gma-miR828a Negatively Regulates Resistance to Tea Leaf Spot Caused by Lasiodiplodia theobromae Through Targeting the CsMYB28-CsRPP13 Module.

Abstract

Leaf spot caused by the fungus Lasiodiplodia theobromae severely affects the quality and production of tea (Camellia sinensis) in plantations across southwestern China. Currently, no effective control measures are available, and the damage to tea leaves is also exacerbated by a lack of understanding regarding the epidemiology of the disease. Previous studies have suggested that gma-miR828a is differentially expressed during L. theobromae infection and may target and cleave the mRNA of CsMYB28. In this study, we characterised CsMYB28 as encoding a transcription factor (TF) that localises to the nucleus, cell membrane, and cytoplasm. This gene was found to be differentially and spatiotemporally expressed in leaf tissues following L. theobromae infection of leaves of the tea plant. Altered CsMYB28 expression, achieved by transient overexpression or stable genetic transformation of Nicotiana benthamiana, or transient silencing using antisense oligonucleotides (AsODN) in the tea plant, indicated that CsMYB28 contributes to resistance against L. theobromae. Using DNA affinity purification sequencing, yeast one-hybrid, and dual-luciferase assays, we also identified that CsMYB28 bound to the AATTAATT motif of CsRPP13, thereby activating the expression of CsRPP13. Additionally, degradome sequencing, β-glucuronidase (GUS) assays, and RNA ligase-mediated rapid amplification of cDNA ends revealed that miR828a cleaved CsMYB28 mRNA, negatively regulating its expression. The results from transient overexpression and stable transformation studies, combined with AsODN-mediated silencing in the tea plant, suggested that miR828a plays a negative regulatory role in modulating the response of the tea plant to L. theobromae infection. This study demonstrates that the miR828a-CsMYB28-CsRPP13 mediates the response of the tea plant to L. theobromae infection.