Effect analysis of S5-interacting genes on rice hybrid sterility using nontransgenic gamete killer

• Published in: Plant science (Limerick) 2025-03, Vol.352, p.112357, Article 112357
• Main Authors: Li, Jie, Huang, Fu, Jiang, Yingxia, Rao, Jianglei, Fan, Yourong, Yang, Jiangyi
• Format: Article
• Language: English
• Subjects: Endoplasmic reticulum stress; Hybrid sterility; Reproductive isolation; Segregation distortion; Speciation
• ISSN: 0168-9452; 1873-2259; 1873-2259
• DOI: 10.1016/j.plantsci.2024.112357

While hybrids between japonica and indica rice exhibit strong heterosis, they often suffer from hybrid sterility (HS). Hybrid fertility of the embryo sac is predominantly regulated by a three-gene system (comprising closely linked ORF3, ORF4 and ORF5) at rice S5 locus. The cooperation of ORF5+ and ORF4+ can result in endoplasmic reticulum (ER) stress and sporophytically kill all embryo sacs, while ORF3+ can gametophytically protect the residing embryo sac. We previously identified four S5-interacting genes (SIGs) using a transgenic line BLORF5+ (Balilla carrying transgenic ORF5+) and a wide compatibility variety Dular (DL or D). Homozygote and hemizygote of ORF5+ transgene had significantly different spikelet fertility (SF), which disturbed the phenotypic effects of SIGs. However, HS effects of SIGs under the endogenous (nontransgenic) gamete killer remained unknown. We formerly constructed a semisterile near isogenic line (NIL) S5-BL/NJ by introgressing S5 fragment of indica rice Nanjing11 (NJ or N, carrying ORF3+ORF4-ORF5+ haplotypes) into the genome of japonica rice Balilla (BL or B, carrying ORF3-ORF4+ORF5- haplotypes). The gamete-protecting effect of ORF3+ in NJ may confuse SF effect of the SIGs, so we knocked out ORF3+ of S5-NJ/NJ and crossed it with BL to get gamete-killing S5-BL/NJΔORF3+, which can kill all (KA) gametes (abbreviated as enS5KA). Compared with the exS5KA line (a NIL carrying ORF5+ transgenic, wihch can kill all gamete), the enS5KA line conferred SIGs a more pronounced SF effect. The enS5KA,SIG-DDDD (four SIGs carry homozygous DL alleles) genotype caused a SF of about 78 %, while SF of exS5KA,SIG-DDDD was only about 62 %. Moreover, all SIGs acted in a sporophytic manner without segregation distortion of genotype. Although enS5KA,SIG-DDDD plants had high SF, the ER stress still existed. The ovule section revealed that enS5KA,SIG-BBBB genotype (four SIGs carry homozygous BL allele, with ER stress and SF < 5 %) caused abnormal degradation of nucellar cells and functional megaspores. In contrast, enS5KA,SIG-DDDD genotype preserved most nucellar cells and functional megaspores. These results lay the foundation for further research on HS mechanism of S5 and SIGs and cloning of candidate genes. •S5-interacting gene (SIGs) are the female gamete protectors of sporophyte action mode.•SIGs can restore over 75 % of infertility caused by the endogenous S5 gamete killer.•SIGs ensures the normal development of the embryo sac by protecting nucellar c