Screening for TaXI-Ⅳ Interacting Proteins in Wheat and Validation of Its Interaction with TaHRLI-4D

doi:10.13560/j.cnki.biotech.bull.1985.2025-0993

Abstract

Abstract:

Objective The wheat xylanase inhibitor TaXI-Ⅳ plays an important role in wheat resistance to Fusarium head blight. It can inhibit both the degradation of cell wall xylan and the cell necrosis induced by Fusarium gramineum xylanase. Screening TaXI-Ⅳ-interacting proteins will provide insights into its molecular regulatory mechanisms during wheat defense against F. graminearum infection, as well as support the discovery of disease resistance-related genes in wheat. Method Bioinformatic analysis and subcellular localization were conducted for the TaXI-Ⅳ protein. The bait vector for TaXI-Ⅳ was constructed using homologous recombination and subsequently tested for functionality and self-activation. TaXI‑Ⅳ‑interacting proteins were screened by yeast two-hybrid (Y2H) assay and verified through yeast retransformation, luciferase complementation (LCA), and bimolecular fluorescence complementation (BiFC) assays. RT‑qPCR was performed to analyze the expressions of the corresponding interacting‑protein genes after F. graminearum infection. Furthermore, transgenic Arabidopsis thaliana lines were then generated and inoculated with F. graminearum via leaf injection to assess disease resistance. Result TaXI-Ⅳ was localized to the plasma membrane and/or cell wall. The recombinant bait vector pBT3N-TaXI-Ⅳ showed no auto-activation in the yeast strain NMY51 and produced functional protein, confirming its suitability for cDNA library screening. A total of 23 proteins that interacted with TaXI-Ⅳ were obtained by the split-ubiquitin membrane yeast two-hybrid system. These proteins were involved in diverse biological processes in plants, including transcriptional regulation, stress response, biotic defensive reaction, oxidative stress response. Notably, TaXI‑Ⅳ interacted with the hypersensitive response-like lesion-inducing protein TaHRLI‑4D, as demonstrated in both yeast and plant systems. The expression of TaHRLI-4D was induced by F. graminearum infection, and its overexpression in A. thaliana enhanced plant resistance to the pathogen. Conclusion This study screened 23 positive TaXI-Ⅳ-interacting proteins, including the pathogenesis-related protein TaNLTP-4B (non-specific lipid transfer protein) and the reactive oxygen metabolism-related protein TaTrx-4A (thioredoxin). It was further verified that the HR-like lesion inducing protein gene TaHRLI-4D participates in the wheat defense response against F. graminearum infection.

Key words: wheat, xylanase inhibitor TaXI-Ⅳ, yeast two-hybrid (Y2H), BiFC, HR-like lesion-inducing protein TaHRLI-4D

LIU Yang, BAI Yi-fan, CHEN Wei, ZHOU Jing-jing, CAI Hua, SI Hong-qi. Screening for TaXI-Ⅳ Interacting Proteins in Wheat and Validation of Its Interaction with TaHRLI-4D[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0993.

Figures/Tables 8

References 34

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