Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system

Thao Phuong Bui; Huy Le; Dong Thi Ta; Cuong Xuan Nguyen; Ngoc Thu Le; Truong Thi Tran; Phuong Van Nguyen; Gary Stacey; Minviluz G Stacey; Ngoc Bich Pham; Ha Hoang Chu; Phat Tien Do

doi:10.1186/s12870-023-04549-5

Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system

BMC Plant Biol. 2023 Nov 3;23(1):533. doi: 10.1186/s12870-023-04549-5.

Authors

Thao Phuong Bui^{1

2}, Huy Le^{1

3}, Dong Thi Ta¹, Cuong Xuan Nguyen^{1

4}, Ngoc Thu Le¹, Truong Thi Tran⁵, Phuong Van Nguyen², Gary Stacey⁴, Minviluz G Stacey⁴, Ngoc Bich Pham^{1

6}, Ha Hoang Chu^{1

6}, Phat Tien Do^{7

8}

Affiliations

¹ Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam.
² University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, Hanoi, Vietnam.
³ Present address: Department of Biology, Washington University in St. Louis, St. Louis, USA.
⁴ Division of Plant Sciences, University of Missouri, Columbia, MO, 65211, USA.
⁵ Legumes Research and Development Center, Vietnam Academy of Agriculture Science, Hanoi, Vietnam.
⁶ Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam.
⁷ Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam. dtphat@ibt.ac.vn.
⁸ Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam. dtphat@ibt.ac.vn.

Abstract

Background: Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance.

Results: A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants.

Conclusions: Our results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Keywords: CRISPR/Cas9; GmMLO genes; Powdery mildew resistance; Soybean; Targeted mutation.

MeSH terms

CRISPR-Cas Systems*
Disease Resistance / genetics
Fungi
Glycine max* / genetics
Mutation
Plant Breeding
Plant Diseases / genetics
Plant Diseases / microbiology
RNA, Guide, CRISPR-Cas Systems

Substances

RNA, Guide, CRISPR-Cas Systems