Transcription Factor CsWRKY65 Participates in the Establishment of Disease Resistance of Citrus Fruits to Penicillium digitatum

Penicillium digitatum is the primary pathogen that causes serious yield losses worldwide. In our previous study, CsWRKY transcription factors (TFs) and some genes associated with immunity were identified in citrus fruits after P. digitatum infection, but little information is available in the literature on the mechanisms of TFs in citrus disease resistance. In this study, the possible mechanisms of CsWRKY65 participating in the establishment of disease resistance were investigated. Results show that CsWRKY65 was a transcriptional activator in the nucleus. The dual-luciferase transient assays and electrophoretic mobility shift assays showed that CsWRKY65 bound with CsRbohB, CsRbohD, CsCDPK33, and CsPR10 promoters to activate gene transcription. Besides, the transient overexpression of CsWRKY65 induced reactive oxygen species accumulation and increased PR gene expression in Nicotiana benthamiana leaves. The transient overexpression of CsWRKY65 in the citrus peel enhanced the disease resistance against P. digitatum. In conclusion, CsWRKY65 is likely to be involved in regulating the disease resistance to P. digitatum of citrus fruits by directly activating the expressions of CsRbohB, CsRbohD, CsCDPK33, and CsPR10. This study provides new information for the mechanism of citrus WRKY TFs participating in the establishment of disease resistance.