R-loop structures (RNA:DNA hybrids) have important functions in many biological processes,including transcriptional regulation and genome instability among diverse organisms.DNA topoisomerase 1 (TOP1),an essential manipulator of DNA topology during RNA transcription and DNA replication processes,can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription.TOP1 is required for plant development,but little is known about its function in preventing co-transcriptional R-loop accumulation in various biological processes in plants.Here we show that knockdown of OsTOP1 strongly affects rice development,causing defects in root architecture and gravitropism,which are the consequences of misregulation of auxin signaling and transporter genes.We found that R-loops are naturally formed at rice auxin-related gene loci,and overaccumulate when OsTOP1 is knocked down or OsTOP1 protein activity is inhibited.OsTOP1 therefore sets the accurate expression levels of auxin-related genes by preventing the overaccumulation of inherent R-loops.Our data reveal R-loops as important factors in polar auxin transport and plant root development,and highlight that OsTOP1 functions as a key to link transcriptional R-loops with plant hormone signaling,provide new insights into transcriptional regulation of hormone signaling in plants.