Creation of thermosensitive male sterility line in rice via a temperature‐sensitive mutation in receptor kinase

In rice agronomy, hybridization is a crucial method to augment crop productivity. The cornerstone of hybrid crop breeding is the utilization of male-sterile lines. Compared to the traditional three-line breeding system, the two-line breeding strategy, which leverages thermo-sensitive genic male sterility (TGMS) and photoperiod-sensitive genic male sterility (PGMS), offers significant benefits by expanding the genetic reservoir available for breeding programs. Currently, two-line hybrid rice occupies 44% of the total hybrid rice cultivation area. However, the availability of TGMS and PGMS germplasm and genetic resources remains severely restricted. Notably, TGMS lines originating from mutations at the tms5 locus account for at least 83.8% of the two-line hybrid rice varieties in China (Zhang et al., 2022). The leucine-rich repeat receptor kinase EMS1 and its ligand, TPD1 peptide, form a critical receptor–ligand complex indispensable for the proper development of the anther tapetum. Mutations in ems1 and tpd1 both result in sterility characterized by an absence of pollen. Previous research has elucidated that EMS1 and the brassinosteroid receptor BRI1 utilize common downstream signalling pathways, allowing for the functional substitution of their kinase domains (Zheng et al., 2019). Among the various BRI1 mutant alleles, bri1-301 is particularly notable due to the G-989-I substitution, which almost completely eliminates kinase activity both in vitro and in vivo; yet surprisingly, it only produces a mild dwarf phenotype compared to more severe or null bri1 alleles (Xu et al., 2008). The phenotypic severity and protein accumulation of bri1-301 are modulated by temperature (Figure 1a), with accelerated degradation occurring at elevated temperatures through an unidentified pathway. At 22°C, bri1-301 protein accumulates normally, whereas at 29°C, its accumulation is markedly compromised (Figure 1b; Figure S1) (Lv et al., 2018; Zhang et al., 2018). Transgenic expression of GFP-tagged bri1-301 retains its sensitivity to high temperatures (Figure 1c). Comparative evaluation of in vitro autophosphorylation activities reveals that bri1-301 loses most of its autophosphorylation capability, and EMS1 demonstrates significantly weaker autophosphorylation activity compared to BRI1 (Figure 1d). Consequently, we attempted to introduce the bri1-301 mutation site into a chimeric EMS1-BRI1 receptor to preserve biological activity while imparting temperature sensitivity (Figure 1e). By employing the EMS1 promoter to drive the expression of the EMS1-BRI1* construct in the ems1 mutant background, we achieved notable phenotypic restoration at 22°C, characterized by the generation of pollen and fertile siliques. Contrarily, this phenotypic amelioration was unattainable at 29°C (Figure 1f,g). Protein quantification assays in floral buds indicated that protein accumulation was hindered at higher temperatures, whereas it was unaffected at lower temperatures (Figure 1h). These findings indicate that transgenic expression of the EMS1-BRI1* chimeric receptor in Arabidopsis can yield temperature-dependent variable fertility plants. In rice, the MSP1 (MULTIPLE SPOROCYTE1) gene encodes a protein homologous to EMS1, and the orthologous gene TDL1A, analogous to arabidopsis TPD1, has been identified and characterized. To confer the temperature-sensitive male sterility trait of EMS1-BRI1* from arabidopsis to rice, we investigated whether the temperature-sensitive mutation site in the arabidopsis bri1-301 protein exhibits a comparable function in rice. By introducing the Atbri1-301 gene into an Osbri1 weak knockout mutant, we observed that Atbri1-301, similar to AtBRI1, can complement the Osbri1 phenotype at 22°C. Conversely, at 29°C, Atbri1-301 fails to complement the rice phenotype, with protein assays indicating significant degradation at elevated temperatures (Figure 1i,j). Examination of the phosphorylation state of transgenic plants' OsBZR1 showed results consistent with the phenotype (Figure S2), indicating that Atbri1-301 retains temperature sensitivity as a functional BR receptor in rice. Utilizing a two-step approach, we generated a complete knockout line of rice MSP1 via gene editing (Figure S3) and subsequently introduced the chimeric mutant gene EMS1-BRI1* from arabidopsis into the MSP1 knockout line under the regulation of the rice MSP1 promoter. As anticipated, the EMS1-BRI1* construct ameliorated the male sterility phenotype of the msp1 mutant at 22°C, exhibiting thermosensitivity by failing to produce seeds at 29°C (Figure 1k,n). EMS1-BRI1*/msp1 plants manifested the same pollen-free induced male sterility phenotype as msp1 under elevated temperatures (Figure 1m,n). Protein quantification in EMS1-BRI1* transgenic plants indicated that protein levels were associated with the fertility phenotype, with reduced expression of tapetum development-related genes at higher temperatures (Figure 1o; Figure S4). Temperature-dependent cultivation demonstrated a progressive decline in fertility across transgenic lines from 22°C to 29°C, with seed set rates positively correlating with protein accumulation (Figure S5). Hybrid recovery assays indicated that the high-temperature sterility phenotype could be fully restored (Figure S6). Comparative analyses of seed set rates under variable temperature regimes revealed significant variances among different transgenic lines (Figure S7), suggesting the feasibility of identifying TGMS lines with lower sterility onset temperatures suitable for agronomic applications within the transgenic population. Collectively, these findings elucidate that a conserved temperature-sensitive mechanism involving the bri1-301 protein can be leveraged to generate temperature-sensitive sterile lines in both Arabidopsis and rice (Figure 1q). The bri1-301 mutation site exhibits high conservation among both dicotyledonous and monocotyledonous taxa. We hypothesized that introducing a similar modification in the rice OsBRI1 gene could replicate the functional characteristics seen in the arabidopsis bri1-301 mutant. To investigate this, we generated an analogous mutation in OsBRI1, termed OsBRI1(G913I). Subsequent in vitro analyses demonstrated a complete loss of autophosphorylation activity. Furthermore, heterologous expression of OsBRI1(G913I) in Atbri1-116 and Osbri1 failed to complement the phenotypic defects, in contrast to Atbri1-301, which retains significant biological functionality (Figure S8). These findings highlight a functional divergence at this mutation site between rice and Arabidopsis. In this investigation, we modulated the tapetum developmental pathway by incorporating a thermosensitive allele serendipitously identified during BRI1 research. This allele demonstrates both thermosensitivity and biological efficacy. By introducing it into the rice msp1 mutant, we achieved the generation of a genetically stable thermosensitive male-sterile line. Our study integrates advanced insights from molecular biology, utilizes precise molecular breeding methodologies and explores novel strategies for the production of thermosensitive male-sterile crop genotypes. This work was supported by the National Natural Science Foundation of China (32100264), Natural Science Basic Research Program of Shaanxi (2023-JC-YB-181 and 2023-JC-QN-0201) and Yan'an University Doctoral Research Initiation Project (YDBK2023-45). The authors declare no conflict of interest. B.W., Q.W., and H.Y. designed the experiments. Q.W., F.H., and J.J. provided technical support. Q.W. performed the data analyses. B.W., A.X., and C.Y. wrote and revised the manuscript. All authors have read and approved the final manuscript. The data that support the findings of this study are available on request from the corresponding author. Figure S1 The bri1-301 protein exhibits biological activity and thermosensitivity. Figure S2 bri1-301 protein is temperature-sensitive and activates the BR signalling pathway by dephosphorylating OsBZR1 in rice. Figure S3 Rice MSP1 gene knockout sequence. Figure S4 EMS1-BRI1* transgenic rice lines show reduced the expression of tapetum development-related genes at high temperature. Figure S5 Seed setting rate of EMS1-BRI1*/msp1 transgenic plants at different temperatures. Figure S6 Temperature-sensitive sterile phenotype restored in F1 hybrids. Figure S7 Phenotypes of multiple transgenic lines of bri1-301 and EMS1-BRI1*. Figure S8 OsBRI1(G913I) mutation loses kinase activity and biological function. 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