V2 Protein Enhances the Replication of Genomic DNA of Mulberry Crinkle Leaf Virus

Mulberry crinkle leaf virus (MCLV), identified in mulberry plants (Morus alba L.), is a member of the genus Mulcrilevirus in the family Geminiviridae. The functions of the V2 protein encoded by MCLV remain unclear. Here, Agrobacterium-mediated infectious clones of a wild-type MCLV vII (MCLV^WT) and two V2 mutant MCLV vIIs, including MCLV^mV2 (with a mutation of the start codon of the V2 ORF) and MCLV^dV2 (5'-end partial deletion of the V2 ORF sequence), were constructed to investigate the roles of V2 both in planta and at the cellular level. Although all three constructs (pCA-1.1MCLV^WT, pCA-MCLV^mV2, and pCA-MCLV^dV2) were able to infect both natural host mulberry plants and experimental tomato plants systematically, the replication of the MCLV^mV2 and MCLV^dV2 genomes in these hosts was significantly reduced compared to that of MCLV^WT. Similarly, the accumulation of MCLV^mV2 and MCLV^dV2 in protoplasts of Nicotiana benthamiana plants was significantly lower than that of MCLV^WT either 24 h or 48 h post-transfection. A complementation experiment further confirmed that the decreased accumulation of MCLV in the protoplasts was due to the absence of V2 expression. These results revealed that MCLV-encoded V2 greatly enhances the level of MCLV DNA accumulation and is designated the replication enhancer protein of MCLV.