Tea Plant miR5368 ‐p5 Negatively Regulates Resistance Against Lasiodiplodia theobromae Through Targeting the CsWRKY57 ‐ CsLRR ‐ RLK Module

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Lasiodiplodia theobromae can cause severe diseases, including leaf spot, leaf necrosis and stem canker in tea plants, leading to substantial losses in both tea leaf production and quality. However, the mechanisms underlying host resistance remain poorly understood. In this study, we identified CsWRKY57 as a nucleus-localised transcription factor whose expression dynamically responds to L. theobromae infection. Transient overexpression and antisense oligonucleotide (AsODN)-mediated silencing in tea leaves, along with stable overexpression in transgenic Nicotiana benthamiana leaves, demonstrated that CsWRKY57 enhanced resistance to L. theobromae. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that CsWRKY57 interacts with CsTIFY5A in the nucleus, further confirming that CsTIFY5A negatively regulates disease resistance through transient overexpression and AsODN-mediated silencing in tea leaves. DNA affinity purification sequencing, electrophoretic mobility shift assay and dual-luciferase assay indicated that CsWRKY57 binds to the AGTCAA motif in the CsLRR-RLK promoter, thereby activating its expression. Transient overexpression and AsODN-mediated silencing assays in tea leaves demonstrated that CsLRR-RLK positively regulates resistance to L. theobromae. Additionally, degradome sequencing, β-glucuronidase and dual-luciferase assays revealed that miR5368-p5 cleaves CsWRKY57 mRNA. Transient overexpression and AsODN assays in tea leaves, as well as stable overexpression of miR5368-p5 in transgenic N. benthamiana, indicated that miR5368-p5 negatively regulates resistance to L. theobromae. Our results suggest that the miR5368-p5-CsWRKY57-CsLRR-RLK module, which also includes CsTIFY5A interacting with CsWRKY57, plays a critical role in regulating the defence response of tea plants to L. theobromae. The results provide valuable insights into the mechanisms governing the response of tea plants to L. theobromae infection.

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