Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1-104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*

Virus isolate G6 was obtained from Hibiscus rosa-sinensis showing yellow and leaf curl symptoms in Guangzhou, Guangdong Province. The complete nucleotide sequence of DNA-A was determined to be 2 737 nucleotides encoding six potential ORFs. Comparison showed that G6 DNA-A had more than 89% sequence identify with all isolates of Cotton leaf curl Multan virus (CLCuMV) and shared the highest sequence identify (96.1%) with CLCuMV isolate 62. G6 DNA-A had 87.1%-89.8% sequence identity with those of CLCuRV isolates, while less than 87% identities with other begomoviruses. Phylogenetic analysis of G6 DNA-A and selected begomoviruses showed that G6 was most closely related to CLCuMV isolates, and they clustered together as a separate branch. Satellite DNA molecule (G6 DNAbeta) was found to be associated with G6 using the primers beta01 and beta02. G6 DNAbeta contains 1346 nucleotides, with a potential functional ORF (C1) in complementary sense DNA. Pairwise comparison indicated that G6 DNAbeta had the highest sequence identities with CLCuMV DNAbeta (92.1%) and CLCuRV DNAbeta (88.7%), but less than 80% sequence identities with other reported satellite DNA molecules. Phylogenetic analysis indicated that G6 DNAbeta was most closely related to CLCuMV DNAbeta and the two DNAbetas clustered together as a separate branch, and formed the main branch with DNAbeta of CLCuRV and MYVV-Y47. It is concluded that G6 infecting Hibiscus rosa-sinensis is an isolate of CLCuMV.
Base Sequence ; DNA, Satellite ; chemistry ; DNA, Viral ; chemistry ; Geminiviridae ; classification ; genetics ; Gossypium ; virology ; Hibiscus ; virology ; Phylogeny

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAbeta) is required to induce typical symptoms in host plants. DNAbeta encodes a single gene (termed betaC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing the betaC1 gene of a DNAbeta associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing betaC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the betaC1 protein showed that the betaC1 protein accumulated in the nuclei of cells. The possible biological function of the betaC1 protein was discussed.
Cell Division ; physiology ; Cell Nucleus ; genetics ; metabolism ; ultrastructure ; Cells, Cultured ; DNA, Viral ; genetics ; Geminiviridae ; genetics ; Plant Diseases ; genetics ; virology ; Plant Leaves ; cytology ; genetics ; growth & development ; metabolism ; Plants, Genetically Modified ; growth & development ; metabolism ; Recombinant Proteins ; metabolism ; Tobacco ; cytology ; growth & development ; metabolism ; ultrastructure ; Viral Proteins ; genetics ; metabolism