Changyan Li, Lei Zhou, Bian Wu, Sanhe Li, Wenjun Zha, Wei Li, Zaihui Zhou, Linfeng Yang, Lei Shi, Yongjun Lin, Aiqing You

Cells; 2022 Aug 16;11(16):2535.  doi: 10.3390/cells11162535.

Abstract

xa13 is a recessive pleiotropic gene that positively regulates rice disease resistance and negatively regulates rice fertility; thus, seriously restricting its rice breeding application. In this study, CRISPR/Cas9 gene-editing technology was used to delete the Xa13 gene promoter partial sequence, including the pathogenic bacteria-inducible expression element. Rice with the edited promoter region lost the ability for pathogen-induced gene expression without affecting background gene expression in leaves and anthers, resulting in disease resistance and normal yield. The study also screened a family of disease-resistant and normal fertile plants in which the target sequence was deleted and the exogenous transgene fragment isolated in the T₁ generation (transgene-free line). Important agronomic traits of the T₂ generation rice were examined. T₂ generation rice with/without exogenous DNA showed no statistical differences compared to the wild type in heading stage, plant height, panicles per plant, panicle length, or seed setting rate in the field. Two important conventional rice varieties, namely Kongyu131 (KY131, Geng/japonica) and Huanghuazhan (HHZ, Xian/indica), were successfully transformed, and disease-resistant and fertile materials were obtained. Currently, these are the two important conventional rice varieties in China that can be used directly for production after improvement. Expression of the Xa13 gene in the leaves of transgenic rice (KY-PD and HHZ-PD) was not induced after pathogen infection, indicating that this method can be used universally and effectively to promote the practical application of xa13, a recessive disease-resistant pleiotropic gene, for rice bacterial blight resistance. Our study on the regulation of gene expression by editing noncoding regions of the genes provides a new idea for the development of molecular design breeding in the future.

See https://pubmed.ncbi.nlm.nih.gov/36010612/

Figure 1. Schematic diagram of promoter editing vector Cas9: P1+P2 and results of the editing of the Xa13 promoter mediated by Cas9: P1+P2 in two conventional rice varieties. (A) Schematic description of the wild-type Xa13 promoter. Two target sites (P1 and P2) were located in the promoter region upstream of the Xa13 CDS region. The 31 bp PXO99-inducible element was located entirely between the two target sites (P1 and P2). (B) PCR products amplified by primers PCX-F/R to detect the results of the Xa13 promoter edited with the CRISPR/Cas9 system in the T₀ generation of KY131. Three transgenic rice materials (KY-PD1-3), which were completely cut between P1 and P2 by Cas9 protein, were obtained. (C) Sequences and chromatograms of the three transgenic plants (KY-PD1-3) after editing. All three transgenic plants (PD1-3) were perfectly connected and repaired after cutting at the two target sites without any base deletion or insertion. (D) PCR products amplified by primers PCX-F/R to detect the results of the Xa13 promoter edited with the CRISPR/Cas9 system in the T₀ generation of HHZ. Four transgenic rice materials (HHZ-PD1-4), which were completely cut between P1 and P2 by the Cas9 protein, were obtained. (E) Sequences and chromatograms of the four transgenic plants (HHZ-PD1-4) after editing. All four transgenic plants (HHZ-PD1-4) were perfectly connected and repaired after cutting at the two target sites without any base deletion or insertion.