Diversification of plant agronomic traits by genome editing of brassinosteroid signaling family genes in rice.

Brassinosteroids (BRs) regulate various agronomic traits such as plant height, leaf angle, and grain size in rice (Oryza sativa L.); thus, BR signaling components are promising targets for molecular rational design. However, genetic materials for BR-signaling genes or family members remain limited in rice. Here, by genome editing using clustered regularly interspaced short palindromic repeats (CRSPR)/Cas9 tools, we generated a panel of single, double, triple, or quadruple mutants within three BR signaling gene families, including GSK3/SHAGGY-LIKE KINASE1 (GSK1)-GSK4, BRASSINAZOLE-RESISTANT1 (OsBZR1)-OsBZR4, and protein phosphatases with kelch-like (PPKL)1-PPKL3, under the same background (Zhonghua11, japonica). The high-order mutants were produced by either simultaneously targeting multiple sites on different genes of one family (GSKs and PPKLs) or targeting the overlapping sequences of family members (OsBZRs). The mutants exhibited a diversity of plant height, leaf angle, and grain morphology. Comparison analysis of the phenotypes together with BR sensitivity tests suggested the existence of functional redundancy, differentiation, or dominancy among the members within each family. In addition, we generated a set of transgenic plants overexpressing GSK2, OsBZR1/2, and PPKL2, respectively, in wild-type or activated forms with fusion of different tags, and also verified the protein response to BR application. Collectively, these plants greatly enriched the diversity of important agronomic traits in rice. We propose that editing of BR-related family genes could be a feasible approach for screening of desired plants to meet different requirements. Release of these materials as well as the related information also provides valuable resources for further BR research and utilization.

The divergence of brassinosteroid sensitivity between rice subspecies involves natural variation conferring altered internal auto-binding of OsBSK2.

Japonica/geng and indica/xian are two major rice (Oryza sativa) subspecies with multiple divergent traits, but how these traits are related and interact within each subspecies remains elusive. Brassinosteroids (BRs) are a class of steroid phytohormones that modulate many important agronomic traits in rice. Here, using different physiological assays, we revealed that japonica rice exhibits an overall lower BR sensitivity than indica. Extensive screening of BR signaling genes led to the identification of a set of genes distributed throughout the primary BR signaling pathway with divergent polymorphisms. Among these, we demonstrate that the C38/T variant in BR Signaling Kinase2 (OsBSK2), causing the amino acid change P13L, plays a central role in mediating differential BR signaling in japonica and indica rice. OsBSK2L13 in indica plays a greater role in BR signaling than OsBSK2P13 in japonica by affecting the auto-binding and protein accumulation of OsBSK2. Finally, we determined that OsBSK2 is involved in a number of divergent traits in japonica relative to indica rice, including grain shape, tiller number, cold adaptation, and nitrogen-use efficiency. Our study suggests that the natural variation in OsBSK2 plays a key role in the divergence of BR signaling, which underlies multiple divergent traits between japonica and indica.

Regulation of Brassinosteroid Signaling and Salt Resistance by SERK2 and Potential Utilization for Crop Improvement in Rice.

The complex roles of the steroid hormone brassinosteroids (BRs) in many different yield- and stress-related traits make it difficult to utilize the hormones for crop improvement. Here, we show that SERK2 as a BR signaling component is a potentially useful candidate for BR manipulation in rice. We generated multiple mutant alleles of SERK2 by CRISPR/Cas9 editing and show that knockout of SERK2 results in a compact structure accompanied with increased grain size. SERK2 is localized on plasma membrane and can interact with OsBRI1, the BR receptor, suggesting its conserved role as co-receptor in BR signaling. Consistently, the mutant has impaired BR sensitivity compared to wild type. Notably, the mutant is highly sensitive to salt stress as evaluated by plant survival rate as well as transcriptome analysis, whereas has slightly increased sensitivity to ABA, the stress hormone. By contrast, overexpression of SERK2 significantly enhances grain size and salt stress resistance, importantly, without affecting plant architecture. Furthermore, while salt suppresses SERK2 transcription, the protein is greatly induced by salt stress. Taken together, we propose that the adverse condition induces SERK2 accumulation to enhance early BR signaling on plasma membrane in favor of the anti-stress response. Our results illustrate the great potentials of specific BR components such as SERK2 for crop improvement by utilizing flexible strategies.