Fig 1.
Maximum likelihood (ML) phylogenetic tree and colour coded matrix of pairwise sequence identity based on full-length sequences of genome components A and B. (A) DNA-A; (B) DNA-B. ML phylogenies were constructed by multiple alignments of complete DNA-A and DNA-B sequences of BleICV and representative begomoviruses. ML method in MEGA 7 was based on the Tamura-Nei model and the tree support was tested by bootstrapping with 1000 replicates. The scale bar represents the genetic distance. The genomic sequence of Tomato pseudo-curly top virus (TPCTV), a topocuvirus, was used as outgrup. For begomovirus acronyms see S1 Table.
Fig 2.
Region of BleICV CP promoter encompassing the three phylogenetic footprints (PhyF) shared with ToChLP.
Upper module. Simplified representation of BleICV CP promoter, illustrating the two repeats with canonical CLEs (letters in red). Lower module. Phylogenetic footprints in the CP promoter of BleICV and ToChLPV (spacing nucleotides between PhyFs were omitted for simplicity). CLE = Conserved Late Element; the arrows denote either direct or inverted repeats; the blue diamond indicates the central nucleotide of the palindromic 23-nt sequence of ToChLPV.
Fig 3.
Symmetric region ACTT-(N7)-AAGT of 30 New World begomoviruses.
GenBank accesion numbers: TYLDV (KU232891), BleICV (JX827487), ToChLPV (DQ347949), MaBYMV (KU058856), PSLDV (KT899302), BYMMV (FJ944023), AbMBV (JF694480), ToMHV (KT099130), OYMMV (HM035059), SiYMoV (HE806448), MacYMV (AJ344452), DaLCV (JN848773), ToMoV (AY965900), MacYSV (KJ939895), VEM-5 (KT099138), SiCVV (KX691405), SiYBV (KX640991), JaMV (KJ174333), TLCSiV (KY064014), CoMoV (JQ805781), VEM-3 (KT099127), CoYSV (DQ875868), SiMAV (KX896421), BDMV (M88179), TGMV-cs (JF694488), AbGMYV (KC430935), SicGMV (JX857691), SiYMV (AY090558), MaYMJV (FJ601917), TbMoLCV (FM160943).
Fig 4.
CP promoter region containing the TACE of 30 Old World begomoviruses.
GenBank accession numbers: MaBYMV (KU058856), JaLCrIV (KM189818), SLCMV (KP455484), TLCJV (KM383747), ToLCRaV (GQ994095), TYLCVV (EU189150), EgGMV (KU569598), PaLCCNV (KU892674), ChLCVV (HM007121), ICMV (Z24758), StLCV (AJ564742), PepLCBV (JN663853), EYVThV (KY373213), CroYVMV (JX270684), RaMV (KX885030), PaLCV (LT009397), CraYVV (FN401520), AYVV (X74516), EuLCGV (AM411424), ChLCAV (KM880103), TYLCMV (LM651401), TYLCV-IL (EF523478), PepYVMV (FM876849), EACMKV (KJ887946), ToLCBV (KM383762), ToLCNV (AM701761), ToLCoV (AJ865341), EACMMV (KP890350), ToLCAntV (AM701767), SiYVVV (KF990601), AYVCNV (HG003652). Note that the first virus, MaBYMV, is an American BGV.
Fig 5.
Alignment of the CLE-TACE region of a curtovirus and four Asian begomoviruses.
Note the 17 bp segment of the BCTV, SyLCV and AEV TACEs, which is 100% identical in sequence. The distance between the CLE and the TACE is also conserved in these geminiviruses. GenBank accession numbers: BCTV-Cfh (X97203), SyLCV (KU933258), AEV (HE861940), TbCSV (KU934097), SbYCrV (AB050781).
Fig 6.
TACE and associated cis-acting elements in the CP promoter of viruses belonging to three Geminiviridae genera.
Coloured boxes: red, CLE; yellow, TACE; green, TATA-box. Nucleotides of TbYLJV and BCTV TACEs which are identical are underlined.
Fig 7.
Effect of multiple copies of CLE on GUS expression driven by a truncated -46 and -90 CaMV 35S promoter.
Several independent lines of transgenic plants harbouring constructs with synthetic multimers of the CLE upstream of truncated 35S promoters were analysed. Total protein was extracted of the different plant sets and assayed for GUS activity. An average expression (dotted line) and standard deviation are shown for each set of plants. (46-3CLE n = 6, 90-3CLE n = 9, 46-6CLE n = 7).
Fig 8.
Tissue specific-expression directed by a synthetic promoter containing six copies of the CLE.
Transgenic tobacco plantlets harboring a [6CLE-35S prom(-46)-GUS] construct displayed high GUS expression in all leaf cells, including trichomes. A seedling harbouring a [35S prom(-46)-GUS] construct did not express the reporter gene in none of the tissues. The name of the binary vector used to transform the plants is indicated: pBI 46S harbors the [35S prom(-46)-GUS] construct; pBI 46S 6CLE harbors the [6CLE-35S prom(-46)-GUS] construct.
Fig 9.
Effect of EuMV infection on GUS expression driven by synthetic promoter containing three copies of the CLE.
Four independent transgenic plant lines harbouring the [3CLE-35Sprom(-46)-GUS] construct were tested in the absence or presence of viral factors. Total protein was extracted from the plants 15 dpi and assayed for GUS activity. A two-tailed paired t-test showed GUS expression differences for most lines at p<0.05*. Mean and standard deviation are shown. (Line1 n = 4, p = 0.0869, Line2 n = 7, p = 0.0125, Line3 n = 11, p = 0.0010, Line4 n = 17, p = 0.0002).
Fig 10.
The CLE is a TrAP responsive element.
Protoplast transient expression assays were performed with different versions of truncated TGMV CP promoter constructs in presence or absence of EuMV DNA-A factors. Total protein of transfected protoplasts was extracted two days post-transformation and assayed for GUS activity. The average and standard deviation of three independent experiments are shown. The red dotted line represents the average expression of empty (promoter-less) vector pBS-GUS in absence of viral factors. This vector was the backbone used for the generation of the different promoter constructs. For comparison purposes a construction with the uidA gene driven by the complete CaMV 35S promoter was included in the experiments.