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390
To the Editor:
Transcription activator–like (TAL) ef-
fectors of Xanthomonas oryzae pv. oryzae
(Xoo) contribute to pathogen virulence
by transcriptionally activating specic
rice disease-susceptibility (S) genes
1, 2
.
TAL effector nucleases (TALENs)—fu-
sion proteins derived from the DNA
recognition repeats of native or custom-
ized TAL effectors and the DNA cleav-
age domains of FokI
3, 4, 5
—have been
used to create site-specic gene mod-
ications in plant cells
6, 7
, yeast
8
, ani-
mals
9, 10, 11, 12
and even human plurip-
otent cells
13
. Here, we exploit TALEN
technology to edit a specic S gene in
rice to thwart the virulence strategy of
X. oryzae and thereby engineer herita-
ble genome modications for resistance
to bacterial blight, a devastating disease
in a crop that feeds half of the world’s
population.
We targeted the rice bacterial blight
susceptibility gene Os11N3 (also called
OsSWEET14) for TALEN-based disrup-
tion. This rice gene encodes a member
of the SWEET sucrose-efux transporter
family and is hijacked by X. oryzae pv.
oryzae, using its endogenous TAL effec-
tors AvrXa7 or PthXo3, to activate the
gene and thus divert sugars from the
plant cell so as to satisfy the pathogen’s
nutritional needs and enhance its per-
sistence
2, 14
. The Os11N3 promoter con-
tains an effector-binding element (EBE)
for AvrXa7, overlapping with another
EBE for PthXo3 and with the TATA
box (Figure 1a and Supplementary Fig-
ure 1). We deployed two pairs of de-
signer TALENs (pair 1 and pair 2) inde-
pendently to induce mutations in these
overlapping EBEs of the Os11N3 pro-
moter and thus to interfere with the vir-
ulence function of AvrXa7 and PthXo3,
but not the developmental function of
Os11N3 (Supplementary Figure 1 and
Supplementary Note). The TALE repet-
itive regions used for nuclease fusions
included the native AvrXa7 and three
designer TALE repetitive regions cus-
tom synthesized using a modular
Published in Nature Biotechnology 30 (2012), pp. 390–392; doi: 10.1038/nbt.2199 Published online May 7, 2012.
Copyright © 2012 Nature Publishing Group. Used by permission.
Supplementary information is presented following the References.
Correspondence
High-efciency TALEN-based gene editing produces
disease-resistant rice
Ting Li,
1
Bo Liu,
1
Martin H. Spalding,
1
Donald P. Weeks,
2
and Bing Yang
1
1. Department of Genetics, Development & Cell Biology, Iowa State University, Ames, Iowa, USA
2. Department of Biochemistry, University of Nebraska–Lincoln, Lincoln, Nebraska, USA
Corresponding author — Bing Yang, email byang@iastate.edu
Figure 1. High-
efciency tar-
geted gene ed-
iting using
TALENs. (a) Over-
lapping elements
targeted by two
pairs (1 and 2) of
designer TALENs
in the Os11N3
promoter. (b–
d) Genotypes of
progeny (T
1
) of
primary trans-
genic plants (T
0
)
derived from
TALEN-express-
ing embry-
onic cells from
three indepen-
dent transforma-
tion experiments
(Exp.). Each of
the two alleles
of an individual
plant are desig-
nated as being
wild type (wt) or
as having a nu-
cleotide inser-
tion (+) or a de-
letion (−) and are
separated top
and bottom by
a dividing line.
The designation
“−55/(−7/+3)”
indicates that
one allele con-
tains a deletion
of 55 bp and
that the other
allele has both a deletion of 7 bp and an insertion of 3 bp. (e) Sequences of Os11N3 mu-
tations induced by the pair 2 TALENs with deletions (dashes) and insertions (red letters).
TALEN-binding sequences are underlined in wt and the overlapping EBEs are shaded in gray.
(f,g) Expression of Os11N3 and Os04g19960 induced by AvrXa7 in plants of different gen-
otypes. Quantitative reverse transcription (RT)-PCR was performed with RNA derived from
treatments of nonpathogenic Xoo strain ME2 and pathogenic ME2(avrXa7). 2
ΔΔCt
is a mea-
sure of transcript abundance for a selected gene (Os11N3 in f or Os04g19960 in g) relative
to the abundance of transcripts produced from a constitutively expressed gene (OsTFIIAg5),
as determined by relative PCR cycle thresholds (Ct). (h) Resistance phenotype displayed by
two T
2
mutant plants compared with the disease susceptibility phenotype of a nontrans-
genic wt rice plant.
Hi g H -e f f i c i e n c y TALen-b A s e d g e n e e d i T i n g p r o d u c e s d i s e A s e -r e s i s T A n T r i c e 391
assembly method
8
. Each designer
TALEN contained 24 repeat units for
recognition of a specic set of 24 con-
tiguous nucleotides at the target sites
(Supplementary Figure 1).
For each pair of TALEN genes, one
TALEN gene (half of the pair) was un-
der the control of the 35S promoter of
cauliower mosaic virus and the other
gene was driven by the maize ubiqui-
tin 1 promoter, comprising a specic
TALEN pair in a single plasmid (Sup-
plementary Figure 2). Each plasmid
also contained a marker gene for hy-
gromycin resistance. These constructs
were introduced into rice embryonic
cells using Agrobacterium tumefaciens,
and individual transformant cells were
selected, propagated and regenerated
into whole plants (T
0
). The Os11N3
promoter regions from a number of in-
dependent hygromycin-resistant cal-
lus lines and the segregating progeny
(T
1
) of self-pollinated T
0
plants were
amplied using the polymerase chain
reaction (PCR) and sequenced to de-
tect potential sequence alterations. For
TALEN pair 1 genes, two of ve ex-
amined callus lines contained bial-
lelic mutations (Supplementary Fig-
ure 3). Of 23 randomly selected T
1
progeny produced from self-pollina-
tion of 7 independent T
0
plants trans-
formed with TALEN pair 1 genes,
about half (48%) carried mono- or bi-
allelic mutations (including the four
mutations detected in the two previ-
ously examined callus lines; Figure
1b). Approximately two-thirds (63%)
of the randomly selected T
1
plants (n
= 30) generated from self-pollination
of 66 independent T
0
plants from the
two independent transformation ex-
periments carried mutations that were
induced by the TALEN pair 2 genes
(Figure 1c,d). In total, 16 distinct mu-
tations, including 6 that were homo-
zygous, were detected in 53 T
1
plants
from TALEN pair 1 and pair 2. The
majority of these mutations were small
deletions that left the TATA box intact,
with the exception of two deletions in
heterozygous lines that also contained
a wild-type allele (Figure 1e and Sup-
plementary Figure 4). Bacterial infec-
tion assays using the leaf-tip clipping
method on other T
1
plants (n = 627)
generated from TALEN pair 2 (experi-
ment 1) and not previously genotyped
demonstrated that approximately 48%
of the treated plants showed resis-
tance to infection by pathogenic Xoo
as evidenced by the length of leaf le-
sions (1–4 cm for resistance versus 10–
14 cm for susceptibility; Supplemen-
tary Figure 5). DNA sequence analyses
of 27 such Xoo-resistant T
1
plants con-
rmed the presence of homozygous
monoallelic or heterozygous biallelic
EBE mutations and revealed 17 ad-
ditional, distinct mutant haplotypes
(Supplementary Figure 6). All mutant
plants were morphologically normal
compared to wild-type plants, indicat-
ing that the developmental function of
Os11N3 was not disrupted.
Forty plants from the second gen-
eration (T
2
) of three self-pollinated
T
1
plants were also genotyped by se-
quencing to determine the heritability
of three TALEN-generated mutations,
all of which, whether homozygous or
heterozygous, were passed on to T
2
plants (Supplementary Figure 7).
To determine the effects of TALEN-
directed mutations, we investigated
whether the pathogenic strain of
Xoo that is dependent on AvrXa7 or
PthXo3 for virulence is able to either
induce the modied Os11N3 gene in
homozygous T
2
plants or cause dis-
ease. The modied Os11N3 gene was
no longer inducible by AvrXa7 or
PthXo3 delivered by the pathogenic
strain of the bacterium (ME2(avrXa7)
or ME2(pthXo3)) in T
2
plants homozy-
gous for either the 9-, 6-, 15- or 4-bp
deletion (Figure 1f for AvrXa7, Sup-
plementary Figure 8a for PthXo3).
The loss of induction was spe-
cic to Os11N3, as the induction of
Os04g19960, a transposon coding
gene collaterally targeted by AvrXa7,
was not prevented (Figure 1g). Simi-
larly, the induction of another S gene
(Os8N3, also known as OsSWEET11)
by PthXo1 in the T
2
mutant plants
remained unaffected (Supplemen-
tary Figure 8b). These TALEN-modi-
ed T
2
plants also showed strong re-
sistance to infection of the AvrXa7- or
PthXo3-dependent Xoo strains but not
the PthXo1-dependent pathogenic Xoo
strain as determined from symptoms
(Figure 1h for AvrXa7) and by quan-
titative measurement of the lengths of
leaf lesions in a standard pathogene-
sis assay described in Supplementary
Methods (Supplementary Figure 9).
We also investigated the possibil-
ity of using genetic segregation to ob-
tain genetically modied rice lack-
ing any selection marker and TALEN
gene. The PCR assay using primers for
amplication of the hygromycin resis-
tance gene and for amplication of the
TALEN genes failed to detect the pres-
ence of either gene in 5 out of 37 T
1
plants that contained the desired ge-
netic modications in the Os11N3 pro-
moter and that were disease resistant
(Supplementary Figure 10). Although
392 Li , Li u , sp A L d i n g , We e k s , & yA n g i n N a t u r e B i o t e c h N o l o g y 30 (2012)
these data clearly demonstrate the ab-
sence of intact TALEN and hygromy-
cin-resistance genes, further sequenc-
ing of the genomes of several mutants
and the Kitake parental line will be
needed to conclusively demonstrate
that all of the transgene fragments
have been removed.
The rice Os11N3 gene is induced by
32 of 40 Xoo strains collected world-
wide (T. Li and B. Yang, unpublished
data). However, polymorphisms in the
Os11N3 gene that prevent induction
by AvrXa7- and/or PthXo3-dependent
Xoo strains and also confer disease re-
sistance have not been identied in
rice germplasm. The approaches de-
scribed here for precisely and ef-
ciently editing the disease suscepti-
bility elements in Os11N3 and for the
subsequent removal of transfer DNA
(T-DNA) sequences by classic genetics
likely can be applied directly to elite
rice varieties to simultaneously or se-
quentially edit multiple susceptibil-
ity genes (for example, Os11N3 and
Os8N3), leading to resistance to the
major forms of bacterial blight. Present
methods using TALEN-based technol-
ogy in rice should be easily modied
for application to other plant species
and, thus, hold substantial promise in
facilitating gene modication–based
research and crop improvement.
Acknowledgments — We thank D.
Wright and C. Yao for helpful sug-
gestions, L. Xue and J. Luo for techni-
cal assistance, and F.F. White and S.
Howell for critical reading of the man-
uscript. This work was supported by
grants from the US National Science
Foundation (0820831 to B.Y., MCB-
0952323 to M.H.S., and MCB-0952533
and EPSCoR grant 1004094 to D.P.W.).
Author contributions — B.Y. and T.L.
conceived the study. T.L. and B.L. per-
formed the experiments. B.Y., T.L.,
M.H.S. and D.P.W. analyzed the data.
All authors contributed to the writing
of the paper.
Competing nancial interests — T.L.,
B.L. and B.Y. are inventors on a pat-
ent application covering TALEN-me-
diated rice engineering for disease
resistance.
References
1. Yang, B., Sugio, A. & White, F.F.
Proc. Natl. Acad. Sci. USA 103,
10503–10508 (2006).
2. Antony, G. et al. Plant Cell 22, 3864–
3876 (2010).
3. Li, T. et al. Nucleic Acids Res. 39, 359–
372 (2011).
4. Christian, M. et al. Genetics 186, 757–
761 (2010).
5. Miller, J.C. et al. Nat. Biotechnol. 29,
143–148 (2011).
6. Mahfouz, M.M. et al. Proc. Natl.
Acad. Sci. USA 108, 2623–2628
(2011).
7. Cermak, T. et al. Nucleic Acids Res.
39, e82 (2011).
8. Li, T. et al. Nucleic Acids Res. 39,
6315–6325 (2011).
9. Sander, J.D. et al. Nat. Biotechnol. 29,
697–698 (2011).
10. Huang, P. et al. Nat. Biotechnol. 29,
699–700 (2011).
11. Tesson, L. et al. Nat. Biotechnol. 29,
695–696 (2011).
12. Wood, A.J. et al. Science 333, 307
(2011).
13. Hockemeyer, D. et al. Nat. Biotech-
nol. 29, 731–734 (2011).
14. Chen, L.Q. et al. Science 335, 207–
211 (2012).
1
SUPPLEMENTARY INFORMATION
High-efficiency TALEN-based gene editing produces disease-resistance rice
Ting Li
1
, Bo Liu
1
, Martin H. Spalding
1
, Donald P. Weeks
2
, Bing Yang
1 *
1
Department of Genetics, Development & Cell Biology, Iowa State University, IA 50011, USA;
2
Department of Biochemistry, University of Nebraska-Lincoln, NE 68588 , USA.
*
To whom correspondence should be addressed. E-mail: byang@iastate.edu
Table of Contents
Materials and Methods
Supplementary Figure 1. TALENs and their DNA targets in the promoter of chromosomal
Os11N3 gene.
Supplementary Figure 2. Schematic diagram of a two-gene expression cassette in a single
binary vector designed for Agrobacterium-mediated rice transformation.
Supplementary Figure 3. Analysis of site-specific mutations within the Os11N3 gene promoter
in T0 callus lines expressing the Pair 1 TALENs.
Supplementary Figure 4. Sequence of Os11N3 gene mutations in T1 plants induced by the Pair
1 TALENs.
Supplementary Figure 5. Disease resistance in transgenic rice T1 plants.
Supplementary Figure 6. Additional haplotypes detected in T2 plants carrying Os11N3 gene
mutations produced with Pair 2 TALENs.
Supplementary Figure 7. Genetic Segregation of forty T2 plants derived from self-pollination
of three T1 plants associated with the Pair 2 TALENs.
Supplementary Figure 8. Expression of Os11N3 (a) and Os8N3 (b), respectively, induced by
PthXo3- and PthXo1-dependent Xoo strains in T2 plants of different genotypes.
Nature Biotechnology: doi:10.1038/nbt.2199
