Plant viral intergenic DNA sequence repeats with transcription enhancing activity

Abstract

Background: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection.

Results: DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components.

Conclusion: Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.

Figure 1

Viral enhancer elements. All viral repeats that produced greater than a 25% increase in 35S min activity are listed. For each active element the accession number, relative enhancement (with standard error), repeat length, repeat separation, source virus (and genus) and viral sequence are shown. Adaptor sequences are listed in the header of the sequence column and with imperfect repeats in bold and partial palindromes within repeats underlined.

Figure 3

Alignment of active repeat elements. Each directly repeated element is offset (at the "/") to align both copies of the repeat. Related elements are additionally aligned as paired repeat alignments. Bases that differ within paired repeats are in lowercase bold and palindromic sub-elements within the repeats are indicated by arrows. Areas of the alignments used to determine a consensus sequence are boxed.

Figure 2

T-DNA map of plasmid 35S min (in pPZP212). T-DNA borders: RB = right border, LB = left border, FiLUC = firefly luciferase, Nos^t = nopaline synthase transcription terminator, PClSV = Peanut chlorotic streak virus promoter, Bar = phosphinothricin acetyl transferase, 35S^t = transcription terminator for the Cauliflower mosaic virus (CaMV) 35S transcript. DNA sequence insert shows the minimal 35S promoter from CaMV, from -46 to +1 (transcription start). Upstream from the minimal 35S promoter are the restriction sites (underlined: HindIII; BamH, overlined: XbaI; KpnI) used to insert test sequences and downstream is the start codon from the luciferase coding region (bold ATG).