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 Arabidopsis

Abstract

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.

Figure 1.

Induction ratio (inoculated/mock) of AtPDR12 determined by RTQ-RT-PCR after inoculation of Arabidopsis plants with the incompatible fungal pathogen A. brassicicola (A) and the compatible fungal pathogens Sclerotinia sclerotiorum (B), Fusarium oxysporum (C), and the compatible bacterial pathogen Pseudomonas syringae pv tomato DC3000. Data shown for each time point were obtained from two independently replicated experiments for A. brassicicola. The highest fold induction values (i.e. 105-, 371-, 11-, and 68-fold) observed also were shown.

Figure 2.

Induction ratios (treated to mock) of AtPDR12 determined by RTQ-RT-PCR after treatment of Arabidopsis plants with SA (A), ethylene (B), and MJ (C) compared with untreated control plants. Data for each time point were obtained from at least two independently replicated experiments. The highest fold-induction values (260.6-, 16.3-, and 39.7-fold) observed during the time course were also shown for each treatment. Please note that the y axis in A differs from those in B and C.

Figure 3.

Diurnal changes at the transcript abundance of AtPDR12 determined by RTQ-RT-PCR in Arabidopsis. Data shown for each time point were obtained from two independently replicated experiments.

Figure 4.

The relative transcript abundance of AtPDR12 in various Arabidopsis tissues collected from the plants at flowering and seed setting stage. Black and gray bars, AtPDR12 transcript levels in untreated and SA-treated (aboveground organs) plants, respectively. The relative transcript abundance shown here for each tissue was normalized using actin expression (see “Materials and Methods”). The transcript abundance values calculated based on 25S RNA levels in these tissues also produced similar results (data not shown).

Figure 5.

The average fold induction of AtPDR12 determined by RTQ-RT-PCR in wild-type and defense signaling mutants at 5 and 15 h after inoculation with A. brassicicola. ** and *, Statistically significant differences between the wild-type and mutant expression values of AtPDR12 at P > 0.01 and P > 0.05, respectively (A). The fold induction of AtPDR12 in npr1 and wild-type (Columbia [Col-0]) plants treated for 12 h with SA (B). Relative transcript levels of AtPDR12 measured by RTQ-RT-PCR analysis in leaf tissue of wild type and the cpr5 mutant plants inoculated (In) with A. brassicicola (5 and 15 h after inoculation) compared with mock-inoculated (M) tissue (C). Each bar = average expression value from three to four independently replicated inoculation experiments. **, Significant difference between mock-inoculated wild-type and mock-inoculated cpr5 plants at both 5- and 15-h time points at P > 0.01.

Figure 6.

The induction ratio (treated to untreated) of AtPDR12 and defense-related genes in the plants treated with the diterpenoid compound sclareol in Arabidopsis as determined by RTQ-RT-PCR analysis. Arabidopsis plants were floated on sterile water containing 100 μm sclareol for 24 h before RNA isolations. Control plants were similarly floated on sterile water containing 0.06% (v/v) dimethyl sulfoxide (DMSO). Lectin, gene encoding lectin-like protein; hevein, gene encoding hevein-like protein; B. chitinase, gene encoding basic chitinase.

Figure 7.

Schematic representation of the genomic organization of AtPDR12 (A). The overall predicted structure of AtPDR12 demonstrating the transmembrane domains and the ABC domain, consisting of an ABC signature and Walker A and B motifs (B, adapted from Davies and Coleman, 2000). The confirmed location of the T-DNA insertion in the AtPDR12 insertion line is also indicated in both A and B.

Figure 8.

Effect of sclareol on germination rate (A) and root lengths (B) in wild-type (Col-0) and AtPDR12 insertion line plants (AtPDR12Ins). Germination rates of seeds (40 seeds each) at various sclareol concentrations were scored 3 d after the plates were placed in a germination chamber. The root lengths of the seedlings from wild type and the AtPDR12 insertion line germinated on a medium containing either 50 μm sclareol (50 s) or no sclareol (0 s) were measured 3 to 6 d after the plates were placed into a germination chamber and incubated vertically under continuous diffuse light at 24°C.

Figure 9.

Effect of sclareol on the development of plants from wild-type (Col-0) and the AtPDR12 insertion line at 6 d after the plates were placed into a germination cabinet. The seeds were germinated on plates containing either 0, 100, 200, or 300 μm sclareol and grown vertically under continuous diffuse light at 24°C.