Independent modulation of individual genomic component transcription and a cis-acting element related to high transcriptional activity in a multipartite DNA virus

Abstract

Background: The genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear.

Results: To understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, -M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR.

Conclusions: Our data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

Keywords: Banana bunchy top virus; Cis-acting element; Independent modulation; Multipartite DNA virus; Transcription.

Fig. 1

Plots of qPCR standard curves for each BBTV component. a The qPCR amplification curve of each component of BBTV genome. The X axis represents the number of cycles, while the Y axis represents the dRn value of fluorescence. b The melt curve of each component of BBTV genome. The X axis represents the temperature value, while Y axis represents the -R’(T) value of fluorescence. c The standard curves of DNA-R, DNA-U3, DNA-S, DNA-M, DNA-C, DNA-N, S2 and Sat4 were plotted. The X axis represents the number of DNA copies, while the Y axis represents the Ct value

Fig. 2

The quantitative analysis of DNA level and RNA level of BBTV genomic components. DNA content of BBTV each genomic components comparing with DNA-R from qPCR in B2 (a) and B4 (b) samples. The X axis represents the name of component, while Y axis represents the copy numbers. RNA content of BBTV each genomic components comparing with DNA-R from qRT-PCR and RNAseq in B2 (c) and B4 (d) samples. The X axis represents the name of component, while Y axis represents the copy numbers. P<0.01 was considered as extremely significantly different

Fig. 3

The diagram and sequence analysis of intergenic regions of BBTV each component. a The diagram of intergenic regions of BBTV each component; b Sequence aligned of major common regions (CR-M) of BBTV each component; c Sequence aligned of stem-loop common regions (CR-SL) of BBTV each component. TATTATTAC: Step-loop of highly conserved nine nucleotide sequences; ‘CGGGA’: Five nt repeat sequences; Reverse complement sequences between AGCGCTGGGG and CCCCAGCGCT or between AGCACGGGGG and CCCCCGTGCT; and the mutated ‘CAGGA’ sequences from ‘CGGGA’

Fig. 4

The transcriptional activity of DNA-S, −M and -N promoters by fusing with GFP. a The diagram of recombinant plasmids construction. GV1300, GV1300-S-pro, GV1300-M-pro, GV1300-N-pro. b The relative RNA amount of GFP transcripts transcribed by the different promoter sequences of DNA-S, −M and -N, respectively. P<0.01 was considered as extremely significantly different. c The green fluorescence was observed by a confocal microscopy. Scale bar, 20 μm