Supporting Information Methods S1 & S2
Methods S1
Generation of talC and tal5 expression constructs.
For transient expression in Nicotiana benthamiana, the coding region of talC was PCRamplified, inserted into pENTR/D-TOPO (Life Technologies GmbH), and transferred by
GATEWAY LR-recombination to the binary vector pGWB2 which facilitates a constitutive
35S-driven expression in planta and adds an N-terminal GFP tag to TalC. For expression in
Xanthomonas, the coding region of the N- and C-terminal parts of talC were amplified as
Golden Gate-compatible modules. N- and C-terminal modules were inserted together with a
repeat region dummy module (hax3 repeat module) into a Golden Gate-compatible
pBBR1MCS-5 derivative, pSKX1, which facilitates a lac promoter-driven expression and
addition of a C-terminal FLAG epitope. The dummy repeat region was then exchanged with a
fragment encoding the talC repeat region by classic cloning using StuI and AatII. DNA
sequencing confirmed the integrity of the final construct. The coding region of tal5 was
extracted from cosmid clone 6B7 (obtained upon screening of a MAI1 genomic DNA library
for clones carrying TAL genes by colony hybridization, Y. Yu, V. Verdier and B. Szurek,
unpublished data) and inserted into pSKX1 and pGWB2 for expression in Xanthomonas and
delivery by Agrobacterium, respectively, via classic cloning using BamHI. One BamHI
restriction site is overlapping with the ATG start codon and the second one is located 150 bp
upstream of the stop codon of the tal5 gene. Most of the talC coding region including the
repeat region was exchanged with the corresponding part of tal5 in the pSKX1-derivative
using BamHI. Sequencing confirmed the right insertion into the destination vector.
Methods S2
Choice of target sites and generation of artificial TAL effectors.
Based on the sequence information given by the Rice Genome Annotation Project (version 7;
http://rice.plantbiology.msu.edu/) the promoter regions (500 to 40 bp upstream of the ATG)
of the OsSWEET genes were analyzed to find two appropriate artificial TAL effector
(ArtTAL) binding sites per OsSWEET gene. Potential binding sites were selected according
to several criteria:
a. ArtTAL binding sites should comprise at least 19 bp (including the initial T), thus
corresponding to at least 17.5 repeats to ensure a high specificity.
b. ArtTAL binding site should explicitly be highly specific within the OsSWEET family.
c. Similar to many natural TAL effector binding sites, ArtTAL binding sites should
include or be near to the TATA element.
d. ArtTAL binding sites should be at least 40 to 60 bp upstream of the potential
translational start site.
e. There should be no additional ATG between the 3´ end of the ArtTAL binding sites
and the potential translational start site.
f. The corresponding artificial TAL effector should contain at least 3 to 4 properly
spaced strong RVDs (HD, NN).
The specificity of the chosen binding sites was checked using the Talvez software
(http://bioinfo.mpl.ird.fr/cgi-bin/talvez/talvez.cgi; Pérez-Quintero et al., 2013). The absence
of common collateral target sites (besides of the OsSWEET gene) was evaluated among the
top-20 predicted targets of each ArtTAL. The ArtTALs were generated as previously
described by using the hax3 backbone and between 17.5 and 21.5 repeats to specify the DNA
target sequence. The broad host range vector pSKX1 was used to express the ArtTALs under
control of a lac promoter. The fusion to a C-terminal FLAG epitope allows detection of the
ArtTALs by Western blotting.