Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus

The mulberry crinkle leaf virus (MCLV) is a representative member of the genus Mulcrilevirus of the family Geminiviridae . Here, the bidirectional promoter activity of the long intergenic region (LIR) of MCLV and the roles of a specific element consisting of five GAAAAA repeats [(GAAAAA) ₅ ] in LIR were investigated using Agrobacterium-mediated transient expression and transfection of Nicotiana benthamiana protoplasts. Transient expression results demonstrated that, similar to other geminiviruses, MCLV's LIR also exhibits bidirectional promoter activity. The promoter activity of the C-sense is significantly higher in the experimental host, N. benthamiana , while slightly higher than that of the V-sense in the natural host, mulberry ( Morus spp .). However, the promoter activity of LIR is inconsistent between the experimental and natural hosts, especially its C-sense promoter activity, which is slightly lower in the experimental host but higher in the natural host than that of the cauliflower mosaic virus (CaMV) 35S. The (GAAAAA) ₅ element does not affect the activity and type of LIR promoter, but its absence significantly reduces the replication of the MCLV genome in protoplasts. Based on the constructed truncated mutants, a fragment of 232 nts (named P232) lacking (GAAAAA) ₅ was deduced to be the minimal sequence required for sustaining LIR promoter activity. These findings lay the foundation for further research on MCLV and transgenic breeding of mulberry plants.