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. 2023 Jul 17:14:1209384.
doi: 10.3389/fpls.2023.1209384. eCollection 2023.

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.)

Jinlian Yang  1 , Yaoyu Fang  1 , Hu Wu  1 , Neng Zhao  1 , Xinying Guo  1 , Enerand Mackon  1 , Haowen Peng  1 , Sheng Huang  2 , Yongqiang He  1 , Baoxiang Qin  1 , Yaoguang Liu  3 , Fang Liu  1 , Shengwu Chen  4 , Rongbai Li  1
Affiliations

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.)

Jinlian Yang et al. Front Plant Sci. .

Abstract

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

Keywords: CRISPR/Cas9; OsSULTR3;6; Pi21; bacterial leaf streak; rice; rice blast.

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Conflict of interest statement

Author SC was employed by the company Guangxi Lvhai Seed Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
CRISPR/Cas9-mediated targeted mutagenesis of Pi21 and OsSULTR3;6-EBE. (A) Target sites of CRISPR/Cas9. One target was chosen in the in the second exon of the Pi21 gene; another target was chosen around effector-binding element (EBE) in the promoter region of OsSULRT3;6 gene; the PAM sequences were marked in red. (B) The expression CRISPR/Cas9 vector. OsU6a and OsU6b, rice promoter; HPT, hygromycin; NLS, nuclear localization signal; Tons, the terminator; LB and RB, left border and right border, respectively.
Figure 2
Figure 2
Enhanced blast resistance of the single Pi21 mutant lines. (A) The percentage of lesion areas of rice blast (n = 3 leaves). (B) Rice mutant lines and wild-type 58B were tested for resistance to M. oryzae at the seedling stage. (C) The resistance to M. oryzae rice mutant lines and wild-type 58B was tested at rice reproductive stage. t-test: **P < 0.01.
Figure 3
Figure 3
Enhanced bacterial leaf streak resistance of the single OsSULTR3;6-EBE mutant lines. (A) The percentage of lesion length of bacterial leaf streak (n = 3 leaves). (B) Rice mutant lines and wild-type 58B were tested for resistance to Xoc at the tillering stage. t-test: **P < 0.01.
Figure 4
Figure 4
pi21/Ossultr3;6-EBE double mutation plants with different editing types (A) The mutated sequences of Pi21 and OsSULTR3;6. The number of base deletion and insertion is shown by the mark of minus (−) and plus (+). (B) Amino acid variations of the Pi21 protein in the mutant. The red line indicates the missing protein. *indicates the termination of translation.
Figure 5
Figure 5
Bacterial leaf streak and rice blast resistance were enhanced in mutants. (A) The percentage of lesion areas of rice blast. (B) Rice mutant lines and wild-type 58B were tested for resistance to M. oryzae at the seedling stage. (C) Rice mutant lines and wild-type 58B were tested for resistance to M. oryzae at the mature stage. (D) The percentage of lesion length of bacterial leaf streak. (E) Rice mutant lines and wild-type 58B were tested for resistance to Xoc at the tillering stage. t-test: **P < 0.01.
Figure 6
Figure 6
Expression analysis of pi21, Ossultr3;6 genes and defense responsive genes. (A) Relative expression of pi21. (B) Relative expression of ossultr3;6. (C) Relative expression level of defense responsive genes after inoculation of rice blast. t-test: *P < 0.05 and **P < 0.01.
Figure 7
Figure 7
The agronomic traits of the mutants and the wild type (WT). (A) Phenotypes of pi21/Ossultr3;6-EBE mutant lines. (B) Plant height. (C) Effective spike number. (D) Panicle length. (E) Grain number per panicle. (F) 1,000-grain weight.
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Grants and funding

This study was supported by the State Key Laboratory for the Conservation and Utilization of Subtropical Agro bioresources (No. SKLWSA‐a201914 and No. SKLCUSA-b202203). This research was funded by the Guangxi Zhuang Autonomous Region Science and Technology Department, grant numbers AA17204070, AB16380066, and AB16380093.