Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (6): 1043-1051.doi: 10.3864/j.issn.0578-1752.2025.06.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Screening and Identification of Pigm-1 Interaction Proteins for Disease Resistance of Rice Blast

JIN YiDan1,2(), HE NiQing1(), CHENG ZhaoPing1, LIN ShaoJun1, HUANG FengHuang1, BAI KangCheng1,2, ZHANG Tao1,2, WANG WenXiao1,2, YU MinXiang1, YANG DeWei1()   

  1. 1 Institute of Rice, Fujian Academy of Agricultural Sciences, Fuzhou 350018
    2 College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2024-12-26 Accepted:2025-01-20 Online:2025-03-25 Published:2025-03-25
  • Contact: YANG DeWei

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Abstract:

【Objective】 Rice blast is one of the most devastating diseases of rice production. A broad-spectrum disease resistance gene Pigm-1 was identified but its functional pathway and interactors are unknown. The screening and identification of key proteins in the Pigm-1 signaling pathway will provide an important theoretical basis for rice disease resistance breeding. 【Method】 In this study, the decoy protein pGBKT7-Pigm-1-CC1-576 vector was constructed to detect the decoy protein self-activation, and the toxicity of the decoy protein was detected by separately transforming the plasmid pGBKT7 and pGBKT7-Pigm-1-CC1-576 into Y2H Gold yeast. The rice disease resistance R protein Pigm-1 was screened by cDNA expression yeast library induced by rice blast fungus. The sequencing results were compared and annotated by Rice Information GateWay (RIGW). The interaction of OsbHLH148 protein was verified by Luc, Co-IP and yeast two-hybrid assays, and the tissue expression of the corresponding gene of the interaction protein OsbHLH148 was analyzed by qRT-PCR. 【Result】 The self-activation test showed that the decoy protein pGBKT7-Pigm-1-CC1-576 did not self-activate when cotransformed with the AD plasmid, and the toxicity analysis showed that the decoy protein had little or no toxicity to yeast cells. A total of 124 proteins that may interact with Pigm-1 were obtained by screening the yeast library, and among these proteins, there are ethylene synthesis related, gibberellin synthesis related, active oxygen species clearly related, enzyme metabolism related, and some function unknown. The interaction between Pigm-1-CC1-576 and OsbHLH148 was verified by Luc, Co-IP and yeast two-hybrid methods. Further analysis showed that OsbHLH148 can be induced by blast fungus infection, and the tissue expression analysis showed that OsbHLH148 expression level was the highest in rice leaves at 6 weeks. 【Conclusion】 In this study, 124 proteins that may interact with Pigm-1 were obtained. One of these proteins, OsbHLH148, was selected and verified to interact with Pigm-1-CC1-576. Suggesting that OsbHLH148 may be involved in Pigm-1 mediated resistance of rice blast.

Key words: rice, rice blast, Pigm-1, interacting protein, OsbHLH148

Fig. 1

Auto-activativity detection and toxicity detection of decoy proteins pGBKT7-Pigm-1-CC1-576 a: Auto-activativity detection of the bait protein; b: Toxicity detection of the bait protein"

Fig. 2

Detection of quality detection and insert fragment size a: Library quality detection; b: Insert fragment size detection. M: Marker; 1-24: Colony PCR product"

Fig. 3

Screening of Y2H library and Verification of partial Y2H clones a: Screening of Y2H library; b: Validation of partially positive clones. M: Marker; 1-17: Colony PCR product"

Fig. 4

Verification of interaction between OsbHLH148 and Pigm-1 a: Yeast two-hybrid assay; b: Luc assay; c: Co-IP assay"

Fig. 5

The spatiotemporal expression pattern of OsbHLH148"

Fig. 6

The expression analysis of OsbHLH148 after M. oryzae infection"

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