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

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 BL^ORF5⁺ (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/NJ^ΔORF3+, 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.