1. TAL Effectors Drive Transcription Bidirectionally in Plants
Li Wang, Fabio C. Rinaldi, Pallavi Singh, Erin L. Doyle, Zoe E. Dubrow, Tuan Tu Tran, Alvaro L. Pérez-Quintero, Boris Szurek, Adam J. Bogdanove 
Molecular Plant 
Volume 10, Issue 2, Pages (February 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1
Reverse-Oriented Designer TAL Effectors (dTALEs) Activate the Bacterial Blight Susceptibility Gene OsSWEET14.
Related to Supplemental Figure 1.
(A) Schematic of the OsSWEET14 gene showing the locations, 5′ to 3′ orientations, and sequences of the EBEs targeted by the forward and reverse dTALE pairs dT1381 and -82 and dT1383 and -84. Blue indicates a forward-strand EBE and orange a reverse-strand EBE. Parallel horizontal lines represent the promoter, and the block arrow represents the coding region. Numbers indicate the position of the rightmost base pair in each displayed sequence, represented by a short vertical line in the promoter, relative to the translational start site (+1). The red rectangle represents the putative TATA box.
(B) Expression of OsSWEET14 in rice (cv. Nipponbare) leaves, measured by quantitative real-time RT–PCR, in response to each dTALE, relative to a control TAL effector construct lacking a central repeat region (encoded on plasmid pAC99), delivered by X. oryzae pv. oryzae strain PXO99A derivative ME2. Tissue was sampled 48 h after syringe infiltration of a bacterial suspension. Each value shown is the average of three technical repeats of two biological replicates. An asterisk indicates a significant difference from the control (p < 0.05).
(C) Lesion lengths and (D) representative images for leaves of 6-week-old rice plants 14 days after inoculation with ME2 expressing the indicated dTALEs or the control protein (pAC99). In C, error bars represent the SD of at least 10 replicate inoculations, and an asterisk denotes a significant difference from the control using a two-tail Student t-test (p < 0.05).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2
Forward- and Reverse-Oriented dTALEs across the Promoter of the Bacterial Leaf Streak S Gene OsSULTR3;6 Vary Similarly in Activity and Reveal a Quantitative Correlation between Expression Level and Disease.
Related to Supplemental Figure 1.
(A) Schematic (as in Figure 1) of the OsSULTR3;6 gene showing the Tal2g EBE and the EBEs targeted by forward and reverse dTALEs. Also shown are representative lesions on leaves of 6-week-old rice (cv. Nipponbare) plants 10 days following side-by-side inoculation of X. oryzae pv. oryzicola BLS256 tal2g mutant M27 harboring the repeat-less TAL effector-encoding control plasmid pAC99 (left side of leaf) and M27 containing a plasmid encoding Tal2g or a dTALE (right side), as labeled below the leaf (for forward TAL effectors) or above it (for reverse ones). Bold font highlights Tal2g and dTALEs that contribute to virulence. An asterisk marks a reverse dTALE that has higher activity than its corresponding forward dTALE.
(B) OsSULTR3;6 expression in rice (cv. Nipponbare) leaves, measured by quantitative real-time RT–PCR, in response to Tal2g and each dTALE, relative to the pAC99-encoded control protein, delivered by M27. Tissue was harvested 48 h after inoculation by syringe infiltration. Each value is the average of two technical repeats of two biological replicates. An asterisk indicates a significant difference from the control (p < 0.05). A dagger indicates a significant difference between the forward and the reverse dTALE in a pair.
(C) Relative ability of each dTALE to restore virulence to M27. Shown for each dTALE is the mean ratio of the lesion length resulting from inoculation of M27 expressing that dTALE to the length resulting from the control inoculation on the other side of the leaf (as represented in A). Error bars show the SE of two biological repeats with at least 10 replicates each. An asterisk denotes a significant (p < 0.05) difference from the negative control, M27 harboring pAC99 relative to itself, using a two-tail Student t-test.
(D) Correlation between OsSULTR3;6 expression and lesion length ratio (data from B and C).
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3
OsSULTR3;6 Transcription Start Sites Observed in Response to Individual dTALEs and Tal2g Vary.
RNA used for the qPCR assays represented in Figure 2, including RNA from leaves infiltrated with Xoc BLS256 mutant M81 harboring control plasmid pAC99, were subjected to 5′ RACE. Sequences obtained are represented by horizontal bars for each treatment, drawn to scale relative to the OsSULTR3;6 promoter, shown at the bottom with the dTALE and Tal2g EBEs indicated by colored arrows. The thickness of each bar reflects the frequency at which that sequence was obtained. For each treatment, the total number of sequences obtained (N) and the fold change in OsSULTR3;6 expression (FC) are given on the right. Sequences that initiate within a plausible window for a transcription start site set by the TAL effector (see text) are colored red, except for Tal2g, for which that window coincides with the transcription start site of the control.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4 TAL Effectors Drive Divergent Transcription.
Related to Supplemental Figure 2.
(A) Schematics of the reporter used to assay divergent transcription, with head-to-head coding sequences (CDS) for firefly luciferase and renilla luciferase, shown with a minimal Bs3 promoter insert oriented with the firefly luciferase CDS and amended with a Tal2g EBE either in the forward (For) or reverse (Rev) orientation. The EBE, with the flanking sequences shown, was placed 35 bp upstream of the TATA box (red block).
(B) Firefly and renilla luciferase activity in Nicotiana benthamiana leaves 48 h after syringe infiltration of Agrobacterium delivering t-DNA harboring either the forward (left) or the reverse (right) EBE reporter construct shown in (A) and a 35S-driven gene for Tal2g or the control dT437, for which neither reporter harbors an EBE. An asterisk indicates a significant difference from the corresponding dT437 result (p < 0.05). Each value shown is the average of three technical repeats of three biological replicates.
(C) Schematic of the reporter carrying the 500 bp immediately upstream of the OsSULTR3;6 CDS, oriented with the renilla luciferase CDS. Line arrows indicate the positions and orientations of the EBEs for Tal2g and two pairs of forward and reverse dTALEs tested.
(D) Firefly and renilla luciferase activity resulting from expression of each TAL effector indicated in (C), or a repeat-less TAL effector control construct (transferred from pAC99), alongside the reporter in N. benthamiana leaves as in (B). An asterisk indicates a significant difference from the control result (p < 0.05).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5
Tal3c of X. oryzae pv. Oryzicola Strain BLS256 Drives Expression of Os09g39810 from a Forward- and a Reverse-Strand EBE.
Related to Supplemental Figure 2.
(A) Schematic (as in Figure 1) of the rice Os09g39810 gene showing the forward- and the reverse-strand Tal3c EBEs. A lower case letter represents a mismatch between that base and the Tal3c RVD at that position.
(B) Os09g39810 expression in rice (cv. Nipponbare) leaves, measured by quantitative real-time RT–PCR, at 48 h after syringe infiltration of wild-type BLS256 or tal3c mutant derivative M81 expressing Tal3c from a plasmid (pTal3c), relative to expression in leaves inoculated with M81 harboring the repeat-less TAL effector control plasmid pAC99. An asterisk indicates a significant difference from M81(pAC99) (p < 0.05). Each value shown is the average of three technical repeats of two biological replicates.
(C) (Top) Schematics of the bidirectional reporter carrying the 582 bp immediately upstream of the Os09g39810 CDS oriented with the firefly luciferase CDS, the construct with the reverse-strand Tal3c EBE scrambled (ScrR), and the construct with the forward-strand EBE scrambled (ScrF). Intact forward- and reverse-strand EBEs for Tal3c are represented by blue and orange line arrows, respectively. The EBE for positive control dTALE1647 is also shown as a blue line arrow. (Bottom) Fold change in firefly and renilla luciferase activity in Nicotiana benthamiana leaves 48 h after syringe infiltration of Agrobacterium delivering each construct (by color, in reference to the schematics above) and a 35S-driven gene for Tal3c or dT1647 relative to negative control dTALE dT437, for which none of the constructs harbors an EBE. Among values for a reporter (renilla luciferase or firefly luciferase), and within a plot, different letters indicate a significant difference (p < 0.05).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions