Screening of Interacting Proteins for the Loquat Sunburn-Responsive Factor EjWRKY15 and Analysis of Its Regulatory Network

Loquat (Eriobotrya japonica), a significant economic fruit tree in southern China, is highly susceptible to sunburn injury during its ripening stage, which typically coincides with periods of high temperature and intense light. Sunburn manifests as fruit peel browning, tissue necrosis, and flavor deterioration, severely undermining both the marketability and economic value of the fruit. WRKY transcription factors, as plant-specific regulators of stress responses, play pivotal roles in abiotic stresses such as high temperature and drought. This study aims to analyze the function and regulatory mechanism of EjWRKY15 under sunburn stress, providing a theoretical foundation and candidate gene resources for elucidating the sunburn response pathways in loquat and developing sunburn-resistant germplasms.

Centering on the transcription factor EjWRKY15, its interacting proteins were screened and validated to systematically characterize its expression profiles and molecular interaction networks under sunburn stress. A sunburn-induced yeast two-hybrid (Y2H) cDNA library of loquat fruit was constructed using SMART technology. The library capacity, titer, and recombination rate were evaluated through plating selection and sequence analysis. Using EjWRKY15 as the bait protein, potential interactors were screened from the library via the Y2H system. Functional annotation of the screened proteins was performed using NCBI, Swiss-Prot, and other databases, focusing on factors related to heat and stress responses. The physical interaction between EjWRKY15 and the candidate protein EjLHP1.2 (Like Heterochromatin Protein 1) was validated both in vitro and in vivo through one-on-one Y2H assays and bimolecular fluorescence complementation (BiFC). Based on transcriptome data and correlation analysis, a molecular interaction network for loquat sunburn response was constructed with EjWRKY15 as the core node.

A high-quality cDNA library of loquat fruit with a large capacity and high recombination rate was successfully constructed. After excluding background interference via bait self-activation assays, 12 candidate proteins interacting with EjWRKY15 were obtained, with functions involving epigenetic regulation, stress signaling, and substance metabolism. Notably, EjLHP1.2, which is closely associated with sunburn response, was identified as a key interactor. One-on-one Y2H and BiFC assays confirmed that EjWRKY15 and EjLHP1.2 interact physically both in vivo and in vitro. Expression analysis revealed that EjWRKY15 and its 12 candidate interactor genes exhibited significant differential expression patterns under sunburn stress. Based on the interaction data and expression correlations, an EjWRKY15-mediated sunburn response network was preliminarily established. The network suggests that EjWRKY15 may precisely regulate downstream heat-responsive genes by recruiting EjLHP1.2 and other factors.

This study identified the interacting proteins of EjWRKY15 and revealed its expression patterns and molecular interaction network in the sunburn response of loquat. The results elucidate the molecular mechanism by which the core transcription factor EjWRKY15 mediates the sunburn response of loquat fruit through its protein interaction with the epigenetic regulator EjLHP1.2 and the precise modulation of the associated interaction network. These findings reveal a novel stress-regulatory pathway involving the synergetic mode of transcription factor-chromatin remodeling protein in loquat under high temperature and strong light, providing critical theoretical support and gene resources for the molecular breeding of sunburn-resistant loquat varieties.