Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 292, Issue 47, Pages 19491-19502Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M117.811935
Keywords
ABC transporter; glycosylation; liposome; membrane reconstitution; plant defense; protein purification; single particle analysis; BY-2 cells; Nicotiana tabacum; terpenes
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Funding
- French National Research Agency through the Investments for the Future [ANR-10-INSB-04]
- Plateforme d'Imagerie Cellulaire et Tissulaire - Infrastructures Biologie Sante et Agronomie (PICT-IBiSA) (Institut Curie, Paris, France)
- Belgian Fund for Scientific Research
- Interuniversity Poles of Attraction Program (Belgian State, Scientific, Technical and Cultural Services)
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Within the plant ATP-binding cassette transporter family, pleiotropic drug resistance (PDR) transporters play essential functions, such as in hormone transport or defense against biotic and abiotic stresses. NtPDR1 from Nicotiana tabacum has been shown to be involved in the constitutive defense against pathogens through the secretion of toxic cyclic diterpenes, such as the antimicrobial substrates cembrene and sclareol from the leaf hairs (trichomes). However, direct evidence of an interaction between NtPDR1 and terpenes is lacking. Here, we stably expressed NtPDR1 in N. tabacum BY-2 suspension cells. NtPDR1 was purified as an active monomer glycosylated at a single site in the third external loop. NtPDR1 reconstitution in proteoliposomes stimulated its basal ATPase activity from 21 to 38 nmol of P(i)mg(-1)min(-1), and ATPase activity was further stimulated by the NtPDR1 substrates cembrene and sclareol, providing direct evidence of an interaction between NtPDR1 and its two substrates. Interestingly, NtPDR1 was also stimulated by capsidiol, a sesquiterpene produced by N. tabacum upon pathogen attack. We also monitored the transcriptional activity from the NtPDR1 promoter in situ with a reporter gene and found that, although NtPDR1 expression was limited to trichomes under normal conditions, addition of methyl jasmonate, a biotic stress hormone, induced expression in all leaf tissues. This finding indicated that NtPDR1 is involved not only in constitutive but also in induced plant defenses. In conclusion, we provide direct evidence of an interaction between the NtPDR1 transporter and its substrates and that NtPDR1 transports compounds involved in both constitutive (diterpenes) and induced (sesquiterpenes) plant defenses.
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