Novel pristinamycin-responsive expression systems for plant cells

Novel gene regulation systems were designed for plant cells responsive to t he streptogramin antibiotic pristinamycin. The pristinamycin-repressible pl ant gene regulation concept (PIPpOFF) is based on a transcriptional activat or (PIT) which consists of the Pip protein, the repressor of the pristinamy cin resistance operon of Streptomyces coelicolor fused to the VP16 transact ivation domain of the Herpes simplex virus. PIT mediates pristinamycin-repr essible 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 35 S promoter (P-CaMV35S). Pristinamycin interferes with induction by negative ly regulating the DNA-binding capacity of the Pip moiety of PIT. A second, streptogramin-inducible plant gene regulation system (PIPpON) was construct ed by combining Pip expression with a plant-specific pristinamycin-inducibl e promoter (P-pPIRON). P-pPIRON consists of a PIR3 module cloned downstream of the strong constitutive plant promoter P-CaMV35S. As in the native Stre ptomyces configuration, Pip binds to its cognate sequence within P-pPIRON i n the absence of regulating antibiotic and silences the chimeric plant prom oter. Upon addition of pristinamycin, Pip is released from the PIR3 operato r and full P-CaMV35S- driven expression of desired plant genes is induced. The PIPpOFF and PIPpON systems performed well in Nicotiana tabacum suspensi on cultures and promise to provide an attractive extension of existing plan t gene regulation technology for basic plant research or biopharmaceutical manufacturing using plant tissue culture. (C) John Wiley & Sons, Inc.