TMS5-mediated R-loop dynamics regulate thermosensitive fertility conversion in rice

Hybrid rice is vital for global food security, with the thermosensitive genic male sterility (TGMS) locus tms5 used in over 95% of the two-line breeding system. Despite its widespread application, the regulatory mechanism underlying tms5-mediated TGMS remains unclear. This study demonstrates that under high-temperature conditions, weakened tapetum function and delayed microspore mother cell development lead to pollen defects and male sterility, whereas under low-temperature conditions, tapetal function is restored and microspore mother cell development proceeds normally, allowing fertility comparable to wild-type Zhonghua11 (ZH11). We further reveal that the knockout of TMS5, which encodes an RNase enzyme, leads to significant accumulation of R-loops under high-temperature conditions, compromising genomic stability and causing pollen abortion. This R-loop accumulation correlates with fertility conversion, as R-loop levels are elevated in tms5 at high temperatures when plants show male sterility, but return to ZH11-comparable levels at low temperatures when fertility is restored. Overexpression of the R-loop resolving protein Local Lesions 1 (OsLS1) partially restores tms5 fertility, further supporting a direct role of R-loop dynamics in tms5 fertility. Our findings elucidate the cellular and molecular mechanisms of tms5-mediated TGMS and provide a basis for improving two-line hybrid rice breeding.