Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (23): 4593-4606.doi: 10.3864/j.issn.0578-1752.2024.23.002

• SPECIAL FOCUS: MINING AND UTILIZATION OF CROP DISEASE RESISTANCE AND INSECT-RELATED GENES • Previous Articles     Next Articles

The Mechanism of Cystathionine-β-Synthase OsCBSX4 in Rice Blast Resistance

DIAO ZhiJuan1(), CHEN LiZhe1(), WANG Xun1, LU Ling1, LIU Yan1, ZHANG Jing1, XIA Na1, TANG DingZhong1(), LI ShengPing1,2()   

  1. 1 College of Life Science, Fujian Agriculture and Forestry University/Plant Immunity Center, Fuzhou 350002
    2 School of Future Technology, Fujian Agriculture and Forestry University/Fujian Provincial Key Laboratory of Crop Breeding by Design, Fuzhou 350002
  • Received:2024-02-27 Accepted:2024-04-15 Online:2024-12-01 Published:2024-12-07

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Abstract:

【Objective】 Rice blast is one of the main diseases that threaten rice yield and quality. OsBSK1-2 has been found to be involved in rice blast resistance regulation. Previously, OsCBSX4, a cysteine sulfide-β-Synthase, was identified by screening the proteins that interact with OsBSK1-2. The purpose of this study was to verify the interaction between OsBSK1-2 and OsCBSX4, and clarify the function and molecular mechanism of OsCBSX4 in rice blast resistance, providing a theoretical basis for rice disease resistance breeding.【Method】 Co-immunoprecipitation, bimolecular fluorescence complementation and luciferase complementation assays were used to determine the interaction between OsBSK1-2 and OsCBSX4. Then, quantitative PCR and agrobacterium- mediated transient transformation in N. benthamiana were used to detect the gene expression pattern and protein localization of OsCBSX4. Subsequently, OsCBSX4-knockingout and OsCBSX4-overexpressing plants were generated via CRISPR/Cas9 technology and Agrobacterium-mediated genetic transformation, respectively, and their resistance to rice blast was determined by inoculation with M. oryzae. Moreover, the immune responses induced by chitin and M. oryzae in the oscbsx4 mutant were analyzed using ROS burst and DAB staining assays respectively. In the end, the interaction between OsCBSX4 and OsRbohB was verified via bimolecular fluorescence complementation and split-luciferase complementation imaging assays, and the impact of the metabolite of OsCBSX4 on rice blast resistance was determined using the detached leaf inoculation method. The studies above will reveal the immune function and molecular mechanism of OsCBSX4.【Result】 The interaction between OsCBSX4 and OsBSK1-2 was verified by CoIP, LCI and BiFC assays. Compared to the wild type, the oscbsx4 mutant showed more disease lesions after inoculation with M. oryzae by the spray method, as well as a larger lesion area and greater fungal growth after inoculation with M. oryzae by the punch method, suggesting that knocking out OsCBSX4 decreased rice blast resistance. Moreover, the expression of the pathogenesis-related genes, OsPR5 and OsPR10, and the H2O2 accumulation induced by M. oryzae infection and the ROS burst induced by chitin treatment were reduced in the oscbsx4 mutant. Compared with the wild type, OsCBSX4 overexpressing plants showed a smaller lesion area and less fungal growth after inoculation with M. oryzae by the punch method, suggesting that overexpression of OsCBSX4 increased rice blast resistance. In addition, we found that OsCBSX4 can interact with OsRbohB, a key regulator of rice ROS production and treating rice with L-cysteine, a metabolite of OsCBSX4, does not affect rice blast resistance. 【Conclusion】 OsCBSX4 is an important component of OsBSK1-2 signaling and positively regulates rice blast resistance. OsCBSX4 may mediate ROS production by interacting with OsRbohB, thereby regulating rice immunity.

Key words: rice, cystathionine-β-synthase, OsCBSX4, OsBSK1-2, OsRbohB, rice blast resistance, co-immunoprecipitation, luciferase complementation assay

Fig. 1

Phylogenetic tree analysis of OsCBSX4 and some other CBS proteins in rice and Arabidopsis"

Fig. 2

Determining the interaction of OsCBSX4 and OsBSK1-2 A: BiFC assay. YN, N-terminal of yellow fluorescent protein. YC, C-terminal of yellow fluorescent protein. Bar=10 μm; B: LCA assay. Nluc, N-terminal of luciferase. Cluc, C-terminal of luciferase; C: CoIP assay. Input, control protein. IP, immunoprecipitation"

Fig. 3

Gene expression pattern and protein subcellular localization of OsCBSX4 A: Expression assay of OsCBSX4 in different development tissues using RT-qPCR. B：Expression assay of OsCBSX4 at different times after M. oryzae infection; C: Subcellular localization assay of OsCBSX4-GFP protein in Nicotiana benthamiana leaves. Bar=10 μm; D: Immunoblotting assay of the subcellular localization proteins. ** P<0.01 significantly different. The same as below"

Fig. 4

Determining rice blast resistance and defense responses after M. oryzae infection and chitin treatment of oscbsx4-1 A: The genotype of oscbsx4-1. B: Phenotypes of ZH11 and oscbsx4-1 diseased leaves 3 d after inoculation with M. oryzae by the spray method. Bar=1 cm; C: Lesion number of ZH11 and oscbsx4-1 diseased leaves 3 d after inoculation with M. oryzae by the spray method, n=15; D: Phenotypes of ZH11 and oscbsx4-1 diseased leaves 7 d after inoculation with M. oryzae by the punch method. Bar=1 cm; E and F: Lesion area (E) and relative fungal growth (F) of ZH11 and oscbsx4-1 diseased leaves 7 d after inoculation with M. oryzae by the punch method, n=15; G and H: Expression assay of OsPR5 (G) and OsPR10 (H) in ZH11 and oscbsx4-1 at indicated times after inoculation with M. oryzae by the spray method; I: Chitin induced ROS burst assay in ZH11 and oscbsx4-1; J: H2O2 accumulation assay in M. oryzae inoculated ZH11 and oscbsx4-1 using DAB staining. Bar=1 cm"

Fig. 5

Determining rice blast resistance of oscbsx4-2 A: The genotype of oscbsx4-2. B: Phenotypes of ZH11 and oscbsx4-2 diseased leaves 3 d after inoculation with M. oryzae by the spray method. Bar=1 cm; C: Lesion number of ZH11 and oscbsx4-2 diseased leaves 3 d after inoculation with M. oryzae by the spray method, n=15; D: Phenotypes of ZH11 and oscbsx4-2 diseased leaves 7 d after inoculation with M. oryzae by the punch method. Bar=1 cm; E: Lesion area of ZH11 and oscbsx4-2 diseased leaves 7 d after inoculation with M. oryzae by the punch method, n=15"

Fig. 6

Determining rice blast resistance of OsCBSX4 overexpressing plants A: Detection of OsCBSX4-HA fusion protein in different OsCBSX4 overexpressing lines using immunoblotting; B：Phenotypes of ZH11 and OsCBSX4 overexpressing plants diseased leaves 7 d after inoculation with M. oryzae by the punch method. Bar=1 cm; C: Lesion area of ZH11 and OsCBSX4 overexpressing plants diseased leaves 7 d after inoculation with M. oryzae by the punch method, n=15"

Fig. 7

Determining the interaction between OsCBSX4 and OsRbohB and the affection of L-Cystathionine on rice blast resistance A: BiFC assay. Bar=10 μm; B: LCI assay; C and D: After treating with L-Cystathionine and water for 24 h respectively, the leaves of wild type plant were inoculated with M. oryzae by the punch method. 7 d later, the disease lesions was photographed. Bar=1 cm (C) and lesion area was measured (D), n=15"

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