TALE mediated gene activation is dependent on surrounding DNA sequences.

<p>(<b>A</b>) Model for the DNA-binding mode of TALEs by using the example of Hax3 aligned to the Hax3-box. TALEs contain a central repeat region (red), two nuclear localization signals (NLS) and an acidic activation domain in the C-terminal part. The amino acid sequence of a Hax3 repeat is shown in single letter code. The repeat variable diresidue (RVD) is shaded in grey. Each RVD specifies one nucleotide in the DNA-target sequence. (<b>B</b>) Sequence overview of the analysed 75 bp long DNA fragments that originate either from the <i>Bs4</i> promoter (<i>pBs4</i>, region from -278 bp to +25 bp) or from the <i>Bs4</i> open reading frame (<i>oBs4</i>). Potential promoter elements that were predicted [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173580#pone.0173580.ref037" target="_blank">37</a>] are labelled in grey (TATA-box) blue (W-box), black (CAAT-box) or italic and underlined (5' untranslated region, UTR). (<b>C</b>) TALE-dependent activation of reporter constructs. Each promoter fragment was placed downstream of the Hax3-box and inserted in front of a promoterless <i>uidA</i> reporter gene. <i>Agrobacterium</i> strains delivering <i>hax3</i> under control of the <i>35S</i> promoter were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves along with an <i>Agrobacterium</i> strain delivering the respective reporter construct. As negative control, all reporter constructs were inoculated with an empty vector construct (-). The quantitative β-glucuronidase measurement was performed 2 dpi, error bars were calculated on the basis of three independent replicates and represent the standard deviation. (4-MU, 4-methyl-umbelliferone).</p

<i>Bs4</i> promoter swaps to identify regions that support TALE activity.

<p>(<b>A</b>) The two 75 bp fragments (A, positive, green and B, negative, red) were subdivided into smaller fragments (A1-4, B1-4). Putative promoter elements that were predicted [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173580#pone.0173580.ref037" target="_blank">37</a>] are labelled in grey (TATA-box), blue (W-box), black (CAAT-box) or italic and overlined (5´UTR). (<b>B</b>) Overview of the analysed promoter swaps. The origin of the fused fragments is marked with green and A or red and B, respectively. The fragments were placed downstream of the Hax3-box (H3-B) and upstream of a promoterless <i>uidA</i> reporter gene. The dashed line indicates the location of the <i>attB1</i> site preceding the <i>uidA</i> reporter gene. TALE-dependent activation of the reporter constructs was determined by β-glucuronidase-measurement. <i>Agrobacterium</i> strains delivering <i>hax3</i> under control of the <i>35S</i> promoter were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves along with an <i>Agrobacterium</i> strain delivering the respective reporter construct. The ß-glucuronidase measurement was performed two dpi and calculated as relative activity based on the activity of the reference fragment A (100%). Error bars represent the standard deviation of three independent replicates (4-MU, 4-methyl-umbelliferone).</p

The use of an alternative Activation Domain (AD) or DNA-binding domain (dCas9 activator) does not change the activation pattern of TALEs at the <i>OsSWEET14</i> promoter in <i>N</i>. <i>benthamiana</i>.

<p>(<b>A</b>) Overview of the binding sites and binding orientation of a selected number of artificial TALEs that were fused to the VP64 AD and the binding sites and binding orientation of the analyzed sgRNAs. The dashed line indicates the location of the <i>attB1</i> site preceding the <i>uidA</i> reporter gene (<b>B</b>) Activity of artificial TALEs (filled bars) in comparison to the TALE-VP64 derivatives (framed bars). <i>Agrobacterium</i> strains delivering the TALE constructs under control of the <i>35S</i> promoter were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves along with an <i>Agrobacterium</i> strain delivering the reporter construct. TAL1ΔAD was used as an internal control. The ß-glucuronidase measurement was performed 2 dpi. Error bars represent the standard deviation of three independent replicates (4-MU, 4-methyl-umbelliferone). (<b>C</b>) The nucleolytically inactive dead Cas9 (dCas9) variant was fused to the C-terminus of Hax3 to generate a dCas9 activator. <i>Agrobacterium</i> strains delivering dCas9 activator constructs and the reporter construct that contains the 1 kb promoter fragment of <i>OsSWEET14</i> fused to a promoterless <i>uidA</i> gene were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves. GUS measurement was performed 2 dpi, error bars represent the standard deviation of three independent replicates (4-MU, 4-methyl-umbelliferone). Please notice the different scales on the TALE and dCas9 activator graphs indicating that TALEs are more potent activators in this example.</p

TALEs enhance transcription from diverse positions in the rice <i>OsSWEET14</i> promoter in <i>N</i>. <i>benthamiana</i> in a partially TATA-box dependent manner.

<p>(<b>A</b>) Overview of the reporter construct containing the <i>OsSWEET14</i> wildtype (WT) promoter fragment (1kb upstream of the ATG) and the <i>OsSWEET14-11</i> promoter mutant (Δ 33 bp; 967 bp upstream of the ATG; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173580#pone.0173580.ref041" target="_blank">41</a>]) fused to the promoterless <i>uidA</i> reporter gene. The dashed line indicates the location of the <i>attB1</i> site preceding the <i>uidA</i> reporter gene. The binding sites and binding orientations of artificial TALEs (coloured) and the natural TALEs TalC and AvrXa7 (white) are marked with arrows, the arrowhead indicates the orientation of the activation domain relative to the <i>uidA</i> gene. Reverse binding TALEs are labeled with "-" in front of the number. RVD sequences and target sites are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173580#pone.0173580.s006" target="_blank">S1 Table</a>. <b>(B)-(E)</b> Activity of the artificial and natural TALEs. <i>Agrobacterium</i> strains delivering TALE constructs under control of the <i>35S</i> promoter were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves along with an <i>Agrobacterium</i> strain delivering the reporter construct. The TALE Hax3 that does not bind to the <i>OsSWEET14</i> promoter was used as negative control to exclude background promoter activity. The ß-glucuronidase measurement was performed 2 dpi. Error bars represent the standard deviation of three independent replicates (4-MU, 4-methyl-umbelliferone). (<b>B</b>) Activity of forward TALEs in combination with the WT reporter. (<b>C</b>) Activity of reverse TALEs in combination with the WT reporter. (<b>D</b>) Activity of forward TALEs in combination with the mutant reporter. (<b>E</b>) Activity of reverse TALEs in combination with the mutant reporter. (D, E) TALEs whose binding sites overlap with the deletion in the mutant promoter are indicated with Δ33 bp. Arrows highlight a strong reduction of TALE activity in comparison to the WT reporter.</p

Artificial TALEs do not always shift the natural Transcriptional Start Site (TSS) at the <i>OsSWEET14</i> promoter in <i>N</i>. <i>benthamiana</i>.

<p>Overview of analyzed artificial and natural TALEs that bind to different positions in the <i>OsSWEET14</i> promoter. The natural <i>OsSWEET14</i> TSS in rice is marked with a red "A", and the AvrXa7-induced TSS is marked with a grey "G" [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173580#pone.0173580.ref020" target="_blank">20</a>]. <i>Agrobacterium</i> strains delivering the <i>35S</i>-controlled TALE constructs were co-inoculated into <i>N</i>. <i>benthamiana</i> leaves along with an <i>Agrobacterium</i> strain delivering the reporter construct. 1 dpi leaf discs were harvested and total RNA was extracted. cDNA was produced and used for 5' RACE. The first nucleotide of each identified TSS is labeled with a capital letter. The distance of the TSS to the 3´end of forward-orientated TALE target sequences and to the 5´end of reverse-orientated TALE target sequences as well as the number of analyzed clones ("x") is indicated to the right. TSSs that overlap with the natural <i>OsSWEET14</i> TSS in rice are marked in red.</p

Reverse binding artificial TALEs can activate <i>OsSWEET14</i> expression in a natural <i>Xoo</i>-rice infection.

<p>(<b>A</b>) Overview of the binding sites and binding orientation of analyzed artificial TALEs (coloured arrows) and of the natural TALE TalC (white arrow). (<b>B</b>) Reverse- and forward-oriented TALEs can both support disease development of <i>Xoo</i> in rice. Leaves of <i>Oryza sativa</i> cv. Nipponbare were inoculated with the <i>Xoo</i> strains BAI3, BAI3Δ<i>talC</i> or BAI3Δ<i>talC</i> carrying an empty vector plasmid (<i>ev</i>), <i>talC</i> or an artificial <i>TALE</i> on a plasmid. Inoculation of rice leaves with MgCl₂ served as negative control. Pictures of phenotypes were taken 5 dpi. Water soaking symptoms are marked with a white arrow. (<b>C</b>) <i>OsSWEET14</i> expression levels following <i>Xoo</i> infection. Leaves of <i>Oryza sativa</i> cv. Nipponbare were inoculated with the same <i>Xoo</i> strains as in (<b>B</b>). 1 dpi leaf material was harvested and the transcript levels of <i>OsSWEET14</i> were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The error bars indicate the standard deviation of three biological replicates. The fold-change of <i>OsSWEET14</i> expression was calculated based on the negative control BAI3Δ<i>talC</i> + <i>ev</i>. An asterisk (*) indicates a significant increase in <i>OsSWEET14</i> expression calculated with the students <i>t</i>-Test.</p

<i>Oryza sativa</i> ssp <i>japonica</i> microarray experiments from PLEXdb used in this paper.

a<p><i>Xoo</i>: <i>X. oryzae</i> pv. <i>oryzae</i>; <i>Xoc</i>: <i>X. oryzae</i> pv. <i>oryzicola</i>.</p

Positional preference of TAL effector target sites relative to the TATA-box (left) and TC-box (right).

<p>The estimated density of positions from the positive set is plotted as a green line, while the density of the negatives is plotted in red. The green points at the bottom of the plots represent the distribution of positions from the positive set along the x-axis, where the points are distributed randomly in y-direction to make individual points distinguishable.</p

Recognition of predicted target sites by AvrXa10.

<p>(A) RVDs of the TAL effector AvrXa10 and predicted target sites. The optimal box is deduced from the known RVD specificites, while box 6, box 38, box 41, and box 98 are TALgetter AvrXa10 target predictions from rice promoters. Mismatches and non-optimal RVD-base pair combinations are shaded in light and dark grey, respectively. (B) AvrXa10 and Hax3 target boxes are cloned upstream of the minimal <i>pBs4</i> promoter and a promoterless <i>uidA</i> reporter gene. The artificial TAL effector ArtBs4 targets the <i>pBs4</i> promoter and is used as control for reporter construct integrity. (C) Specific recognition of target boxes. Reporter constructs are codelivered via <i>A. tumefaciens</i> into <i>N. benthamiana</i> with (+) and without (−) constructs producing TAL effectors, respectively, and GUS reporter activity was determined two days post inoculation. Error bars indicate standard deviation ( samples). 4-MU, 4-methyl-umbelliferone. Leaf disks are stained with X-Gluc (5-bromo-4-chloro-3-indolyl--D-glucuronide). A blue color indicates reporter gene activity.</p

Data sets of putative target genes of expressed TAL effectors obtained from comparative studies (in alphabetical order).

<p>Data sets of putative target genes of expressed TAL effectors obtained from comparative studies (in alphabetical order).</p