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) 35 S. 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.