Functional characterization of the replication-associated protein (Rep) promoter of an alpha-satellite associated with Ageratum enation virus

Ageratum enation virus (AEV) (Begomovirus agerati), belonging to the genus Begomovirus, is associated with DNA components known as alphasatellite and betasatellites. The alphasatellite encodes the replication-associated protein (alpha-Rep), which is essential for self-replication but relies on helper viruses for transmission between plants. In this study, we propose that the alpha-Rep promoter exhibits bidirectional promoter activity and can drive the expression of heterologous genes in plants. Using Agrobacterium-mediated transient and stable transgenic techniques, we identified the minimal promoter sequence of the Rep gene from the Ageratum enation virus-associated alpha satellite (AEA) (Colecusatellite agerati) that initiates β-glucuronidase (GUS) reporter gene transcription in Nicotiana benthamiana plants. Promoter deletion analysis showed varying promoter activities. Especially, all essential cis-elements for strong promoter activity were located within the 331 nucleotides upstream of the translation start codon, displaying robust and consistent transient GUS expression compared to the control promoter (CaMV35S). The findings lay the groundwork for developing a versatile viral vector system suitable for reverse genetics and genome editing across a broad host range, enhancing our understanding of viral-host interactions and their applications in biotechnology.