| Systemic phosphate (Pi) starvation signaling is regulated by the conserved central regulator, transcription factor AtPHRl and its homolous in other plants including rice (OsPHR2). The previous work has demonstrated a negative feedback regulation of the central regulator by SPX1protein (refered as proteins with exclusively SPX domain). The further work also indicates that the interaction between SPX1&2and PHR2inhibits the binding ability of PHR2to its cis-element P1BS (PHR1-binding sequence), but how SPX1&2negatively regulate PHR2function is still unclear. In this study, we explore how SPX1&2negatively control PHR2in vitro.We optimized prokaryotic expression conditions (induce16hours with IPTG concentration1mmol/L and20℃induction temperature) and purified GST-SPX1and GST-SPX2in vitro with constructed SPX1and SPX2fusion GST-Tag prokaryotic expression vector (pGEX-4T-l-SPXl and pGEX-4T-1-SPX2). GST-SPX1and GST-SPX2proteins were used for EMS A (Electrophoretic mobility shift assay). Results from Pull-down experiments showed that P1BS displaced SPX1&2interaction with PHR2in a P1BS-dosage manner only in absence of Pi(-Pi). EMSA experiments showed that SPX1&2reciprocally inhibited PHR2binding to P1BS in a dose-dependent manner, but only in the presence of Pi. In addition, the phosphate (Pi) concentration dosage experiment showed that with Pi concentration increase, the competitive binding ability of SPX1&2to PHR2also increased. Furthermore, this negative effect of+Pi on interaction between SPX1and PHR2is also found by phosphite (Phi), an anolog of Pi but can not be used in metabolism in plants, but not by nitrogen (N) and sulohate (S), suggesting the specific effect of Pi on the interaction between PHR2and SPX1&2. Taken together, this study identified SPX1&2feedback regulate central regulator PHR2by competing its binding with P1BS is dependent on Pi concentration,which help rice accurately respond to phosphate starvation. |
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