tomato is associated with intercellular salicylic acid (SA) accumulation and the transition to flowering. Forward and reverse genetic screens were performed to identify genes required for ARR and to investigate the mechanism of the ARR response. Infiltration of SA into the intercellular space of the ARR‐defective mutant iap1‐1 (important for the ARR pathway) partially restored ARR function. Inter‐ and intracellular SA accumulation was reduced in the mutant iap1‐1 compared with the wild‐type, and the SA regulatory gene EDS1 was also required for ARR. Combining microarray analysis with reverse genetics using T‐DNA insertion lines, four additional ARR genes were identified as contributing to ARR: two plant‐specific transcription factors of the NAC family [ANAC055 (At3g15500) and ANAC092 (At5g39610)], a UDP‐glucose glucosyltransferase [UGT85A1 (At1g22400)] and a cytidine deaminase [CDA1 (At2g19570)]. These four genes and IAP1 are also required for ARR to Hyaloperonospora parasitica. IAP1 encodes a key component of ARR that acts upstream of SA accumulation and possibly downstream of UGT85A1, CDA1 and the two NAC transcription factors (ANAC055, ANAC092).

INTRODUCTION

The interaction of plants and pathogens has led to the evolution of many complex defence pathways (Maor and Shirasu, 2005). An initial line of defence encountered by pathogens is referred to as ‘basal resistance’, in which the recognition of microbe‐associated molecular patterns (MAMPs), such as bacterial flagellin, leads to basal defence induction (Eulgem, 2005; Felix et al., 1999; Heath, 2000; Kunze et al., 2004). Activation of the basal resistance pathway leads to callose deposition in the cell wall (Kim et al., 2005) and the production of reactive oxygen species (Nurnberger et al., 2004) and the expression of defence‐related genes (Navarro et al., 2004). Some pathogens have evolved the ability to suppress or elude plant basal resistance (Speth et al., 2007) and plants have responded by evolving additional resistance responses, such as R gene‐mediated resistance (Maor and Shirasu, 2005), in which a specific resistance (R) receptor interacts directly or indirectly with a specific pathogen‐derived avirulence product, leading to defence and the hypersensitive response (HR) (Heath, 2000).

Age‐related resistance (ARR) has been observed in diverse species, including rice, pepper, cowpea, tobacco and Arabidopsis thaliana (reviewed in Develey‐Rivière and Galiana, 2008). As some plants mature, they become increasingly resistant to normally virulent pathogens. ARR has been associated with the transition to flowering in some cases or senescence in others, and with the accumulation of secondary metabolites or defence proteins. Whether ARR affords broad‐spectrum resistance to different pathogens has not been addressed for most plants; however, mature tobacco plants become more resistant to the oomycetes Peronospora tabacina (Wyatt et al., 1991) and Phytophthora parasitica (Hugot et al., 1999) and to Tobacco mosaic virus(Fraser, 1981; 1991, 1993). Moreover, recent studies have demonstrated that ARR is induced in mature Arabidopsis in response to virulent Pseudomonas syringe pv. tomato (Pst) and pv. maculicola (Kus et al., 2002), as well as Hyaloperonospora parasitica (Rusterucci et al., 2005). During ARR in Arabidopsis, Pst‐induced chlorotic disease symptoms are reduced and an HR‐like response is not observed (Kus et al., 2002). Virulent Pst growth is reduced 10‐ to 100‐fold (Kus et al., 2002), whereas H. parasitica conidiospore production is reduced five‐ to 50‐fold, in mature ARR‐competent relative to young plants. The absence of an HR‐like response (Kus et al., 2002) suggests that ARR is not a form of R gene‐mediated resistance. Moreover, ARR provides resistance to two pathogen types (bacterium and oomycete), suggesting that it is not a form of basal resistance in which molecules common to a particular pathogen type are recognized by MAMP receptors (Eulgem, 2005). The ability to manifest ARR in Arabidopsis is associated with flowering in plants both delayed and accelerated in the transition to flowering (Rusterucci et al., 2005). In standard ARR experiments, Col‐0 plants become competent for ARR at the transition to flowering, which occurs at approximately 6 weeks of age in plants grown under short‐day conditions (Kus et al., 2002).

Salicylic acid (SA) accumulation is required during many R gene‐mediated and basal resistance responses and during systemic acquired resistance (SAR) (1994, 1995; Uknes et al., 1993; Vernooij et al., 1994). There is mounting evidence that, during SAR, SA acts as a signalling molecule by inducing the oxidoreduction of key cysteines, leading to the translocation of reduced non‐expressor of pathogenesis‐related 1 (NPR1) into the nucleus, where it interacts with reduced TGA transcription factors, which subsequently up‐regulate defence genes, such as pathogenesis‐related 1 (PR1) (reviewed in Fobert and Despres, 2005). SA accumulation is also required for the Arabidopsis ARR response (Cameron and Zaton, 2004; Kus et al., 2002), as demonstrated by the ARR‐defective phenotype of SA‐deficient NahG (Gaffney et al., 1993) sid1 and sid2 (Nawrath and Métraux, 1999). However, ARR occurs in the npr1‐1 (non‐expresser of PR1) mutant, and PR1 gene expression is reduced during ARR, suggesting that SA does not play its NPR1‐dependent signalling role during ARR, as is seen in other resistance responses, such as SAR and basal resistance (Cameron and Zaton, 2004; Kus et al., 2002).

During ARR, SA accumulation in the intercellular space has been associated with anti‐Pst activity (Cameron and Zaton, 2004). In addition, the reduction of SA in the intercellular space using salicylate hydroxylase impairs ARR, whereas the addition of SA to the intercellular space enhances ARR in the wild‐type and restores ARR in the sid2 mutant (Cameron and Zaton, 2004). Taken together, these data suggest that intercellular SA may act as an antimicrobial agent against Pst during ARR in Arabidopsis. Our knowledge of the genes involved in ARR is limited. Therefore, a classical genetic screen for ARR‐defective mutants and a microarray/reverse genetics approach were implemented to identify the genes required for ARR.

RESULTS

Identification of iap1‐1 (important for the ARR pathway), an ARR‐defective mutant

Classical mutant screening was employed to identify the genes involved in ARR using the standard ARR assay, in which Arabidopsis grown under short‐day conditions is delayed in the transition to flowering and becomes ARR competent at the transition to flowering or mature stage at approximately 6 weeks of age (Kus et al., 2002). Approximately 5000 M2 fast neutron‐mutagenized Col‐0 seeds (Lehle Seeds, Round Rock, TX, USA) were grown for 6 weeks and inoculated with virulent Pst[106 colony‐forming units (cfu)/mL]. Plants displaying disease symptoms similar to those of the ARR‐defective NahG transgenic line, as compared with wild‐type Col‐0 ARR‐competent plants, were selected as potential mutants (M2 generation) and were re‐screened in the M3 and M4 generations. The iap1‐1 mutant supported high levels of bacterial growth (approximately 107 cfu/leaf disc) and was therefore compromised in ARR. iap1‐1 was genetically characterized by backcrossing with Col‐0 wild‐type to produce the F1 and F2 generations. Mature F1 plants supported intermediate levels of bacterial growth and symptoms (approximately 4.0 x 106 cfu/leaf disc) compared with wild‐type ARR‐competent plants (approximately 6 x 105 cfu/leaf disc). F2 progeny segregated in a 1 : 2 : 1 ratio (chi‐squared P > 0.5) of ARR‐competent (<1.0 x 105 cfu/leaf disc) to intermediate ARR [(2.0–8.0) x 105 cfu/leaf disc] to ARR‐defective (1.0 x 106 to 3.0 x 107 cfu/leaf disc) respectively, suggesting that iap1‐1 is a semi‐dominant mutation. Typical disease symptoms are shown in Fig. 1. iap1‐1 mutant plants displayed no obvious developmental abnormalities. A homozygous iap1‐1 plant line was isolated in the F3 generation and was used in all subsequent experiments.

Figure 1.

Figure 1

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Disease symptoms in Col‐0 and iap1‐1 (homozygous and heterozygous). Leaves were inoculated with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) and photographed at 3 days post‐inoculation (dpi).

iap1‐1 is not compromised in basal resistance

Pst suppresses basal resistance in young Arabidopsis plants (Speth et al., 2007); however, this suppression is not complete, such that Arabidopsis exhibits some resistance to Pst. For example, basal defence mutants sid2 and eds1 (enhanced disease susceptibility 1) are more susceptible than the wild‐type to Pst (Falk et al., 1999; Nawrath and Métraux, 1999). To determine whether IAP1 is required not only for the ARR response in mature plants, but also for basal resistance in young plants, young [3 weeks post‐germination (wpg)] and mature (6 wpg) iap1‐1 and Col‐0 were inoculated with Pst. Young iap1‐1 and Col‐0, plus mature iap1‐1, supported high Pst levels (>107 cfu/leaf disc) compared with mature ARR‐competent Col‐0 (Fig. 2a). Bacterial levels in young iap1‐1 and Col‐0 were not significantly different (Student's t‐test), indicating that basal resistance is not affected by the iap1‐1 mutation.

Figure 2.

Figure 2

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In planta bacterial levels in iap1‐1, Col‐0 and NahG. (a) Col‐0, iap1‐1 and NahG were inoculated with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) at 3 and 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. This experiment was repeated four times with similar results. (b) In planta bacterial growth in mature iap1‐1 and Col‐0 plants (6 wpg) was measured over 3 dpi (106 cfu/mL Pst) and is presented as the mean of three samples ± SD. The bacterial density was significantly lower (Student's t‐test) in Col‐0 than iap1‐1 by day 3. This experiment was repeated three times with similar results.

In standard ARR assays, bacterial levels are measured at 3 days post‐inoculation (dpi); therefore, bacterial growth was monitored over 3 days to determine when the ARR defect in iap1‐1 is first observed. Bacterial growth in Col‐0 and iap1‐1 was similar at 0, 1 and 2 dpi, demonstrating that the number of bacteria infiltrated and Pst growth over 2 days was similar in both. At 3 dpi, Col‐0 plants displayed ARR, whereas Pst levels were 10‐fold higher (P= 0.0042, Student's t‐test) in ARR‐defective iap1‐1 (Fig. 2b).

ARR to H. parasitica is compromised in iap1‐1

Previous studies have demonstrated that ARR in Arabidopsis is also effective against the oomycete pathogen H. parasitica, virulent isolate Noco 2 (Rusterucci et al., 2005). To determine whether IAP1 function is also required for ARR to H. parasitica, ARR assays were performed using mature plants inoculated with H. parasitica isolate Noco 2 by spraying leaves with 106 sporangia/mL. Spore numbers per leaf were quantified at 7 dpi. Col‐0 plants supported little H. parasitica growth (∼10 000 spores/leaf) and therefore displayed ARR; however, ∼180 000 spores/leaf were observed on iap1‐1 plants, suggesting that functional IAP1 is also required for the ARR response to H. parasitica (Fig. 3a,b).

Figure 3.

Figure 3

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Hyaloperonospora parasitica infection of iap1‐1. (a) Six‐week‐old Col‐0 and iap1‐1 plants were sprayed with the virulent H. parasitica isolate Noco 2. Photographs were taken 7 days after infection. (b) The number of H. parasitica spores per infected leaf was quantified using a haemocytometer. This experiment was repeated with similar results.

SA partially rescues the iap1‐1 ARR defect

Previous studies in our laboratory have suggested that the ARR pathway leads to an accumulation of SA in the intercellular space, where it may act as an antimicrobial agent during ARR (Cameron and Zaton, 2004; Kus et al., 2002). If IAP1 is upstream of SA accumulation in the ARR pathway and SA accumulation is a key component of ARR, then SA addition to the intercellular space prior to Pst inoculation should rescue the ARR defect in iap1‐1, as previously observed in sid2 (Cameron and Zaton, 2004). SA (0.1 mM) was infiltrated into mature iap1‐1 and Col‐0 plants, 5 and 24 h prior to inoculation with Pst (106 cfu/mL). SA is still present in the intercellular space at 5 h post‐infiltration, whereas it has been absorbed into the cell by 24 h post‐infiltration (Cameron and Zaton, 2004). ARR was enhanced 20‐fold in Col‐0 plants inoculated with Pst at 5 h post‐SA infiltration (hpSAi) compared with those inoculated at 24 hpSAi or water‐infiltrated controls (t‐test <0.05; Fig. 4a). Pst levels were reduced two‐fold in iap1‐1 plants challenged with Pst at 5 hpSAi compared with those inoculated at 24 hpSAi or water‐infiltrated controls (t‐test <0.05; Fig. 4a). In two additional experiments, Pst levels were reduced two‐ and four‐fold in iap1‐1 inoculated with Pst at 5 hpSAi compared with water‐infiltrated controls (data not shown). ARR was enhanced in Col‐0 and iap1‐1 plants only when SA was still present in the intercellular space (5 hpSAi). Pst levels were reduced in iap1‐1 to a lesser extent compared with Col‐0; therefore, the ARR defect in iap1‐1 was partially restored by intercellular SA application.

Figure 4.

Figure 4

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Salicylic acid (SA) application and SA levels in iap1‐1 and Col‐0. (a) Leaves of 5‐week‐old plants were inoculated with Pseudomonas syringe pv. tomato (Pst) at 5 or 24 h post‐SA infiltration (hpi; with 0.1 mM). In planta bacterial levels [colony‐forming units/leaf disc (cfu/ld)] were monitored 3 days after Pst inoculation (SA, Pst) and compared with control treatments (H, Pst), where water infiltration was followed by inoculation with Pst 24 h later. Asterisks indicate a significant decrease in bacterial density in SA‐treated plants vs. the corresponding water‐treated plants (Student's t‐test). The mean ± standard deviation (SD) of three replicate samples is shown. Experiments in which Pst was inoculated at 5 hpSAi were repeated twice with similar results. (b) Intercellular washing fluids (IWFs) were collected from mature leaves that were either mock‐inoculated or inoculated with Pst (106 cfu/mL). SA levels were determined and are presented as ng/mL of IWF collected. Asterisks indicate a significant difference in SA levels in mock‐inoculated leaves compared with leaves inoculated with Pst (Student's t‐test). The mean ± SD of three replicate samples is shown. This experiment was repeated twice with similar results. (c) Mature leaves were mock‐inoculated or inoculated with Pst (106 cfu/mL). IWFs were removed and total SA levels (SA plus SA glucosides) were determined in leaves and presented as ng/g fresh weight. Asterisks indicate a significant difference in SA levels in mock‐inoculated leaves compared with leaves inoculated with Pst (Student's t‐test). The mean ± SD of three replicate samples is shown. This experiment was repeated with similar results.

Intercellular SA accumulation is reduced in iap1‐1 in response to Pst

The ARR‐defective phenotype of iap1‐1 was partially rescued by intercellular SA infiltration, suggesting that IAP1 lies upstream of intercellular SA accumulation in the ARR pathway, leading to the prediction that iap1‐1 plants will accumulate little intercellular SA relative to wild‐type plants. To address this question, intercellular washing fluids (IWFs) were collected from mature Col‐0 and iap1‐1 plants at 24 h post mock‐inoculation with 10 mM MgCl2 or inoculation with Pst (106 cfu/mL). This time point was chosen, as previous work has demonstrated that intercellular SA levels increase in IWFs by 24 h post‐inoculation (hpi) (Cameron and Zaton, 2004). Gas chromatography‐mass spectrometry (GC‐MS) analysis was employed to measure free SA levels in IWFs collected from ARR‐competent Col‐0 (in planta Pst∼ 105 cfu/leaf disc at 3 dpi) and ARR‐incompetent iap1‐1 (in planta Pst∼ 107 cfu/leaf disc at 3 dpi) plants. Free SA was measured, as previous experiments support the hypothesis that unconjugated SA acts in the intercellular space as an antimicrobial agent during ARR (Cameron and Zaton, 2004; Kus et al., 2002); moreover, experiments in tobacco indicate that conjugated SA is not biologically active (reviewed in Ryals et al., 1994). IWFs collected from Col‐0 leaves inoculated with Pst accumulated ∼190 ng/mL SA, compared with ∼60 ng/mL in IWFs collected from mock‐inoculated leaves (t‐test <0.05; Fig. 4b). No significant difference in intercellular SA accumulation was observed in IWFs collected from iap1‐1 leaves that were mock‐inoculated or inoculated with Pst (Fig. 4b). These data demonstrate that iap1‐1 does not accumulate intercellular SA in response to Pst, and suggest that IAP1 is upstream of intercellular SA accumulation in the ARR pathway.

Intracellular SA accumulation is reduced in iap1‐1 in response to Pst

The iap1‐1 mutation results in a loss of accumulation of SA in the intercellular space in response to Pst, suggesting that IAP1 is involved in SA biosynthesis or perhaps in the transport of SA to the intercellular space. If iap1‐1 is involved in SA biosynthesis, intracellular SA accumulation in response to Pst should also be reduced in iap1‐1. Mature Col‐0 and iap1‐1 intracellular SA levels were determined in leaves from which the IWFs were removed at 24 h post mock‐inoculation or inoculation with Pst (106 cfu/mL). This time point was chosen, as previous work has demonstrated that intercellular SA levels increase by 24 hpi during ARR (Cameron and Zaton, 2004). GC‐MS analysis was employed to measure total SA levels (SA plus SA glucosides) in ARR‐competent Col‐0 (Pst∼ 105 cfu/leaf disc at 3 dpi) and ARR‐incompetent iap1‐1 (Pst∼ 107 cfu/leaf disc at 3 dpi) plants. Col‐0 leaves inoculated with Pst accumulated ∼10 000 ng/g fresh weight SA compared with ∼3000 ng/g fresh weight from mock‐inoculated leaves (Fig. 4c). Total intracellullar SA levels were reduced to 1000 ng/g fresh weight in iap1‐1 leaves inoculated with Pst or mock‐inoculated (Fig. 4c). These data demonstrate that iap1‐1 does not accumulate intracellular SA in response to Pst, and suggests that IAP1 is upstream of intracellular SA accumulation in the ARR pathway.

eds1‐1 is ARR defective

EDS1 is an essential regulator necessary for the accumulation of SA, which, in turn, acts as a signalling molecule to up‐regulate downstream defences (Falk et al., 1999; 2001, 2005). As EDS1 is involved in SA accumulation, it was hypothesized that it may play a role in ARR. eds1‐1 and wild‐type Ws plants were assayed for ARR by inoculating young and mature plants with Pst. A significant decrease in bacterial growth (t‐test <0.05) was not observed in eds1‐1, as the bacterial density remained high (∼107) in both young and mature plants (Fig. 5a). Conversely, in planta bacterial levels were reduced 17‐fold in mature relative to young wild‐type plants. These data suggest that EDS1 is required for the ARR response.

Figure 5.

Figure 5

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(a) In planta bacterial levels in eds1‐1, jar1‐1, jin1‐1, Col‐0 and Ws. Plants were inoculated with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) at 3 and 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (cfu/ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. This experiment was repeated once for jin1 and twice for eds1‐1 and jar1‐1 with similar results. (b) eds1‐1 and Col‐0 plants (5‐week‐old) were infiltrated with water (H, Pst‐5) or salicylic acid (SA) (0.1 mM) (SA, Pst‐5) , followed by inoculation with Pst 5 h later, as in Fig. 4. This experiment was repeated twice with similar results.

As EDS1 is involved in SA accumulation and intercellular SA infiltration partially rescues the iap1‐1 ARR defect, a similar experiment was performed with eds1‐1. The addition of SA to the intercellular space of eds1‐1 prior to inoculation with Pst resulted in a two‐fold reduction (t‐test not statistically significant) in bacterial density relative to water‐infiltrated control plants; however, these plants still supported high levels of Pst (∼2 x 107 cfu/leaf disc) (Fig. 5b). It is interesting to note that, in one of two other experiments, there was a statistically significant reduction (t‐test <0.05) in Pst growth in SA‐infiltrated eds1‐1 plants; however, Pst levels remained high (107 cfu/leaf disc, data not shown). Thus, the addition of SA to the intercellular space of eds1‐1 plants did not result in the rescue of the eds1‐1 ARR defect.

The ARR response occurs in jar1‐1 and jin1‐1

Jasmonic acid (JA) and related metabolites are lipid‐derived compounds which act as signals in defence to some pathogens (often necrotrophic) and in the wound response to insects, as well as in plant growth and development (reviewed in Wasternack, 2007). Work to date suggests that SA acts as an anti‐Pst agent in the intercellular space during ARR, rather than as a signal for the up‐regulation of defence genes such as PR1 (Cameron and Zaton, 2004; Kus et al., 2002). Therefore, other signalling molecules, such as JA, may be required for ARR signalling. To test this hypothesis, two JA mutants, jar1‐1 (jasmonate‐resistant 1) and jin1‐1 (jasmonate‐insensitive 1), were assayed for ARR competence.

JAR1 encodes a JA‐amino synthetase that activates JA for optimal signalling in Arabidopsis by conjugating it to amino acids, such as isoleucine (Ile) (Staswick and Tiryaki, 2004). Therefore, jar1‐1 mutants do not accumulate activated JA‐Ile and are defective for downstream JA‐Ile‐induced defence signalling (Staswick and Tiryaki, 2004). Col‐0 and jar1‐1 mutants were assayed for ARR competence by inoculating both young and mature plants with Pst (Fig. 5a). A 10‐fold decrease in bacterial density was observed in mature relative to young Col‐0 and jar1‐1 plants. This significant decrease in bacterial growth (t‐test <0.05) in mature plants suggests that jar1‐1 mutants maintain a functional ARR pathway despite the disruption in JA signalling.

Studies have indicated that the phytotoxin coronatine, produced by the P. syringae group of pathovars (including Pst), acts as a molecular mimic of JA‐Ile (Brooks et al., 2005; Krumm et al., 1995; Staswick and Tiryaki, 2004) to elicit the JA signalling pathway downstream of JAR1 and JIN1, leading to the suppression of SA‐mediated defences, and thereby favouring Pst growth (Laurie‐Berry et al., 2006). Therefore, although studies with jar1‐1 and Pst will reveal whether a JA‐dependent response requires JA‐Ile, it cannot establish whether an intact JA signalling pathway is required (Laurie‐Berry et al., 2006). Instead, studies with jasmonate‐insensitive mutants, such as jin1‐1, are necessary. JIN1 is a MYC2 transcription factor (Lorenzo et al., 2004) that functions downstream of JAR1‐synthesized JA‐Ile and Pst‐produced coronatine, and therefore is required for JA defence signalling, even in the presence of coronatine‐producing pathogens (Laurie‐Berry et al., 2006). Young and mature Col‐0 and jin1‐1 plants were tested for their ability to display ARR by inoculation with Pst (Fig. 5a). A significant 10‐fold decrease (t‐test <0.05) in bacterial levels in mature relative to young Col‐0 and jin1‐1 plants confirms that jin1‐1 mutants have an intact ARR pathway, suggesting that JIN1‐dependent JA signalling is not required for ARR.

Identification of ARR‐associated genes using microarray analysis

A microarray experiment was conducted to identify genes that are differentially regulated during ARR. Gene expression was compared between mature Col‐0 plants that were mock‐inoculated or inoculated with Pst. It was possible to analyse only one time point after inoculation, and therefore samples were collected at 12 hpi on the basis of previous intercellular SA reduction experiments using salicylate hydroxylase, which suggested that intercellular SA negatively affects Pst growth during ARR between 5 and 24 h after inoculation (Cameron and Zaton, 2004). RNA extraction and cDNA synthesis were performed on three replicates of each treatment, and each replicate was analysed using an Affymetrix GeneChip microarray (see Supporting Information). The signal strength of each gene was averaged over the three replicates of each treatment, and the average signal strength of each gene was compared between samples that were mock‐inoculated or inoculated with Pst. Significance analysis of microarrays (SAM; Tusher et al., 2001) determined which genes were significantly up‐ or down‐regulated. SAM identifies fewer false‐positive up‐ or down‐regulated genes than an average t‐test when dealing with microarray data (Tusher et al., 2001). A delta value of 1.483 resulted in the identification of 231 differentially expressed genes with a corresponding false discovery rate (q value) of <15%. SAM identified 125 significantly up‐regulated and 105 significantly down‐regulated genes. The complete list of genes identified in this microarray is available at http://bar.utoronto.ca/affydb/cgi‐bin/affy_db_proj_browser.cgi. Genes up‐regulated greater than two‐fold are listed in Table 1 and significantly down‐regulated genes are listed in Table S2 (see Supporting Information).

Table 1.

Microarray of age‐related resistance (ARR)‐associated genes (up‐regulated ≥ two‐fold)*.

AGI no.† Probe ID no. (affynames)‡ Transcript signal ratio§ q value (%) Functional annotation**
At2g30770 267567_at 3.5 3.8 CYP71A13: cytochrome P450 71A13 may be involved in defence response, camalexin biosynthesis, indoleacetaldoxime dehydratase activity
At5g24780
At5g24770 245928_s_at 2.8 3.0 VSP1 and VSP2: vegetative storage protein 1 shows putative phosphatase activity, defence, jasmonic acid responses; vegetative storage protein 2 shows putative phosphatase activity, anti‐insect activity, induced by abscisic acid, jasmonic acid, salt, water deficiency, senescence, insect feeding and wounding
At1g75750 262947_at 2.7 3.0 GASA1: GA‐responsive GAST1 may be responsive to gibberellic acid, abscisic acid, brassinosteriods; may be localized to the cell wall
At3g47420 252414_at 2.4 3.0 Glycerol‐3‐phosphate transporter, putative cell membrane location, carbohydrate transport, sugar:hydrogen symporter activity
At3g01970 258975_at 2.3 3.8 WRKY45: putative WRKY transcription factor
At5g39610 249467_at 2.3 3.7 ANAC092: NAC transcription factor
At3g15500 258395_at 2.3 3.5 ANAC055: NAC transcription factor
At5g56870 247954_at 2.2 3.7 β‐GAL4: β‐galactosidase 4, putative carbohydrate metabolism
At5g13080 245976_at 2.1 3.8 WRKY75: putative WRKY transcription factor involved in nutrient import, lateral root development, response to stress, nutrients levels
At2g19570 265943_at 2.1 3.7 CDA1: cytidine deaminase 1
At1g22400 261934_at 2.1 3.8 UGT85A1: UDP‐glucosyl transferase 85A1
At3g50770 252136_at 2.0 3.0 CML41: calmodulin‐related protein 41, putative calcium ion binding
At1g70690 260179_at 2.0 3.7 Kinase receptor‐like protein, putative function in response to pathogens
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*

Gene expression in mature plants was compared between mock‐ and Pseudomonas syringe pv. tomato (Pst)‐inoculated plants at 12 h post‐inoculation.

†

Gene AGI number.

‡

Probe ID number.

§

Transcript signal ratio represents the fold change in transcript signal after Pst inoculation compared with mock inoculation.

q value represents the false discovery rate assigned by significance analysis of microarrays (SAM).

**

Functional annotation according to The Arabidopsis Information Resource 8 (TAIR 8), May 16, 2008.

Three members of the CYTOCHROME P450 (CYP) gene family were differentially regulated in the microarray. The most highly up‐regulated microarray gene, CYP71A13 (∼3.5‐fold up‐regulation, Table 1), plays a role in camalexin production in Arabidopsis (Nafisi et al., 2007). CYP85A2 was also up‐regulated (∼1.4‐fold, http://bar.utoronto.ca/affydb/cgi‐bin/affy_db_proj_browser.cgi), and is involved in brassinosteroid (BR) biosynthesis, which is important for leaf and flower development, flowering time and defence to pathogens (Kim et al., 2005; Nakashita et al., 2003; Nomura et al., 2005); little is known about the biochemical role of CYP72A15 (∼0.77‐fold, http://bar.utoronto.ca/affydb/cgi‐bin/affy_db_proj_browser.cgi).

Members of the NAC (petunia NO APICAL MERISTEM and Arabidopsis ATAF1, and CUP‐SHAPED COTYLEDONS) and WRKY plant transcription factor families were up‐regulated in the microarray (ANAC055 and ANAC092, ∼2.3‐fold; ANAC029, ∼1.8‐fold; WRKY45, ∼2.3‐fold; WRKY75, ∼2.1‐fold) (Table 1, http://bar.utoronto.ca/affydb/cgi‐bin/affy_db_proj_browser.cgi). The NAC and WRKYgene families are implicated in a number of biotic and abiotic stress responses (Olsen et al., 2005; Rushton and Somssich, 1998). ANAC055 and ANAC092 are expressed in response to NaCl and abscisic acid (ABA) treatments (He et al., 2005; Tran et al., 2004). In addition, ANAC055 gene expression is up‐regulated in response to methyl jasmonate (MeJA) treatments in a COI1‐dependent manner (Bu et al., 2008; He et al., 2005). ANAC029 appears to be a positive regulator of the onset of leaf senescence (Guo and Gan, 2006). NAC and WRKY genes may contribute to ARR gene regulation.

A CYTIDINE DEAMINASE (CDA) gene family member (∼2.1‐fold) was up‐regulated and two URIDINE DIPHOSPHATE‐GLUCOSYLTRANSFERASE (UGT) gene family members were identified in the microarray (Table 1). CDA genes play a role in RNA editing by converting cytidine to uridine (Faivre‐Nitschke et al., 1999; Gott and Emeson, 2000; Vincenzetti et al., 1999); therefore, post‐transcriptional gene regulation may be important during ARR. UGT85A1 was up‐regulated (∼2.1‐fold: Table 1), whereas UGT73B2 was down‐regulated (∼0.8‐fold: http://bar.utoronto.ca/affydb/cgi‐bin/affy_db_proj_browser.cgi). UGT85A1 expression is observed in leaf, stem, flower and root tissue (Woo et al., 2007), whereas UGT73B is expressed in response to some pathogens, oxidative stress, wounding and SA or MeJA application (Langlois‐Meurinne et al., 2005). Arabidopsis contains ∼120 putative UGTs (Li et al., 2001) that are involved in the conjugation of glucose to proteins and secondary metabolites, including SA (Jones et al., 1999; Li et al., 2001; Yalpani et al., 1992). The ARR‐associated UGTs may be involved in the modification of SA or another metabolite that is important for ARR.

Microarray/reverse genetics identifies genes required for ARR to Pst and H. parasitica

By combining the ability to identify ARR‐associated genes using microarray analysis with reverse genetics using T‐DNA insertion lines (Arabidopsis Biological Resource Center, ABRC), genes that are not only up‐regulated during ARR, but whose function contributes to ARR, can be identified. A number of ARR microarray genes, ANAC055 (SALK_014331), ANAC092 (SALK_090154), CDA1 (SALK_036597) and UGT85A1 (SALK_085809), β‐galactosidase (At5g56870, SALK_093071) and putative glycerol‐3‐phosphate permease (At3g47420, SALK_096228) were listed as containing T‐DNA insertions by ABRC. Plants containing homozygous T‐DNA insertions in their respective genes were identified by polymerase chain reaction (PCR). Reverse transcriptase (RT)‐PCR with gene‐specific primers was performed to confirm that the T‐DNA insertion reduced the expression of these genes (Fig. S1, see Supporting Information). RT‐PCR analysis demonstrated that both ANAC092 and ANAC055 gene expression was abolished in the anac092 and anac055 mutants, confirming the study by He et al. (2005) with anac092 (Fig. S1a). However, expression of UGT85A1 and CDA1 in the ugt85A1 and cda1 mutants was not completely abolished; rather expression was reduced compared with the wild‐type (Fig. S1b,c). These T‐DNA mutants were assayed for ARR by comparing in planta Pst levels in young and mature plants compared with Col‐0 control plants. T‐DNA insertion lines in β‐galactosidase (SALK_093071) and the putative glycerol‐3‐phosphate permease (SALK_096228) displayed a wild‐type ARR response (data not shown). In contrast, mature T‐DNA insertion mutants anac055, anac092, cda1 and ugt85A1 were more susceptible to Pst compared with the wild‐type ARR‐competent Col‐0 (Fig. 6a). Both anac092 and anac055 were compromised in ARR, as demonstrated by a modest 10‐ and 14‐fold reduction in Pst levels in mature relative to young plants, respectively (Expt #1 and #2, Fig. 6a). In contrast, a full ARR response was observed in mature relative to young wild‐type Col‐0 (154‐fold Pst reduction). The cda1 mutant was compromised in ARR, but to a lesser extent, as it still displayed a 65‐fold reduction in Pst density in mature relative to young plants (Expt #3, Fig. 6a). Although the ARR response was rather modest in Col‐0 in the ugt85A1 experiment, there was little reduction in Pst density in mature relative to young ugt85A1 plants, indicating that ugt85A1 is compromised in ARR (Expt #4, Fig. 6a).

Figure 6.

Figure 6

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(a) In planta Pseudomonas syringe pv. tomato (Pst) levels in young and mature Col‐0, anac092, anac055, cda1, ugt85A1 and sid2. Plants were inoculated with 106 colony‐forming units (cfu)/mL Pst at 3, 5 or 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (cfu/ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. Col‐0 controls are presented for each independent experiment. Significant differences between mature Col‐0 and mutants are indicated by an asterisk (P < 0.05 Student's t‐test). Different letters represent significant differences between mature genotypes in Expt. 3 (Tukey's honest significant difference, P < 0.05). These experiments were repeated at least twice with similar results. (b) Mature (6 wpg) Col‐0, anac092, anac055, cda1 and ugt85A1 were spray inoculated with Hyaloperonospora parasitica isolate Noco 2. The number of H. parasitica spores per infected leaf was quantified using a haemocytometer at 7 dpi. This experiment was repeated twice with similar results.

Another way to quantify ARR is to compare Pst levels in mature ARR‐competent Col‐0 vs. each mutant (Fig. 6a, Expt #1–#4), such that higher Pst levels in the mutants equals greater susceptibility or compromised ARR. Compared with mature Col‐0, mature anac055 and anac092 plants were seven‐fold more susceptible, mature cda1 plants were two‐fold more susceptible and mature ugt85A1 plants were five‐fold more susceptible than their respective Col‐0 controls (t‐test, P < 0.05 between each Col‐0/mutant pair in Fig. 6a). In at least two other experiments (data not shown), similar results were observed (anac055 and anac092, two‐ to seven‐fold more susceptible; cda1, two‐ to six‐fold more susceptible; ugt85A1, three‐ to five‐fold more susceptible). These data suggest that anac055, anac092, cda1 and ugt85A1 mutants are compromised in ARR. However, mature mutant plants still supported a 10–65‐fold decrease in Pst density relative to young mutant plants (Fig. 6a), suggesting that ARR is not completely abolished in these plants.

Basal resistance to Pst in young plants was also monitored in these ARR mutants to determine whether these ARR genes are also required for basal defence. Young anac055, anac092, cda1 and ugt85A1 plants supported high in planta Pst levels, similar to Col‐0 (∼107 cfu/leaf disc, Fig. 6a), suggesting that functional ANAC055, ANAC092, CDA1 and UGT85A1 are not required for basal resistance.

The ARR response in Arabidopsis is also effective in reducing the growth of H. parasitica (Rusterucci et al., 2005). Therefore, to determine whether ANAC055, ANAC092, CDA1 and UGT85A1 are also required for ARR to H. parasitica, mature Col‐0, anac055, anac092, cda1 and ugt85A1 plants were inoculated with H. parasitica isolate Noco 2, and the number of plants that supported H. parasitica sporulation at 8 dpi was determined (Table 2, Fig. 6b). Only 50% of mature Col‐0 plants supported H. parasitica sporulation, whereas 100% of anac092, 91% of ugt85a1, 81% of anac055 and 71% of cda1 plants supported H. parasitica sporulation (Table 2), suggesting that ANAC055, ANAC092, CDA1 and UGT85A1 contribute to the ARR response to H. parasitica. Spores per infected leaf were also quantified for these plants. Mature Col‐0 plants supported 4.0 x 103 spores per infected leaf, whereas spore levels were six‐fold higher on anac092, 2.5‐fold higher on anac055 and five‐fold higher on cda1 plants (Fig. 6b, t‐test P < 0.05). Spore levels were similar on ugt85a1 and Col‐0, suggesting that ANAC055, ANAC092 and CDA1 are more important for ARR to H. parasitica than is UGT85A1.

Table 2.

Growth of Hyaloperonospora parasitica in age‐related resistance (ARR) mutants.

Genotype No. of plants inoculated* No. of leaves inoculated* Infection (%)†
Col‐0 10 168 50
anac055 11 181 81
anac092 10 164 100
cda1 8 118 71
ugt85a1 12 188 91
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*

Hyaloperonospora parasitica isolate Noco 2 suspension (106 conidiosporangia/mL) was sprayed on to Col‐0 and ARR mutant leaves [6 weeks post‐germination (wpg)], and a plant was considered to be infected if sporangiophores were observed on most leaves (∼20 leaves/plant).

†

Infection (%) was calculated as the number of infected plants divided by the total number of plants inoculated. This experiment was repeated twice with similar results.

ARR gene expression analysis in Col‐0 and iap1‐1

The semidominant loss‐of‐function nature of iap1‐1 suggests that IAP1 may act as a positive regulator of downstream genes in the ARR pathway. RT‐PCR was used to determine whether SA‐associated genes (ICS1, SID1, EDS1) and the ARR genes identified in this study were expressed differentially in iap1‐1 compared with Col‐0. Leaves were collected after inoculation with Pst (106 cfu/mL, 7–24 hpi) or left untreated. Expression of ANAC055, ANAC092, CDA1, UGT85A1, ICS1, SID1 and EDS1 was similar in iap1‐1 and Col‐0 (Fig. S2a,b, Table S1, see Supporting Information), suggesting that IAP1 is not upstream of these genes in the ARR pathway. EDS1 was constitutively expressed in untreated leaves and its expression was not increased in response to virulent Pst, as observed previously (Falk et al., 1999). It is interesting to note that ICS1 (SID2), the penultimate enzyme in the chorismate biosynthesis pathway (Wildermuth et al., 2001), and SID1, a putative chloroplast SA transporter (Nawrath et al., 2002), were expressed early at 7 and 14 hpi (Fig. S2a,b), but these genes were not identified in the microarray, which represents ARR gene expression at 12 hpi. Plants used in the microarray experiment were grown in growth chambers without added humidity in the winter (60% humidity), whereas all other experiments presented in this paper were performed in chambers with added humidity (humidity maintained between 70 and 85%). Pst growth is enhanced under conditions of higher humidity (Agrios, 2005); therefore, in higher humidity chambers, Pst would begin the infection process quickly, leading to earlier initiation of ARR (Fig. S3a,b, see Supporting Information).

As ANAC055, ANAC092, CDA1 and UGT85A1 contribute to ARR in mature plants and do not appear to be required for basal resistance in young plants (Fig. 6a), ARR gene expression patterns may differ in young ARR‐incompetent plants relative to mature ARR‐competent plants. RT‐PCR was performed on leaves collected from young (in planta Pst levels ∼107 cfu/leaf disc) and mature (in planta Pst levels ∼105 cfu/leaf disc) Col‐0 plants at <5 min post‐inoculation (mpi) and at 12, 24 and 48 hpi with Pst (Figs 7, S3). CDA1 and UGT85A1 were similarly expressed in young and mature plants. ANAC055 was highly expressed at 48 hpi in young relative to mature plants (Figs 7, S3). Weak expression of ANAC092 was observed in young plants until 48 hpi; however, ANAC092 was expressed to similar levels throughout ARR in mature plants (5 mpi, 12, 24 and 48 hpi).

Figure 7.

Figure 7

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Age‐related resistance (ARR) gene expression analysis. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of leaves collected from 3 and 6 weeks post‐germination (wpg) Col‐0 at <5 min post‐inoculation (mpi) or 12, 24 and 48 h post‐inoculation (hpi) with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst); 28 PCR cycles were used for ANAC055, ANAC092, CDA1, UGT85A1 and ACTIN primers. This experiment was replicated once with similar results.

DISCUSSION

Classical mutant screening identified the ARR‐defective mutant iap1‐1. Young and mature iap1‐1 plants are equally susceptible to virulent Pst, unlike ARR‐competent wild‐type plants. In addition, iap1‐1 is defective in ARR to H. parasitica. Together, these data indicate that IAP1 is required for the ARR response to both a bacterial and oomycete pathogen, suggesting that ARR may provide broad‐spectrum resistance, as has been documented for ARR in tobacco (Fraser, 1981; Hugot et al., 1999; Wyatt et al., 1991; 1991, 1993).

SA infiltration prior to the Pst inoculation of iap1‐1 resulted in a two‐fold decrease in bacterial growth, partially restoring ARR, suggesting that SA accumulation may be impaired in iap1‐1 and that IAP1 may be upstream of SA accumulation. Subsequent analysis indicated that iap1‐1 is deficient in both intra‐ and intercellular SA accumulation during ARR, suggesting that IAP1 functions upstream of SA biosynthesis. During SA infiltration experiments, ARR was enhanced to a greater extent in Col‐0 than in iap1‐1, perhaps because iap1‐1 has lower basal intracellular SA levels than Col‐0, creating a large concentration gradient between inter‐ and intracellular compartments. Therefore, infiltrated SA remained in the intercellular space for an insufficient length of time to rescue the iap1‐1 ARR defect. This phenomenon has been observed previously in NahG plants (Cameron and Zaton, 2004) and may explain why the eds1‐1 ARR defect was not rescued by SA infiltration. Alternatively, it is possible that infiltration of SA into the intercellular space via a needle‐less syringe is not as effective as the natural SA delivery method that is hypothesized to include SA transport to the cell wall in the vicinity of cell wall‐associated bacterial colonies (Cameron and Zaton, 2004). In addition, the fact that SA addition alone did not rescue the iap1‐1 defect suggests that intercellular accumulation of other antimicrobial compounds may be required for a successful ARR response.

EDS1 was tested for its involvement in ARR because it is required for SA accumulation in response to virulent Pst (Feys et al., 2001). Mature eds1‐1 plants did not exhibit ARR, indicating that functional EDS1 is required for ARR. Phytoalexin‐deficient 4 (PAD4) function is also required for ARR (Cameron and Zaton, 2004), and EDS1 and PAD4 are thought to act together in a complex that leads to SA accumulation and defence signalling during basal resistance to virulent Pseudomonas (Feys et al., 2001; Wiermer et al., 2005). Therefore, it is possible that the EDS1–PAD4 complex is a component of both basal resistance and ARR in Arabidopsis.

ARR microarray analysis at 12 hpi identified over 200 differentially expressed genes. The most highly expressed gene in the ARR microarray analysis is a P450, CYP71A13, which has recently been shown to be involved in the biosynthesis of the phytoalexin camalexin (Nafisi et al., 2007). However, ARR occurs in the pad3‐1 camalexin mutant (Kus et al., 2002), suggesting that the phytoalexin camalexin is not required for ARR. A putative T‐DNA insertion mutant in CYP71A13 is currently being characterized to determine whether this P450 produces a secondary metabolite that is required during ARR. Another cytochrome P450 gene, CYP85A2, was also up‐regulated in the microarray and has been implicated in the BR signalling pathway, suggesting that BR signalling may be involved in ARR. BR signalling has been implicated in defence, as demonstrated by BR‐induced resistance in potato and tomato to a variety of pathogens (reviewed in Krishna, 2003). Increased resistance in potato was associated with elevated levels of ABA and ethylene, but not SA, suggesting that BR‐induced defence may be independent of SA (Krishna, 2003). As our accumulated work suggests that intercellular SA accumulation, rather than NPR1/SA signalling, is required for ARR, it seems probable that other signalling molecules, such as BR, may be involved in the ARR pathway.

A number of genes that contribute to the ARR response have been identified, indicating that a single time point (12 hpi) snap shot of ARR gene expression is an inexpensive yet effective method of gene discovery. ARR gene discovery is ongoing in our laboratory as new T‐DNA insertions in additional ARR microarray genes become available. T‐DNA insertion mutants in ANAC055, ANAC092, CDA1 and UGT85A1 were compromised in ARR to Pst. However, ARR was not completely abolished, suggesting that other members of each respective gene family may be compensating for the mutations in anac055, anac092, cda1 and ugt85A1, or there may be other signalling pathways that contribute to ARR. ANAC092 is expressed at early time points after the inoculation of mature ARR‐competent relative to young ARR‐incompetent plants, suggesting that early expression of ANAC092 is required for a successful ARR response.

ANAC055 and ANAC092 belong to the large plant‐specific NAC gene family, consisting of 105 and 75 putative members in Arabidopsis and rice, respectively (Ooka et al., 2003). These genes encode transcription factors with a conserved N‐terminal DNA‐binding NAC domain (1997, 1999; Duval et al., 2002; Hegedus et al., 2003; Taoka et al., 2004; Vroemen et al., 2003; Xie et al., 2000), and a variable C‐terminal transactivation domain (Duval et al., 2002; Hegedus et al., 2003; Taoka et al., 2004; Xie et al., 2000). Studies have indicated that ANAC055 and ANAC092 regulate the expression of genes during abiotic stress responses (He et al., 2005; Tran et al., 2004) and ANAC055 regulates the expression of JA‐related genes during fungal infection (Bu et al., 2008). The identification of targets of the putative ANAC055 and ANAC092 transcription factors will provide insights into the ARR signalling pathway. In addition, ANAC055 and ANAC092 are members of the same gene family and may play redundant roles during ARR; therefore, the analysis of anac055anac092 double mutants is currently underway (Fig. S1a).

The jar1‐1 and jin1‐1 JA pathway mutants were competent for the ARR response, strongly suggesting that this branch of the JA signalling pathway is not required for the ARR response. This is interesting in light of the fact that a number of JA‐associated genes were up‐regulated in the ARR microarray (VSP1, ANAC055), and suggests that the Pst‐produced JA mimic, coronatine (Staswick and Tiryaki, 2004), may be acting to up‐regulate the expression of these genes. Alternatively, it is possible that VSP1 and ANAC055 are expressed during ARR via a JAR1‐ and JIN1‐independent branch of the plant JA pathway.

The ARR‐defective phenotypes of eds1 and iap1‐1 indicate that these genes are necessary for a successful ARR response, whereas the partially ARR‐defective phenotypes of anac055, anac092, cda1 and ugt85A1 suggest that these genes contribute to differing degrees to the ARR response to Pst. In addition, ARR to H. parasitica was also compromised in anac055, anac092, cda1 and, to a lesser extent, ugt85a1, suggesting that these genes, together with IAP1, are important for ARR to both pathogens. Our expression analysis indicates that IAP1 may lie downstream of ANAC055, ANAC092, CDA1, UGT85A1, ICS1 and EDS1, but upstream of SA accumulation in the ARR pathway. Mapping of IAP1 and iap1‐1 is ongoing and will provide insight into the role of IAP1 during the ARR response. The absence of the HR (Kus et al., 2002) suggests that ARR is not a form of R gene‐mediated resistance. Moreover, ARR provides resistance to two pathogen types (bacterium and oomycete), suggesting that it is not a form of basal resistance in which molecules common to a particular pathogen type are recognized by MAMP receptors (Eulgem, 2005). We speculate that ARR is a developmentally controlled response that occurs at the transition to flowering, allowing mature Arabidopsis to recognize the presence of Pst and H. parasitica to elicit ARR signalling and intercellular antimicrobial SA accumulation.

EXPERIMENTAL METHODS

Plant growth conditions

The A. thaliana mutant iap1‐1[IAP1 registered on The Arabidopsis Information Resource (TAIR) gene symbol/name web page] was used in conjunction with controls, wild‐type Columbia (Col‐0) and Wassilewskija (Ws), transgenic NahG (K. Lawton, Syngenta, Research Triangle Park, NC, USA) and sid2 (C. Nawrath, University of Fribourg, Fribourg, Switzerland). Additional mutants examined included eds1‐1 (TAIR), jar1‐1 and jin1‐1 (B. Kunkel, Washington University, St. Louis, MO, USA), anac055 (SALK_014331), anac092 (SALK_090154), cda1 (SALK_036597), ugt85A1 (SALK_085809), glycerol‐3‐PO4 permease (SALK_096228) and β–galactosidase (SALK_093071) (Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH, USA; Alonso et al. (2003). Fast neutron‐mutagenized M2 Col‐0 (glabrous) seeds were obtained from Lehle Seeds and used in the classical ARR mutant screen. Seeds were surface sterilized, germinated and grown as described previously (Rusterucci et al., 2005). The humidity ranged between 75% (winter) and 85% (summer). Plants were grown under a short‐day 9‐h photoperiod at 22–24 °C. Arabidopsis is delayed in the transition to flowering when grown under short‐day conditions (Piñero and Coupland, 1998). In standard ARR experiments, Col‐0 plants become competent for ARR at the transition to flowering stage, which occurs at approximately 6 weeks of age (Kus et al., 2002).

Pst and H. parasitica ARR assays

Arabidopsis plants were inoculated with virulent Pst strain DC3000 (rifampicin and kanamycin resistant) obtained from Dr Andrew Bent (University of Wisconsin, Madison, WI, USA) (Whalen et al., 1991). Pst was grown to mid‐logarithmic phase in King's B medium and kanamycin (50 µg/mL) overnight, shaken at room temperature (22–25 °C) and then diluted to 106 cfu/mL in 10 mM MgCl2. Three to four leaves of young (3 wpg) and four to six leaves of mature plants (5 or 6 wpg) on 7 to 12 plants were inoculated with virulent Pst, and in planta bacterial levels were determined as described previously (Kus et al., 2002; Wolfe et al., 2000). Mutants tested for ARR competency included iap1‐1, eds1‐1, jar1‐1, jin1‐1, anac055, anac092, cda1, ugt85A1, glycerol‐3‐PO4 permease and β‐galactosidase. Photographs of leaves were taken with a Hewlett Packard (Palo Alto, CA, USA) 5.6 MP 56X zoom digital camera.

Infection with H. parasitica isolate Noco 2 was performed by spraying an asexual suspension (106 conidiosporangia/mL) onto leaves of mature plants (6 wpg). Mature plants possessed approximately 20 leaves and were scored as susceptible if most leaves displayed sporangiophores at 7 or 8 dpi. In addition, spores from the leaves of 8–12 plants (110–180 total leaves) were collected, and the spore number per leaf was quantified using a haemocytometer.

SA infiltration experiments, IWF collection and SA determination

SA infiltration experiments and IWF collections were performed as described previously (Cameron and Zaton, 2004; Kus et al., 2002). IWFs were filter sterilized to remove Pst. SA levels were also determined in leaves after IWF removal. SA levels were measured using GC‐MS (Schmelz et al., 2004; Supporting Information).

Microarray analysis and gene expression using RT‐PCR

See Supporting Information.

Supporting information

Fig. S1 Age‐related resistance (ARR) gene expression analysis in T‐DNA insertion mutant plants. (a) Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers for ANAC055 and ANAC092 was performed on untreated Col‐0 and anac055anac092 leaf samples and leaves collected from mature [6 weeks post‐germination (wpg)] anac055 and anac092 plants at 12 hpi with Pseudomonas syringe pv. tomato (Pst). (b) RT‐PCR with primers for CDA1 was performed on untreated Col‐0 and cda1 leaf samples (left panel), leaves collected from untreated (un) mature (6 wpg) Col‐0 and cda1 plants and leaves collected at 48 hpi with Pst (right panel). (c) RT‐PCR with primers for UGT85A1 was performed on untreated Col‐0 and ugt85a1 leaf samples (left panel), leaves collected from untreated (un) mature (5 wpg) Col‐0 and ugt85a1 plants and samples collected at 48 hpi with Pst (right panel). ACTIN was used as a loading control. The number of PCR cycles used is shown.

Fig. S2 Age‐related resistance (ARR) gene expression analysis in iap1‐1 and Col‐0. (a) Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis was performed to monitor ICS1, SID1, EDS1, ANAC055, ANAC092, CDA1 and UGT85A1 gene expression in Col‐0 and iap1‐1. Mature [6 weeks post‐germination (wpg)] Col‐0 and iap1‐1 leaves were collected from 7 to 24 h post‐inoculation (hpi) with Pseudomonas syringe pv. tomato (Pst) [106 colony‐forming units (cfu)/mL] and from untreated controls (UN). ACTIN was used as a loading control; 28 cycles of RT‐PCR were used for all primers. This experiment was repeated twice more with similar results. (b) One replicate is shown; 28 cycles of RT‐PCR were used for all primers in this replicate.

Fig. S3 Replicate age‐related resistance (ARR) gene expression analysis in young and mature Col‐0. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis on leaves collected from (a) young [3 weeks post‐germination (wpg)] and (b) mature (6 wpg) Col‐0 plants at 5 min post‐inoculation (mpi) and 12, 24 and 48 h post‐inoculation (hpi) with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst). ANAC055, ANAC092, CDA1, UGT85A1 and ACTIN primers were used. The number of PCR cycles used is indicated.

Table S1 Forward and reverse primer sequences in reverse transcriptase‐polymerase chain reaction (RT‐PCR).

Table S2 Most down‐regulated genes in the age‐related resistance (ARR) microarray.

Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Supporting info item

Click here for additional data file. (274KB, doc)

ACKNOWLEDGEMENTS

We thank ABRC for T‐DNA insertion lines, B. Kunkel for jin1‐1 and jar1‐1 seeds (Washington University) and R. Sarkar for starting our ongoing ARR mutant screen. This work was supported by grants to R. Cameron [Natural Science and Engineering Research Council (NSERC) of Canada Discovery Grant, Premier's Research Excellence Award of Ontario], start‐up funding and growth chamber maintenance support from McMaster University and by an NSERC Discovery grant to K. Yoshioka and N. Provart. Genome Canada and the Ontario Genomics Institute support the BAR facility at the University of Toronto.

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Associated Data

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Supplementary Materials

Fig. S1 Age‐related resistance (ARR) gene expression analysis in T‐DNA insertion mutant plants. (a) Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers for ANAC055 and ANAC092 was performed on untreated Col‐0 and anac055anac092 leaf samples and leaves collected from mature [6 weeks post‐germination (wpg)] anac055 and anac092 plants at 12 hpi with Pseudomonas syringe pv. tomato (Pst). (b) RT‐PCR with primers for CDA1 was performed on untreated Col‐0 and cda1 leaf samples (left panel), leaves collected from untreated (un) mature (6 wpg) Col‐0 and cda1 plants and leaves collected at 48 hpi with Pst (right panel). (c) RT‐PCR with primers for UGT85A1 was performed on untreated Col‐0 and ugt85a1 leaf samples (left panel), leaves collected from untreated (un) mature (5 wpg) Col‐0 and ugt85a1 plants and samples collected at 48 hpi with Pst (right panel). ACTIN was used as a loading control. The number of PCR cycles used is shown.

Fig. S2 Age‐related resistance (ARR) gene expression analysis in iap1‐1 and Col‐0. (a) Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis was performed to monitor ICS1, SID1, EDS1, ANAC055, ANAC092, CDA1 and UGT85A1 gene expression in Col‐0 and iap1‐1. Mature [6 weeks post‐germination (wpg)] Col‐0 and iap1‐1 leaves were collected from 7 to 24 h post‐inoculation (hpi) with Pseudomonas syringe pv. tomato (Pst) [106 colony‐forming units (cfu)/mL] and from untreated controls (UN). ACTIN was used as a loading control; 28 cycles of RT‐PCR were used for all primers. This experiment was repeated twice more with similar results. (b) One replicate is shown; 28 cycles of RT‐PCR were used for all primers in this replicate.

Fig. S3 Replicate age‐related resistance (ARR) gene expression analysis in young and mature Col‐0. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis on leaves collected from (a) young [3 weeks post‐germination (wpg)] and (b) mature (6 wpg) Col‐0 plants at 5 min post‐inoculation (mpi) and 12, 24 and 48 h post‐inoculation (hpi) with 106 colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst). ANAC055, ANAC092, CDA1, UGT85A1 and ACTIN primers were used. The number of PCR cycles used is indicated.

Table S1 Forward and reverse primer sequences in reverse transcriptase‐polymerase chain reaction (RT‐PCR).

Table S2 Most down‐regulated genes in the age‐related resistance (ARR) microarray.

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