Plant viral intergenic DNA sequence repeats with transcription enhancing activity

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.


Background
Traditionally, analyses of viral promoter structure-function relationship have involved directed deletion or disruption of promoter structure, followed by determination of resulting changes in transcription, if any, resulting from the alterations [1]. A relatively small subset of the promoter elements identified in this way have been subsequently isolated and tested for their ability to influence transcription when inserted into alternative, well defined, basal promoters [2]. As an alternative to so-called 'promoter bashing' approaches to the study of promoter structure, we have instead chosen to 'mine' specific regions of viral DNA for sequence elements that, when combined with a minimal plant promoter, are able to enhance transcription of a reporter gene in planta.
To test the enhancer mining approach we chose to examine a collection of geminivirus and nanovirus intergenic sequences obtained from GenBank. There are a relatively large number of available sequences for these DNA viruses and due to conserved genomic organization they contain easily identifiable intergenic regions [3]. Additionally, several studies have demonstrated in planta promoter activity using isolated or modified geminivirus or nanovirus intergenic sequences [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Although some areas of sequence similarity exist within the intergenic regions of the geminiviruses [22], very few of these common sequence elements have been experimentally shown to contribute to transcriptional activity. We specifically avoided using any test for evolutionary conservation of candidate elements, hoping to identify unique elements that may not necessarily be shared by large groups of related viruses. For this first broad screen, the experimental rational used made two basic assumptions; 1} that viral intergenic regions contain an enrichment of DNA transcriptional regulatory elements; and 2} that important regulatory sequence elements are often duplicated within promoters, either directly repeated, or as inverted copies of sequence segments [22].
The described enhancer mining of viral sequences is not intended to be a comprehensive analysis of viral promoter structure since by design it is limited to identification of promoter elements that up-regulate gene expression and that make use of endogenous plant transcription factors available within the un-infected test plant. However, based upon their iteration, location within intergenic regions, and ability to enhance transcription in planta, any elements identified using this approach are likely to contribute to regulation of in vivo viral gene expression during plant infection. By allowing relatively large numbers of viral sequences to be examined using a defined system, the approach has the potential of generating data useful in comparing positively acting viral promoter elements within and between viral families. In addition, identifica-tion of elements that are active in planta in the absence of viral infection provides results pertinent to understanding virus-host interactions at the level of gene control. Finally, the resulting list of active and inactive viral sequences provides a valuable starting points for subsequent, more detailed, analysis of transcription regulation of individual viruses.

Search for candidate elements
The initial search for sequence repeats was performed on the major intergenic regions of 29 different geminivirus or nanovirus genomic sequences (Figure 1 and Additional file 1). The search was arbitrarily halted after 105 candidate repeats were identified and was not intended to provide a comprehensive representation of all duplicated sequences within any of the viral sequences examined. Although generated using different search criteria than those employed by Arguello-Astorga et al [22], the resulting collection of geminivirus sequence repeats contains some sequences similar or identical to the described "iterons" (it should be noted that functional testing of nearly all of the "iterons" listed has not yet been reported in the literature).

Functional testing of elements
Of the 105 repeats tested (Figure 1 and Additional file 1), 14 (13%) reproducibly resulted in increases of at least 25% above that of the 35S min construct (p < 5% by Student's T-test, the T-test was used only as a guide since by the nature of the assay used, individual data sets are small) (Figure 1 and Additional file 1). The remaining 91 (87%) failed to produce any measurable enhancement of reporter gene activity (see Additional file 1). All the positive elements identified by the in vivo assay were subsequently tested using an in vitro dual-luciferase ® system from Promega Corp. and produced levels of enhancement very similar to those obtained using the in vivo assay (the enhancement values and standard error reported in Figure  1 and Additional file 1 include both in vivo and in vitro data normalized to 35S min = 1.0). The observed enhancement of promoter activity (~2 fold) is relatively modest compared to other viral transcriptional enhancers that have been isolated and tested (e.g., G-box [23] and AS-1 [24] type elements enhance 35S min activity 8-10 fold using this assay, data not shown). This outcome may reflect limitations of the original search parameters (only repeated elements were tested). However, several of the geminiviral elements identified in this screen have been subsequently found to display clear and unique synergistic effects when combined or multimerized (Cazzonelli, Burke and Velten, manuscript in preparation), supporting their potential to contribute to viral gene regulation during infection.
Since all assays were performed on tobacco plants that had been neither infected with any of the viruses screened, nor transfected with any viral components, it is unlikely that elements strictly dependent upon virally encoded regulatory factors, or factors not native to N. tobacum, would be identified. In addition, the screen was limited to those elements that increase gene expression, and no effort was made to confirm data suggesting that an element might be a 'repressor' (e.g., the 11 elements that show 'enhancement' values less than, or equal to, one third of the 35S min activity, see Additional file 1). Considering these limitations, the finding that 13% of the sequences tested produced measurable up-regulation of transcription supports the original assumption that basic transcription regulatory elements are enriched within repeated sequences from the viral intergenic regions. Despite having tested approximately equal numbers of inverted sequence repeats (IR) and direct sequence repeats (DR), 11 of 14 active elements were members of the DR set, with the remaining 3 positives being palindromic (inverted repeats with no sequence between the repeats). This is somewhat surprising since many of the iterated DNA sequence elements within geminivirus intergenic regions are found as both direct and inverted repeats [22], and as such could have been present in either the DR or IR set of elements. Although the numbers tested are small, and the screen was performed using a single plant species, these results suggest that directly repeated sequences within geminivirus and nanovirus intergenic repeats have a higher probability of positively influencing transcription levels than do the inverted sequence structures. It is possible that this bias may reflect the presence within the intergenic region of DNA elements responsible for viral replication [25], including a conserved inverted repeat structure with a ubiquitous central-loop sequence [26]. Seven of the IR elements tested in this study are part of predicted replication hairpin structures (see Additional file 1) and did not, in this test system, result in any measurable enhancement of reporter gene expression.
Manual alignment of all the active DR sequences produced three classes of related elements and several unique individuals ( Figure 3). Five of the 14 positive DR elements contain an already identified geminiviral transcription control element, the "conserved late element" or CLE {GTGGTCCC, [22,27]}. The CLE sequence had been previously shown to affect expression from a minimal 35S promoter, and to be up-regulated by the viral AC2 gene product [27]. The two remaining grouped elements include a pair of "CT" rich repeats (DR08 and DR13) and two related, nearly-palindromic direct repeats from beet curly top virus (BCTV, elements DR19 and DR30). Despite the lack of an exact G-box core sequence {ACGT, [28]}, the nearly palindromic structure of the DR19 and DR30 elements {aaACTTc} is reminiscent of duplicated G-box type geminiviral elements noted by Arguello-Astorga et al [22] and later proposed as functional components within tomato golden mosaic virus (TGMV) and subterranean clover stunt virus (SCSV) promoters [11,20]. When scanned against the online PlantCARE promoter element database {[29,30]} no clear consensus emerges regarding similarity of the discovered viral elements with characterized plant cis regulatory elements (the most common hits were against light or stress responsive elements, although that may simply represent the distribution of plant elements contained within the database). 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.

Genus
Sequences tested: Adaptors: Left=AAGCTTCTAGA / *AAGCTT, Right=GGATCCTCGAG / *GGATCC "^" represents a common stuffer sequence (GAAGATAATC) Partial internal palindromes = underlined, imperfect repeats = . Alignment of active repeat elements 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 3). The results of the searches are displayed in Table 1. Each frequency value (cHits/Mbp) represents the number of hits per million base pairs, corrected for the database base composition using empirically determined G/C and A/T ratios for each of the databases examined (see Materials and Methods). To facilitate comparison, the resulting cHits/Mbp from the Arabidopsis upstream databases (-3000 to -1001, -1000 to -501, and -500 to -1 bp) were normalized relative to the value obtained for each element's occurrence within the A. thaliana coding sequence database (CDS value set to 1.0). In addition to the predicted frequency values, in each case, the element's observed frequency was also compared to a value generated using the average of 18 random oligomers having the same length and base composition as the element tested (in parenthesis, Table 1

Discussion
Except for the CLE elements, none of the active elements identified in this work have been experimentally reported as regulatory components of viral promoters. This is likely a reflection of both the limited number of geminivirus and nanovirus promoters that have been examined in detail [4,5,11,12,14,20,27,42,43], and the alternative approach of examining individual isolated elements used in this study. The mapped promoter components within the intergenic region of Tomato golden mosaic virus (TGMV) sub-genome A (TGMV-A) [14,20] provide a useful benchmark for comparison of results from this enhancer screen. Application of the repeated sequence screen to the TGMV (component B) intergenic region identified a single TGMV Direct repeat, DR38, and a single palindrome (PAL20), both of which were found to be inactive in our assay. This is consistent with published work that indicates most of the defined regulatory sequences within the TGMV-A intergenic region appear to occur as single copies [14,20]. The screen of intergenic repeats reported in this paper did, however, identify the CLE element, one copy of which has been shown to be part of the TGMV-A rightward promoter [14,20]. It is clear that testing only repeated elements will not identify all components of a promoter region, and when focusing on a specific promoter, testing of non-repeated elements (perhaps identified by evolutionary conservation) should be combined with other techniques such as insertion scanning [44]. Recently a collection of plant-functional  (3.42) promoters and terminators were isolated from the set of 7 Subterranean clover stunt virus (SCSV1-SCSV7) subgenomic circles. The collection of sequence repeats tested in this study included 11 inverted or direct repeats from SCSV circles, only one of which (DR08 from SCSV2) showed any enhancing activity. It will be interesting to see how these tested repeated elements behave when examined in the context of the remainder of the SCSV promoter components.

Conclusion
This screen of viral intergenic repeats was undertaken to specifically identify general transcriptional enhancing elements contained within intergenic regions of a subset of geminivirus and nanovirus genomes. The screen was successful in demonstrating transcriptional enhancer activity from one proven viral promoter element and several previously unidentified elements. The occurrence of the repeated elements within intergenic regions, combined with the clear enrichment within viral sequences and Arabidopsis upstream sequences for at least the CLE and TCrich (DR08/13) classes of elements, strongly supports participation of the enhancers in viral gene expression.
The technique of testing isolated elements represents an alternative to normal promoter-by-promoter dissection and provides a useful tool for screening promoter regions for potential functional elements that have been implicated by any number of possible criteria (e.g. copy number, evolutionary conservation, comparison of promoters with similar function, microarray data, etc.).
Although the number of elements tested is relatively small and, so far, only representative of promoters from the geminiviruses and nanoviruses classes of plant viruses, there is a clear trend suggesting that directly repeated elements (including those containing small internal palin-dromic sequences) are more likely to play significant roles in the enhancement of transcription than inverted repeats. This work represents one of the first attempts to directly screen for individual plant promoter elements that are isolated from their native promoter context. It is therefore, difficult to gauge the actual contribution of any of the elements identified to viral gene regulation and biological activity. These results do, however, provide a useful starting point for more detailed analyses of not only geminivirus and nanovirus promoters, but also overall plant promoter structure-function relationships.

Identification of sequence repeats
The search for repeated DNA sequences was performed by visual inspection of computer-generated dot matrix comparisons (criteria: ≥ 66% identity, 10 base window, GeneWorks v2.5.2, Oxford Molecular Group Inc.). Dot matrices generated using each viral plus strand plotted against itself were used to identify direct repeats while inverted repeats were found by plotting each plus strand against its complement.

Production of sequence repeat test constructs
The identified repeats were synthesized as DNA cassettes containing the duplicated elements in their original orientation, either directly repeated with spacer sequence ('DR', 41 elements), inversely repeated with spacer sequence ('IR', 45 elements), or palindromic inverted repeats without spacer ('PAL', 20 elements). In order to limit the tested component to only the repeated elements themselves, any sequence occurring between the viral repeats (ranging from 0 to 146 bp, median separation = 9 bp) was replaced with a 10 bp randomized stuffer sequence (GAAGA-TAATC). The resulting cassettes were inserted immediately upstream from a minimal promoter (-46 to +1 relative to

PClSV Bar FiLUC
Nos t 35S t RB LB AAGCTTCTAGAAGATAATCGGATCCTCGAGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACTAAACCATG ATG -46 +1 transcription start, 35S min) reporter system derived from the cauliflower mosaic virus (CaMV) 35S promoter fused to an intron-modified firefly luciferase (FiLUC) gene (Figure 2, [45]). The resulting test constructs were generated as part of a modified pPZP211 [46] binary plant transformation vector ( Figure 2) and were introduced into the Agrobacteria tumefaciens strain, EHA105 [47] by electroporation [48]. The final Agrobacteria strains each contain, in addition to the test plasmids, a second, compatible, binary transformation vector expressing an intron-modified version of the Renilla reniformis luciferase gene (RiLUC) [49] under control of the constitutive Super-promoter [50]. The FiLUC and RiLUC enzymes can be independently assayed, making the co-transferred constitutive RiLUC gene a useful marker for gene transfer and for normalization of FiLUC values between individual elements [45].

Lucifrease assays
Agrobacteria harboring the test and normalization binary plasmids were grown at 28°C in LB media containing the appropriate antibiotic selection (25 µg/mL kanamycin sulfate or 100 µg/mL spectinomycin) until an OD 600 of 0.8 was achieved. The resulting cultures were centrifuged at 3000 rpm for 15 minutes, washed and re-suspended in an equal volume of infiltration media (50 mM MES, 0.5% glucose, 2 mM NaPO 4 , 100 µM Acetosyringone) before being mechanically infused (5 ml syringe) into multiple individual tobacco (N. tobacum, cv. SR1) leaves (2-4 leaves per test construct). Assays were performed in groups of 4-8 constructs and the resulting luciferase activities (both FiLUC and RiLUC) determined after 3-4 days using an in vivo floating leaf-disk assay developed for this enhancer screen [45]. Test constructs were assayed from 1 to 6 times, with each assay consisting of 2-4 disks (3 mm diameter) per infusion. The disks used in vivo assays were each measured for light production in separate wells of a white-walled 96 well microtiter plate (FLUOstar Optima luminometer ® from BMG Lab Technologies Inc.) and all elements that tested positive in the in vivo assay were subsequently confirmed using the in vitro dual-luciferase ® from Promega Corp (assays performed according to the manufacturers instructions, separate leaf disks from the same leaf infusions were used for the in vivo assays). Each test group included an infusion containing the 35S min construct (lacking any viral test element). In order to compare the various assay systems, all activities were normalized to the activity of the 35S min construct included within each assay set (35S min activity arbitrarily set to 1.0).

Arabidopsis databases
Since the element enrichment approach works best when applied to relatively short, core consensus sequences [31], database searches were limited to those viral enhancers that displayed a clear core consensus (CLE, BCTV DR19/ 30, CT-rich, Figure 3). Results from the viral enhancer searches were compared to values obtained using previously reported plant promoter elements (DRE, ABRE-like, and G-box), and a short DNA sequence that is part of a highly conserved geminiviral replication loop stem sequence (CGCGNCCA) that is identical in base composition and length to the CLE consensus ( Table 1). The short sequence elements were each tested for their frequency of occurrence within a set of DNA sequence databases. One database consists of all entries for geminiviruses plus nanoviruses ([36], as of May, 2004) and all others are from the A. thaliana genomic sequence at the TAIR, PatMatch web site [32]. The geminivirus/ nanovirus BLAST searches were set for short exact matches (the statistical significance threshold set to 1000 and word size set at the element's length), returning the number of occurrences of exact matches for the full length element within the database. The TAIR PatMatch searches (default settings: Max hits, 7500; both strands; mismatch = 0; minimum hits/seq = 1; maximum hits/seq = 100) were performed against sub-datasets representing Arabidopsis coding sequences {"GI CDS (-introns, -UTRs)"}, and various lengths of upstream regions {"Locus Upstream Sequences", -1 to -500, -1 to -1000 and -1 to -3000}. Results from the -500 search were subtracted from the -1000 results, to generate hits from -501 to -1000 and -1000 results subtracted from the -3000 data to calculate hits from -1001 to -3000. In order to allow direct comparison between searches in different databases, using sequence elements of differing length and base composition, the number of database hits was corrected for the size of the database (number of hits divided by the database size in mega-base pairs {Mbp}) and base composition (hits/Mbp divided by the predicted number of hits per Mbp using upon the element sequence and base composition of each search database). The dataset base compositions were determined from downloaded sequence files and are: A. thaliana CDS: A/T = 55.8%, G/C = 44.2%; A. thaliana upstream (-1 to -500): A/T = 67.43%, G/C = 32.57%; A. thaliana upstream (-501 to -1000): A/T = 66.24%, G/C = 33.76%; viral: A/T = 56.2%, G/C = 43.8%. The resulting frequency of occurrence is a corrected number of hits per mega-base pairs (cHits/Mbp). For ease of comparison between elements, all of the cHits/Mbp values have been normalized to the corresponding cHits/ Mbp number from the A. thaliana CDS database (set arbitrarily to 1.0). Correction of the element's frequency using the calculated random probability of occurrence does not account for the possible impacted by intrinsic base-order bias that may occur within each sequence database, specifically the coding region database. These biases can potentially shift cHits/Mbp numbers markedly from those calculated using simple random base composition fre-quencies. To help confirm the significance of any observed enhancement in an elements frequency, mean cHits/Mbp values for 18 randomly generated sequences that match each test sequence for base composition and length were determined to provide a baseline value for comparison to that of the test element (shown in parenthesis, Table 1). A total of 18 sequences were used to produce the reported baseline as mean cHits/Mbp values were found to routinely level off at n values of between 8-12 random sequences examined (data not shown).