Novel pristinamycin-responsive expression systems for plant cells

Novel gene regulation systems were designed for plant cells responsive to the streptogramin antibiotic pristinamycin. The pristinamycin-repressible plant gene regulation concept (PIPpOFF) is based on a transcriptional activator (PIT) which consists of the Pip protein, the repressor of the pristinamycin resistance operon of Streptomyces coelicolor, fused to the VP16 transactivation domain of the Herpes simplex virus. PIT mediates pristinamycin-repressible activation of a synthetic plant promoter (P(pPIR)) in tobacco cells consisting of a nine Pip-binding site-containing artificial operator (PIR3) placed upstream of a TATA-box derived from the cauliflower mosaic virus 35S promoter (P(CaMV35S)). Pristinamycin interferes with induction by negatively regulating the DNA-binding capacity of the Pip moiety of PIT. A second, streptogramin-inducible plant gene regulation system (PIPpON) was constructed by combining Pip expression with a plant-specific pristinamycin-inducible promoter (P(pPIRON)). P(pPIRON) consists of a PIR3 module cloned downstream of the strong constitutive plant promoter P(CaMV35S). As in the native Streptomyces configuration, Pip binds to its cognate sequence within P(pPIRON) in the absence of regulating antibiotic and silences the chimeric plant promoter. Upon addition of pristinamycin, Pip is released from the PIR3 operator and full P(CaMV35S)-driven expression of desired plant genes is induced. The PIPpOFF and PIPpON systems performed well in Nicotiana tabacum suspension cultures and promise to provide an attractive extension of existing plant gene regulation technology for basic plant research or biopharmaceutical manufacturing using plant tissue culture.