NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense

Abstract

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

Figure 1.

NpPDR1 is expressed in the leaf trichomes, the root, and the petals. A, Microsomal fractions were extracted from the whole leaf (L) or leaf epidermis (E) of N. plumbaginifolia rosette or flower-stalk leaves at different developmental stages or of the indicated size (width [cm]/length [cm]) and subjected to western blotting using anti-NpPDR1 or anti-H⁺-ATPase antibodies. Five to ten leaves were pooled per stage. The apparent molecular masses of the bands are indicated on the right. B and C, The epidermis was peeled from mature N. plumbaginifolia stalk leaves and prepared for in situ immunolocalization (see "Materials and Methods") using anti-NpPDR1 (B) or anti-H⁺-ATPase antibodies (C). D, The epidermis of a transgenic N. tabacum plant expressing NpPDR1-gusA was peeled off and processed for histochemical analysis; GUS-positive structures are stained blue. Bars = 20 μm. E, Microsomal fractions extracted from the indicated root sections of a flowering plant grown in the soil (left section) or from the whole root of a plant grown in vitro in Murashige and Skoog solid medium, soil, or hydroponic culture (right section) were submitted to western blotting using the indicated antibodies. The apparent molecular masses of the bands are indicated on the right. F, Roots of a transgenic N. tabacum plant expressing the NpPDR1-gusA construct were processed for histochemical analysis. The left section shows a secondary root emerging from a primary root. The middle section shows absence of GUS expression in the tip. GUS-positive structures are stained blue. The right section shows a root cross section after GUS staining. G, Microsomal fractions were extracted from the indicated flower organs and submitted to western blotting using the indicated antibodies. The petal top (colored) and bottom parts were separated and individually tested.

Figure 2.

NpPDR1 expression in the leaf following bacterial infection. A, Mature stalk leaves from a flowering N. plumbaginifolia plant were infiltrated through the stomata with the indicated Pseudomonas strain. After the indicated period of time, the infiltration site (lanes marked 1), a 5 mm zone surrounding the infiltration site (lanes marked 2), or a zone remote from the site (lanes marked 3) was used to prepare a microsomal fraction, which was used for western blotting using the indicated antibodies. B, Mature leaves of a transgenic plant expressing NpPDR1-gusA were left untreated (−) or infiltrated (+) with P. syringae pv tabaci LMG 5393, then, after 52 h, a leaf section remote from the infiltration site was examined by GUS histochemical analysis. C, Total RNA extracted from whole leaves treated as in A (28 h for P. syringae pv syringae and P. syringae pv tabaci and 52 h for P. fluorescens and P. marginalis) was subjected to RT-PCR analysis using primers specific for the indicated genes. D, Mature leaves of a wild-type plant were infiltrated with water (H₂O), 800 μm JA, 1 mm SA, or 400 μm amino cyclopropane carboxylic acid (ACC), an ethylene precursor, then, after 24 h, the infiltrated zones were used to prepare a microsomal fraction which was used for western blotting using the indicated antibodies.

Figure 3.

NpPDR1 silencing by RNA interference. A, Microsomal fractions prepared from the leaf epidermis (L_E) or from a leaf section 16 h after sclareol infiltration (L_Scl) of wild-type or NpPDR1-silenced plants were subjected to western blotting using the indicated antibodies. wt, Wild-type; 3, 6, 8, 9, PDR1-silenced lines. B, RNA was prepared from a leaf section 96 h after P. syringae pv tabaci infiltration and subjected to northern-blot analysis using primers specific for the indicated genes. C, Microsomal fractions prepared from the samples analyzed in B were subjected to western blotting using the indicated antibodies.

Figure 4.

NpPDR1-silenced cells display increased susceptibility to sclareol. A, Protoplasts were isolated from stalk leaves of wild-type and NpPDR1-silenced (PDR1-s) plants and incubated for 9 h with the indicated sclareol concentrations, then protoplast viability was examined by fluorescence microscopy using fluorescein diacetate. The figure shows representative pictures. Bars = 10 μm. B, Group data for four independent experiments examining protoplast viability for each treatment. Protoplast viability is expressed as the percentage (mean ± standard error) of fluorescent protoplasts compared to the total number of protoplasts.

Figure 5.

Spontaneous infection of NpPDR1-silenced plants by B. cinerea. A, A silenced plant (PDR1-s) spontaneously infected by B. cinerea is shown. The frame shows the region enlarged in B. C, An age-matched wild-type (wt) plant is shown for comparison. D, Quantitative data showing for four silenced lines the number of plants that spontaneously became infected and died before seed production. For each of the three experiments (numbered 1, 2, and 3), six plants of the wild-type and the indicated lines were grown in soil pots in a growth chamber.

Figure 6.

NpPDR1-silenced plants are more susceptible to B. cinerea infection. A, Microsomal fractions extracted from a leaf disk from an NpPDR1-silenced plant incubated for 10 d with or without B. cinerea (10⁶ spores/mL) were subjected to western blotting using the indicated antibodies. The apparent masses of the bands are indicated on the right. B, Sixteen leaf disks were cut from 2-month-old in vitro wild-type (wt) and NpPDR1-silenced (PDR1-s) plants, and infected in vitro by spotting with 5 μL of the indicated concentration of B. cinerea spores. Leaf disks were observed after 14 d. This experiment was performed three times. C, After 2 weeks of acclimation to the compost substrate, 14 wild-type (wt) and 14 NpPDR1-silenced (PDR1-s) plantlets were infected with 1 mL of B. cinerea spores (10³/mL) and observed after 14 d.