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First published online October 2, 2003; 10.1104/pp.103.024182 Plant Physiology 133:1272-1284 (2003) © 2003 American Society of Plant Biologists Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis1Cooperative Research Centre for Tropical Plant Protection (E.J.C., P.M.S., K.K., J.P.A., D.J.M., P.R.E., J.M.M.) and Department of Biochemistry and Molecular Biology (E.J.C., P.M.S., J.P.A., D.J.M., P.R.E.), The University of Queensland, St. Lucia, Queensland 4072, Australia; Commonwealth Scientific and Industrial Research Organization Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Queensland 4067, Australia (K.K., J.M.M.); and Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium (I.A.M.A.P., B.P.A.C)
The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.
Plants have multiple defense mechanisms to fight against microbial pathogens. Some of these defenses involve preformed chemical and physical barriers, which impede pathogen entry into the host plant, whereas others are stimulated in response to pathogen attack and subsequently limit further pathogen growth. Successful recognition of pathogen-derived signals can ultimately result in the hypersensitive response or programmed cell death, which acts to stop the spread of an attempted infection by a biotrophic pathogen. Pathogen challenge also activates a number of signaling pathways that coordinately regulate expression of many genes encoding various transcriptional regulators, enzymes functioning in the synthesis of phytoalexins and other secondary metabolites, pathogenesis-related proteins, and a number of other antimicrobial molecules (Schenk et al., 2000  ). At least three chemical signal molecules are known to regulate the signaling pathways associated with plant defense responses. These are salicylic acid (SA), jasmonic acid (JA) and its methyl ester, methyl jasmonate (MJ), and ethylene (Dong, 1998; Reymond and Farmer, 1998). Substantial cross talk also occurs among these signaling pathways for mounting a coordinated defense response that may be dependent on the type of the challenging pathogen (for review, see Feys and Parker, 2000; Thomma et al., 2001; Kunkel and Brooks, 2002). The recent use of large-scale gene expression analyses (e.g. cDNA microarrays) suggests that potentially a large number of genes are associated with plant defense responses (Maleck et al., 2000; Schenk et al., 2000). However, so far, only a small number of plant genes identified in these microarray experiments have been functionally characterized at the molecular level.
ATP-binding cassette (ABC)-type membrane proteins (ABC transporters) function as ATP-driven efflux pumps that export a wide variety of compounds (Davies et al., 2000
Our interest is in the identification and functional characterization of genes that are associated with interactions of Arabidopsis with necrotrophic fungal pathogens such as Alternaria brassicicola (Schenk et al., 2000
Identification of AtPDR12 by cDNA Microarray Analysis
To identify plant genes that may be specifically induced during the Arabidopsis-A. brassicicola interaction, we first constructed a subtractive cDNA library from Arabidopsis leaf material collected at various time points after inoculation (see "Materials and Methods"). In total, 2,000 anonymous cDNA clones from this library were PCR amplified, and amplification products were spotted in duplicate on microarray slides. These slides were then hybridized simultaneously with Cy3- and Cy5-labeled cDNA probes prepared from two independent biological replicates each of A. brassicicola-inoculated and uninoculated plants and from plants treated with signaling compounds SA, MJ, or ethylene at various time points after inoculation/treatment. These analyses identified 195 cDNAs that were expressed at least 2-fold or higher in RNA samples from inoculated plants. We fully or partially sequenced 53 such clones, which identified 30 unique genes. Seven of these genes had previously described defensive functions. For instance, genes encoding antimicrobial or pathogenesis-related proteins (e.g. PDF1.2 and PR-1) and genes encoding enzymes involved in the synthesis of secondary metabolites/phytoalexins (e.g. a lipoxygenase, a glutathione S-transferase) and defense signaling compounds (e.g. 12-oxophytodienoate reductase) were represented among these genes. The microarray analysis also led to the identification of 23 genes (to be described elsewhere) with either unknown functions or functions that were not previously associated with plant defense responses. One of the cDNAs strongly induced in microarray analysis (i.e. showed >2-fold induction at 3, 6, 12, and 24 h after inoculation) by A. brassicicola was identified as the AtPDR12 gene (At1g15520). BLASTP searches using the deduced amino acid sequence of AtPDR12 demonstrated particularly high amino acid sequence identities between AtPDR12 and annotated PDR-like ABC transporter proteins from other plant species. The amino acid sequence of AtPDR12 was 69%, 68%, and 65% identical to NtPDR12 of tobacco (Nicotiana tabacum), NpABC1 of N. plumbaginifolia, and SpTUR2 of S. polyrrhiza, respectively. The other proteins that also showed particularly high amino acid identities to AtPDR12 were recently annotated PDR subfamily of ABC transporters from rice (Oryza sativa) including PDR7 (CAD59570, 65%), PDR8 (BAB21279, 65%), PDR9 (BAB21276, 67%), PDR10 (BAB21273, 65%), PDR11 (BAB21275%–66%), BAB93292 (63%), and SpTUR2 (65%; see also Jasinski et al., 2003
Searching the Arabidopsis genome for AtPDR12-like sequences revealed at least 13 other PDR-like ABC transporter genes with strong nucleotide sequence similarities (75%–85%) to the AtPDR12 gene. This indicated the likelihood that cross-hybridization from such related sequences could complicate the confirmation of microarray results by any other hybridization-based procedure. Therefore, we used RTQ-RT-PCR with AtPDR12-specific primers for subsequent expression analyses. RTQ-RT-PCR is a specific and sensitive method that is now widely used particularly for transcript profiling of individual members of gene families in plants (Charrier et al., 2002
We also tested the induction patterns of the AtPDR12 gene after treatments with SA, MJ, and ethylene using plants grown and sampled under identical conditions as those used in pathogen inoculation assays. In these experiments, SA treatment strongly induced expression of AtPDR12 up to 260-fold at 12 h after treatment (Fig. 2A). Similarly, treatments with ethylene and MJ resulted in a 16- and a 40-fold induction of this gene as early as 3 and 6 h after treatment, respectively (Fig. 2, B and C). Overall, these results suggested that AtPDR12 was coordinately regulated by multiple defense-associated signals with distinct induction kinetics.
Interestingly, although we observed strong and consistent induction of this gene by pathogen inoculation or treatments with chemical signal compounds, fold-induction values observed for some time points were more variable than the others (for instance, see the 1-, 24-, and 48-h time points after SA, ethylene, and MJ treatments in Fig. 2). To better understand the possible reason(s) underlying this phenomenon, we examined the transcript abundance of AtPDR12 in uninoculated plants grown and sampled under identical growth conditions using two independently replicated experiments (i.e. RNA collected from plants grown and inoculated in separate occasions). This expression profiling revealed steadily reduced levels of the AtPDR12 transcript abundance during the light period, suggesting an influence of the diurnal cycle on AtPDR12 expression (Fig. 3). Interestingly, the time points with more variable induction values coincided with the early hours of light period during which we observed a rapid reduction in the AtPDR12 transcript abundance.
We also analyzed the transcript abundance of AtPDR12 in various Arabidopsis tissues taken from the plants at flowering and seed-forming stage. Varying levels of AtPDR12 transcripts were detected in stems, cauline leaves, roots, developing siliques, and flowers, with expression in stems and cauline leaves being significantly higher than in other tissues (Fig. 4). Treating the aboveground organs of these plants with SA induced AtPDR12 expression in the stems, cauline leaves, and the roots, whereas it did not have any effect on AtPDR12 transcript levels in siliques and flowers (Fig. 4).
To further characterize which signaling pathway(s) plays a major role in regulating AtPDR12 expression, we examined the induction kinetics of AtPDR12 in various Arabidopsis signaling mutants at 5 and 15 h after A. brassicicola inoculation (Fig. 5A). To reliably detect potential quantitative differences in AtPDR12 expression in each of the mutants, we replicated these inoculation experiments at least three times at different occasions. Because our analyses showed that the expression of AtPDR12 was more strongly induced by SA than by any other chemical treatment, it is expected that the SA signaling pathway would have particular importance for the induction of AtPDR12 after pathogen challenge. To examine the role of SA accumulation in regulating AtPDR12 expression, we analyzed the induction patterns of AtPDR12 in the eds5 and npr1 mutants at 5 and 15 h after inoculation with A. brassicicola. The pathogen-induced expression of AtPDR12 was clearly reduced at the 5-h time point in the eds5 mutant as compared with induction of AtPDR12 in pathogen inoculated wild-type plants (Fig. 5A). The eds5 mutant is known to have reduced levels of SA accumulation; thus, the abolishment of AtPDR12 expression in this mutant at a relatively early stage of infection suggests that pathogen-responsive expression of AtPDR12 may require accumulation of sufficient amount of SA. Despite this initial delay, expression of AtPDR12 was restored to wild-type levels at a later time point (
We further examined whether pathogen-responsive expression of AtPDR12 requires a functional NPR1 protein. As shown in Figure 5A, the AtPDR12 expression was significantly reduced at both 5 and 15 h after inoculation with A. brassicicola in this mutant, suggesting that a functional NPR1 protein is required for pathogen responsiveness of AtPDR12. We subsequently treated both the wild-type and npr1 plants with SA and sampled the plants 12 h after treatment because the results presented in Figure 2A showed that AtPDR12 expression was maximal in SA-treated wild-type plants around this time point. These assays did not reveal any significant difference in AtPDR12 expression between the treated wild type and the npr1 mutant (Fig. 5B), suggesting that SA may regulate the expression of this gene in an NPR1-independent manner, although NPR1 is required for pathogen-responsive expression of AtPDR12.
In an effort to determine the presence of any conserved promoter sequence elements that may be responsible in mediating the SA-responsive expression of AtPDR12, we examined approximately 1 kb of the putative AtPDR12 promoter sequence using the PLACE database (available at http://www.dna.affrc.go.jp/htdocs/PLACE/). This analysis identified four W boxes (TTGAC), two ASF-1 elements (TGACG), and three GT-1 (GAAAAA) motifs in the AtPDR12 promoter. All these motifs are conserved sequence elements commonly found in the promoters of SA-regulated genes (Du and Chen, 2000
To determine whether the other major signaling pathways play any role in regulation of expression, we tested the pathogen responsiveness of the AtPDR12 gene in jasmonate and ethylene signaling mutants. Low or no significant induction of this ABC transporter gene was observed in ethylene signaling mutants (etr1 and ein2) and in jasmonate signaling mutant (jar1) at 5 h after inoculation as compared with the wild-type plants (Fig. 5A). Despite this initial delay in induction, 15 h after pathogen challenge, AtPDR12 was induced in etr1 and ein2 to levels that were not significantly different from those observed in wild-type plants. Taken together, these results suggested that, in addition to SA, both JA- and ethylene dependent-signaling pathways are required for rapid induction of this gene after pathogen challenge.
Finally, we examined the transcript abundance of AtPDR12 in the cpr5 mutant. Interestingly, the AtPDR12 transcript levels in the uninoculated cpr5 mutant were as high as AtPDR12 expression observed in the locally inoculated tissue of wild-type plants (Fig. 5C). Hence, similar to constitutive expression of other defense genes such as PR1 and PDF1.2 (Bowling et al., 1997
The close similarity of the AtPDR12 protein to the related ABC transporters SpTUR2 and NpABC1 implicated in sclareol transport has led to the suggestion that AtPDR12 may also function in transporting sclareol or related terpenoids (van den Brûle and Smart, 2002
To gain some insights into the role of AtPDR12, we analyzed an Arabidopsis insertion line (SALK_005635) in which the AtPDR12 genomic sequence was disrupted with a T-DNA insertion. We first confirmed the predicted location of T-DNA insertion within the second putative transmembrane domain from the carboxyl terminus of (Fig. 7, A and B) of AtPDR12 by a DNA sequencing strategy (see "Materials and Methods"). To test whether plants from this insertion line would have enhanced susceptibility to compounds containing an indole ring or SA, we germinated the seeds from wild type and the AtPDR12 insertion line on plant growth medium containing indole acetic acid, abscisic acid, sclareolide, sclareol, Rhodamine 6G, or SA. At 3 d after germination on medium containing 50 µM sclareol, seeds from the AtPDR12 insertion line showed only 52% of the wild-type germination rate. Higher sclareol concentrations (e.g. 100, 200, and 300 µM) affected the germination rates of both the wild-type and AtPDR12 insertion line plants. Particularly, at 300 µM sclareol concentrations, none of the seeds from AtPDR12 insertion line was germinated at 3 d postincubation, whereas a substantial portion of the wild-type plants germinated (i.e. radicle emerged from the seeds; Figs. 8A and 9), but these wild-type seedlings showed extremely slow growth rates. Seeds from both wild type and the AtPDR12 insertion line germinated at similar rates and grew normally on plant growth medium devoid of sclareol (Figs. 8, A and B, and 9). In addition, germinated seedlings from the AtPDR12 insertion line showed a reduced root length after germination as compared with wild-type plants treated similarly. This effect was quantifiable after germination of seeds on a medium containing 50 µM sclareol (Fig. 8B) Germination assays using other indole ring-containing compounds or SA did not reveal any difference between wild type and insertion line, at least at the concentrations tested (data not shown), suggesting that the observed effect was specific to sclareol. Overall, this result suggested that AtPDR12 is functionally related to NpABC1 and SpTUR2, which were previously shown to transport sclareol (Jasinski et al., 2001
If the putative substrate transported by AtPDR12 acts as an endogenous signal for defense gene induction and/or has direct antimicrobial effects on pathogen growth, then the possibility exists that the plants with an inactive copy of this gene would have compromised (i.e. enhanced susceptibility) disease resistance response. To test this hypothesis, we performed two independently replicated inoculation experiments on wild-type and AtPDR12 insertion line plants using fungal (F. oxysporum) and bacterial (P. syringae pv tomato DC3000) pathogens of Arabidopsis. However, the results from these inoculation experiments did not show any altered susceptibility in AtPDR12 insertion line plants (data not shown). Similarly, inoculation with incompatible strains of A. brassicicola and P. syringae pv tomato 1065 did not reveal any enhanced sensitivity to these pathogens, measured as trypan blue staining of the inoculated leaves (data not shown). Taken together, these results suggest that the basal plant defenses operating against at least these two pathogens were still sufficiently intact in the AtPDR12 insertion line plants, although further testing against various other pathogens may be warranted before any conclusions can be made about the role of this gene in plant defense.
Recently, cDNA microarray analyses have been successfully used in Arabidopsis to identify genes associated with various physiological conditions, such as plant responses to biotic and abiotic stresses (Maleck et al., 2000 ; Schenk et al., 2000; Desikan et al., 2001; Seki et al., 2001). However, application of this technology is somewhat limited in most other plant species where well-characterized expressed sequence tag (EST) collections have not yet been assembled fully. Even in plants with large numbers of EST collections, it is possible that certain genes with low or unique expression patterns will not be represented in the available EST collections (Richmond and Somerville, 2000). In the present paper, the screening strategy (microarray analysis of anonymous cDNA clones from a subtractive library) was not dependent on the availability of any of the previously assembled EST collections or prior sequence information. The genes found to be induced after microarray hybridization analysis were partially or fully sequenced and identities of these genes determined. This permitted identification of novel genes that were not previously reported to be associated with plant defense responses.
One of the pathogen-inducible genes identified in this analysis was the AtPDR12 gene, encoding a PDR-like ABC transporter protein. To date, the AtPDR12 gene has not been represented in any of the EST collections available from Arabidopsis, possibly due to very low transcript levels of this gene in the absence of an inducing agent such as pathogen challenge. The PDR subfamily of Arabidopsis contains 13 genes with high DNA sequence homology (Jasinski et al., 2003
The quantification of the transcript abundance of AtPDR12 also revealed that this gene was responsive to diurnal cycles with steadily reduced transcript levels in uninoculated plants at time points where we observed higher variability in induction ratios. It is possible that transcription factors involved in regulating the expression of AtPDR12 under these seemingly different conditions may be competing, contributing to the variation we observed for certain time points. Other examples of pathogen-responsive genes that are also diurnally regulated are known (Schaffer et al., 2001
In addition to pathogen signals, AtPDR12 was also responsive to defense-associated chemical signals SA, ethylene, and MJ. SA-induced expression of AtPDR12 was substantially stronger and sustained for longer periods than the ethylene- and MJ-induced expression of AtPDR12, suggesting that SA may have a predominant effect on the regulation of AtPDR12. The strong effect of SA on the induction of this gene was also evident in various tissues (except siliques) analyzed. Similar to AtPDR12, AtPDR12-homologous NtPDR12 and NpABC1 were found to be responsive to MJ (Sasabe et al., 2002
The NPR1-independent induction of AtPDR12 after SA treatment is in agreement with the notion that an SA-dependent, NPR1-independent defense pathway is functional in Arabidopsis. The presence of this pathway was first proposed based on the observations that the loss-of-function mutations in NPR1 did not result in complete loss of PR gene expression (Rate et al., 1999
Although AtPDR12 is predominantly regulated by the SA signaling pathway, our results from mutant analyses indicated that the functional JA and ethylene pathways are also required for full and/or early induction of this gene. Defense gene induction through overlapping action of SA, JA, and ethylene signaling pathways also has been shown in various Arabidopsis mutants. For instance, defense gene expression and resistance against pathogens are regulated synergistically by SA and JA/ethylene pathways in the hrl1 mutant (Devadas et al., 2002
Recent studies have revealed that ABC transporters from Arabidopsis can be involved in a variety of functions (Coleman et al., 1997
Although enhanced susceptibility to sclareol observed in the AtPDR12 insertion line indicates potential involvement of AtPDR12 in diterpenoid transport, so far there has been no report of diterpenoids or sclareol synthesized in Arabidopsis. In contrast, both monoterpenes and sesquiterpenes are synthesized in Arabidopsis (Chen et al., 2003 Determining the exact substrate of AtPDR12 protein along with its subcellular localization would also reveal more information about the putative function of this ABC transporter protein. Overexpression of AtPDR12 in transgenic plants and evaluation of diterpenoid partitioning and composition could be useful to determine the precise role of this ABC transporter.
Plant Inoculations/Treatments
For the construction of a subtracted cDNA library, Arabidopsis (Col-0) plants were grown in a growth chamber (20°C daytime temperature and 18°C night temperature with a 12-h photoperiod [100 µE m–2 s–1]). Four week-old plants were inoculated with Alternaria brassicicola (strain MUCL 20297) by applying 5-µL drops of a spore suspension (5 x 105 spores mL–1in distilled water) on four to six lower rosette leaves. The plants were placed in a propagator flat with a clear polystyrene lid and kept at high humidity to stimulate infection by hyphal germlings. Control plants were not inoculated, but were otherwise treated the same way. At 3, 6, 9, 12, 18, 24, 48, 56, and 64 h after inoculation, leaves from infected plants (both treated and non-treated leaves from the same plant) were collected. For cDNA microarray and/or RTQ-RT-PCR experiments, seeds of Arabidopsis ecotype Col-0 (wild type) and of defense signaling mutants npr1 (Arabidopsis Biological Resource Center stock no. CS3726), eds5 (CS3735), cpr5 (CS3770), jarl (CS8072), ein2 (CS8844), and etr1 (CS273; all in Col-0 background) were planted in autoclaved soil and cold treated at 4°C for 4 d. Plant were then grown to eight- to 12-leaf stage in controlled environment rooms with a photoperiod of 8 h light (180 µE m–2 s–1 at 24°C with 75% relative humidity and at 20°C/95% relative humidity at night time) and treated with either chemical signal compounds or fungal pathogens. For treatments with SA, plants were sprayed with a 5 mM solution (pH 7) containing 0.1% (w/v) ethanol. MJ treatment was carried out by taping a cotton ball containing 200 µL of a 0.5% (w/v) solution in ethanol onto the wall of a 20-L airtight container. For fungal inoculations, 5-µL drops of a spore suspension (5 x 105 spores mL–1 in water) of A. brassicicola (isolate UQ4273, freshly grown on agar plates containing clarified vegetable juice [Campbell's, Camden, NJ]) were pipetted onto two to four leaves per plant. Agar blocks (4 x 4 mm) containing a lawn of fungal hyphae were used for inoculation with Sclerotinia sclerotiorum (UQ isolate 3833 kindly provided by Ken C. Goulter, Cooperative Research Centre for Tropical Plant Protection, Queensland, Australia) grown on agar plates containing oat flakes. Fusarium oxysporum (isolate 5176 kindly provided by Roger Shivas of Department of Primary Industries, Queensland, Australia) was maintained on either sterile filter paper or on carnation leaf agar and grown on one-half-strength potato dextrose agar for approximately 1 week before inoculations. Five plugs were taken from these plates under sterile conditions with a cork borer and placed into 50 mL of potato dextrose broth containing 100 µg mL–1 streptomycin and 15 µg mL–1 tetracycline. The flasks containing the inoculum were placed onto a shaker for approximately 3 to 4 d and drained through Miracloth (Calbiochem, San Diego) before quantification with a hemocytometer. A spore concentration of 1 x 106 spores mL–1, diluted with sterile distilled water, was used to inoculate the plants. The plants used in the inoculation were grown in autoclaved sandy soil for approximately 3 to 4 weeks before inoculation. Before inoculations, the plants were carefully removed from the soil, and the roots were rinsed with sterile water and then dipped for 20 s into the fungal inoculum. The inoculated plants were sealed with a clear autoclave bag in the first 2 d postinoculation. Ninety plants from each of the wild-type and AtPDR12Ins plants were inoculated in two independent inoculation experiments. The disease symptoms were scored approximately 8 to 11 d postinoculation by counting the number of dead leaves and wilted plants. The amount of fungal mass in inoculated plants was also estimated using RT-Q-RT-PCR using a primer pair (5'-CGCCAGAGGACCCCTAAAC-3' and 5'-ATCGATGCCAGAACCAAGAGA-3') specific (i.e. no homology to Arabidopsis sequences) for amplification of the 18S transcribed internal spacer (GenBank accession no. AY237110). The amount of fungal RNA in the plant tissue was normalized using expression from the Arabidopsis
The bacterial pathogen Pseudomonas syringae pv tomato DC3000 (compatible strain) and P. syringae pv tomato 1065 (incompatible strain) kindly provided by Peter Mohr (Deakin University, Victoria, Australia) were grown on King's B media at pH 7.0 (10 g L–1 Bacto peptone, 1.5 g L–1 K2HPO4, and 15 g L–1 glycerol) containing 50 µg mL–1 each of rifampicin and kanamycin for DC3000, whereas only rifampicin was used for 1065. The cultures were shaken for 2 d at 28°C in the dark and resuspended in sterile 10 mM MgSO4 to the OD600 value of 0.05. The inoculum was then injected into the extracellular spaces of leaves from 3- to 4-week-old plants using a 10-cc syringe. Three leaf discs (6 mm in diameter) from each plant were collected beginning from d 1 of the inoculation and for each of the following 3 to 4 d. The bacterial growth was quantified according to Mohr and Cahill (2003 For sclareol treatments, plants (six- to eight-leaf stage) were floated (roots were immersed into the solution while the abaxial surfaces of the lower leaves were in contact with the solution) on sterile water containing 100 µM sclareol (dissolved in DMSO, Aldrich, Milwaukee, WI) for 24 h before collection of leaves for RNA isolations. Control plants were floated on sterile water containing 0.06% to 0.25% (v/v) DMSO. Control plants were grown in parallel and treated exactly the same way as described above except for the addition of fungal inoculum or chemicals.
For sclareol resistance assays, seeds from wild type and AtPDR12 T-DNA insertion line (40 each) were germinated on petri plates incubated vertically under continuous light at 24°C on one-half-strength B5 medium (Sigma, St. Louis) containing various concentrations of sclareol for 7 d, and root and plant growth were observed daily. Germination assays were also conducted in the presence of (3aR)(+) sclareolide (100 µM; Aldrich Chemicals), indole-3-acetic acid (1 µM; Sigma), abscisic acid (50 µM; mixed isomers, Sigma), SA (100 µM), or rhodamine 6G (1 µM; Sigma). The concentrations of sclareol and sclareolide used in these experiments were in the range of those reported by van den Brûle et al. (2002
Total RNA was extracted by the phenol-LiCl method according to Eggermont et al. (1996
A total of 2,000 plasmid clones from the subtracted cDNA library were collected, and the cDNA inserts were amplified by PCR. For control purposes, cDNA inserts representing negative controls (i.e. not induced after pathogen challenge) and cDNA inserts representing positive controls from the EST collection described by Schenk et al. (2000
Total RNA (500 ng–5 µg) isolated from treated and control plants was denatured at 70°C for 5 min followed by a quick chill on ice in a 13-µL reaction containing 10 ng of anchored oligo(dT) (23 mers), 4.5 ng of random hexamer primers (Invitrogen), and 0.5 µL of 20 mM dNTPs. After the addition of 4 µL of 5x reaction buffer (Invitrogen) and 2 µL of 0.1 M dithiothreitol, the reaction was preheated to 42°C for 2 min before adding 1 µL (200 units) of Superscript II RT (Invitrogen) followed by incubation at 42°C for another 50 min. After terminating the reaction at 70°C for 15 min, the resulting cDNA was subsequently taken up in a volume of 500 µL, and PCR fragments were amplified by using gene-specific primers designed from the coding sequence and over a RNA splice junction of ATPDR12 (5'-CTTTCGCTCAGGTTTTCATCG-3' and 5'-CTTCACCGCCGTCCACTC-3') using the Primer Express 1.5 software (Perkin-Elmer Applied Biosystems, Foster City, CA). The following similarly designed primer pairs were used to detect expression from PDF1.2 (At5g44420, 5'-TTGCTGCTTTCGACGCA-3' and 5'-TGTCCCACTTGGCTTCTCG-3') and from the genes encoding a lectin-like protein (At3g15356, 5'-GTTTCGTCTCTGGGTCATGGA-3' and 5'-GCAGCAACTTGTTATTCCTTGGA-3'), a hevein-like protein (At3g04720, 5'-TGCTACATCCAAATCCAAGCCT-3' and 5'-CGGCAAGTGTTTAAGGGTGAAG-3'), and a basic chitinase (At3g12500, 5'-ATCAGCGCTGCAAAGTCCTTC-3' and 5'-GTGCTGTAGCCCATCCACCTG-3'). RTQ-RT-PCR using the ABI PRISM 7700 Sequence Detector and SYBR Green Master Mix (Perkin-Elmer Applied Biosystems) was carried out using primers at a final concentration of 0.28 µM each and 1 µL of cDNA (the equivalent of 10 ng of total RNA) as template. PCR cycling conditions comprised an initial polymerase activation step at 95°C for 10 min followed by 45 cycles at 95°C for 15 s and 59°C for 1 min. Real-time DNA amplification was monitored and analyzed using the Sequence Detector 1.7 program (Perkin-Elmer Applied Biosystems). 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, each cycle representing a 2-fold change in template abundance. Expression detected from three
All cDNA samples were assayed in duplicate, with cycle threshold (Ct) values generally within 0.5 to 1 cycles of each other. The following statistical analysis of RT-QPCR was performed on experiments replicated more than two times (e.g. AtPDR12 expression in defense signaling mutants). First, the
The location of the T-DNA insertion in the AtPDR12 insertion line (available through Arabidopsis Stock Centre, Ohio State University, Columbus) was originally identified at the Salk Institute Genomic Analysis Laboratory (La Jolla, CA) using the protocol described by Siebert et al. (1995
We thank Iain Wilson for his help in preparation of microarray slides; B. Simpson for advice on RTQ-RT-PCR analysis; Ken Goulter, Peter Mohr, and Roger Shivas for S. sclerotiorum, P. syringae pv tomato, and F. oxysporumstrains, respectively; Peter Mohr, Roger Shivas, and Leanne Forsyth for advice on pathogen inoculations; and the Arabidopsis Stock Centre (Ohio State University, Columbus) for seeds of the AtPDR12 insertion line and mutant lines of Arabidopsis plants. Received March 25, 2003; returned for revision May 18, 2003; accepted July 27, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.024182.
1 This work was supported by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (postdoctoral fellowship to I.A.M.A.P.) and by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen and the Cooperative Research Centre for Tropical Plant Protection (grant to I.A.M.A.P. supporting a research stay at the University of Queensland, Australia). * Corresponding author; e-mail p.schenk{at}tpp.uq.edu.au; fax 61–7–3365–4771.
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ABSTRACT
RESULTS
15 h) in eds5. Consistent with this observation, treatment of the eds5 plants with SA demonstrated AtPDR12 induction, which is similar to the levels normally observed in SA-treated wild-type plants (data not shown). 
-actin genes as described below. 
Ct values were calculated from the average Ct value (PCR cycle no. during exponential amplification) for each sample that was assayed in duplicate, where 
