letters

A gene cluster encoding lectin receptor kinases confers

broad-spectrum and durable insect resistance in rice
Yuqiang Liu1,3, Han Wu1,3, Hong Chen1, Yanling Liu1, Jun He1, Haiyan Kang1, Zhiguang Sun1, Gen Pan1,
Qi Wang1, Jinlong Hu1, Feng Zhou1, Kunneng Zhou1, Xiaoming Zheng2, Yulong Ren1, Liangming Chen1,
Yihua Wang1, Zhigang Zhao1, Qibing Lin2, Fuqing Wu2, Xin Zhang2, Xiuping Guo2, Xianian Cheng1,
Ling Jiang1, Chuanyin Wu2, Haiyang Wang2 & Jianmin Wan1,2

The brown planthopper (BPH) is the most destructive pest of broken down in just a few years owing to the rapid adaptation of
© 2014 Nature America, Inc. All rights reserved.

rice (Oryza sativa) and a substantial threat to rice production,
causing losses of billions of dollars annually1,2. Breeding
of resistant cultivars is currently hampered by the rapid
breakdown of BPH resistance2. Thus, there is an urgent
need to identify more effective BPH-resistance genes.
Here, we report molecular cloning and characterization of
Bph3, a locus in rice identified more than 30 years ago that
confers resistance to BPH. We show that Bph3 is a cluster
of three genes encoding plasma membrane–localized lectin
receptor kinases (OsLecRK1-OsLecRK3). Introgression
of Bph3 into susceptible rice varieties by transgenic or
marker-assisted selection strategies significantly enhanced
resistance to both the BPH and the white back planthopper.
Our results suggest that these lectin receptor kinase genes
function together to confer broad-spectrum and durable
insect resistance and provide a resource for molecular
breeding of insect-resistant rice cultivars.
BPH (Nilaparvata lugens Stål, Hemiptera, Delphacidae) is a monopha-
gous, phloem-sucking herbivore. It sucks the sap from the rice phloem
using its stylet, and causes direct damage to rice plants. BPH can
also cause indirect damage to rice plants through the transmission of
viruses including the rice ragged stunt virus and grassy stunt virus2.
Repeated overapplication of pesticides for BPH management has
heavily polluted the environment3. Breeding of resistant cultivars
is the most cost-effective and environmentally responsible strategy
for BPH management but developing insect resistant cultivars
by traditional breeding approaches is extremely difficult and time
consuming owing to a paucity of knowledge about BPH resistance
genes and germplasm.
To date, 28 BPH resistance loci have been identified from cultivated
and wild species of Oryza2,4,5. Only two of these resistance genes,
Bph14 and Bph26 have been cloned to date6,7. In addition, BPH resist-
ance of IR26 and IR36, two widely cultivated rice varieties that harbor
the BPH resistance loci Bph1 and bph2, respectively, was quickly
the BPH8. Thus, there is still an urgent need to identify new types of
resistance genes and germplasm for developing efficient approaches
to breed broad-spectrum and durable BPH-resistant rice cultivars.
Notably, the Bph3 locus, originally identified in the Sri Lankan
indica cultivar Rathu Heenati9, displayed resistance to four BPH bio-
types (BPH biotypes refer to specific populations of BPH classified
according to their virulence on different BPH resistance genes) 2,10.
Furthermore, rice varieties harboring Bph3 deployed more than
30 years ago in the Philippines are still resistant to BPH8. However,
the molecular basis of this broad-spectrum and durable resistance of
Bph3 against BPH remains unknown.
We observed that Bph3-containing Rathu Heenati infested with
BPH of mixed biotypes (biotype 2 being the dominant one) collected
from a field in Nanjing were not visibly damaged, whereas BPH infes-
tation caused 100% mortality in the susceptible japonica cultivar 02428
(Fig. 1a,b) and indica cultivar Taichung Native 1 (Supplementary
Fig. 1a,b). In addition, fewer BPHs were found on Rathu Heenati
than on 02428 and Taichung Native 1 in a feeding preference experi-
ment (Fig. 1c,e and Supplementary Fig. 1c). When infested with a
fixed number of BPHs on Rathu Heenati, 02428 and Taichung Native
1 seedlings in enclosed cages, the BPH population on 02428 and
Taichung Native 1 increased steadily over time. In contrast, the BPH
population on Rathu Heenati decreased dramatically within a few
hours of infestation (Fig. 1e and Supplementary Fig. 1d). In addition,
both the honeydew excreted by BPH and the BPH survival rate on
Rathu Heenati in a no-choice feeding test were much lower than
those on 02428 and Taichung Native 1 (Fig. 1f,g and Supplementary
Fig. 1e,f). The reduced amount of honeydew excretion from BPH on
Rathu Heenati is consistent with the previous finding that BPH is less
effective in ingesting sieve sap on Rathu Heenati based on an electrical
penetration graphs (EPGs) study11. Collectively, these observations
indicate that Rathu Heenati displays a strong resistance to BPH prob-
ably through interfering with BPH feeding.
Bph3 was previously mapped to the short arm of chromosome 4
(refs. 12,13). Using 6,514 BC2F2:3 and 13,256 BC3F2:3 lines derived

1National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University,
Nanjing, China. 2National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences,
Beijing, China. 3These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.W. (wanjm@njau.edu.cn
or wanjianmin@caas.cn).

Received 7 September 2013; accepted 15 October 2014; published online 8 December 2014; doi:10.1038/nbt.3069

nature biotechnology  advance online publication 

 letters

Figure 1  Comparison of BPH resistance in Rathu a b c

Heenati (RH) and 02428. (a) Representative 100

Seedling mortality rate (%)

image of RH and 02428 infested with BPH.
80
Scale bar, 1 cm. (b) Seedling mortality rate of
RH and 02428 infested with BPH. Data were 60
collected 10 days post infestation (dpi). Error
bars, mean of 3 replicates ±s.e.m. (about 40
25 seedlings each). (c) Representative images
20
of leaf sheaths of 02428 and RH attacked by
**
02428 RH

BPH, showing many fewer BPH on RH. Scale 0

bars, 1 mm. (d) Dynamic changes of the BPH 02428 RH 02428 RH 02428 RH
populations on 02428 (solid circles) and d e f g
RH (open circles) in the feeding preference 02428 RH 02428 RH

Number of BPH per plant

Number of BPH per plant
140

Honeydew per 24 h (mg)

300 3.0 100
** ** **

BPH survival rate (%)

experiment. (e) Dynamic changes of the BPH 250 ** 120
** 2.5
** 80
populations on single seedling of 02428 and RH 200
100 * ** 2.0
80 60
infested with 90 BPH in a closed container. 150 1.5
** 60
(f) Measurement of honeydew excreted from BPH 100 1.0 ** 40
40
on RH and 02428. (g) BPH survival rate on RH 50
** 0.5 20 **
20
and 02428 in a non-choice feeding experiment. 0 0 0 0
Error bars, mean ± s.e.m., by Student’s t-test 0 3 6 12 24 36 72 0 612 24 48 72 96 120 02428 RH 02428 RH
(d, e, g, n = 3; f, n = 5, *P < 0.05, **P < 0.01). Hours after infestation Hours after infestation
© 2014 Nature America, Inc. All rights reserved.

from a cross between Rathu Heenati and 02428, we further mapped
Bhp3 to a 79-kb genomic interval. This region contains seven anno-
tated genes, including three tandem duplicated genes (Os04g0201900,
Os04g0202350 and Os04g0202500) predicted to encode lectin recep-
tor kinases. In addition, there is another lectin receptor kinase
gene (Os04g0202800) located immediately outside of the 79-kb
interval (Fig. 2a). These four genes form a gene cluster encoding
putative lectin receptor kinases, and we named them OsLecRK1 (for
Oryza sativa Lectin Receptor Kinase 1, Os04g0201900), OsLecRK2
(Os04g0202350), OsLecRK3 (Os04g0202500) and OsLecRK4
(Os04g0202800), respectively. These four OsLecRK proteins all belong
to the G-type LecRK family (Supplementary Fig. 2a)14, which has a
domain structure characteristic of LecRK proteins, including an extra-
cellular bulb-type lectin domain, a plant PAN/APPLE-like domain, a
transmembrane domain and an intracellular serine/threonine kinase
domain (Fig. 2b and Supplementary Fig. 2b). As several lectin recep-
tor kinases have been implicated in disease and insect resistance in
several plant species15–18, these four OsLecRK genes were selected for
further investigation as the candidate genes for Bph3.
Sequence comparison of the OsLecRK gene cluster in Rathu
Heenati and 02428 revealed several nucleotide polymorphisms
in 02428, such as premature termination caused by a 7-bp dele-
tion in OsLecRK2, multiple nonsynonymous nucleotide substitu-
tions in OsLecRK1 and OsLecRK3, and a >8-kb insertion upstream
of OsLecRK3 (Supplementary Fig. 3). We were unable to obtain
OsLecRK4 PCR products using four pairs of specific primers, sug-
gesting that OsLecRK4 may be missing or highly diverged in 02428.
Further analysis of the genomic sequences of the OsLecRK gene
cluster in Ptb33 and IR72 (two indica cultivars known to harbor
Bph3)19, and four other newly identified Bph3-containing accessions
(Supplementary Fig. 4) revealed that all these resistant varieties have
identical amino acid sequences for OsLecRK1-OsLecRK3 as in Rathu
Heenati, whereas the BPH-susceptible varieties 02428, Nipponbare
and 9311 have a number of nucleotide polymorphisms in these genes,
causing either amino acid substitutions, protein truncation or reading
frame shift (Fig. 2b). However, for OsLecRK4, polymorphisms were
found not only between the resistant and susceptible varieties, but
also between the resistant varieties (Fig. 2b). Thus, the nucleotide
polymorphisms in OsLecRK1–OsLecRK3 might be mainly responsible
for the differential BPH resistance observed in those resistant and
susceptible varieties.
To test whether the OsLecRK gene cluster confers Bph3 resist-
ance, we transformed a susceptible japonica variety, Kitaake, with
genomic sequences of the Rathu Heenati OsLecRK genes individually
or in combination. Consistent with our mapping results (Fig. 2a),
transgenic plants carrying OsLecRK1, OsLecRK2 or OsLecRK3 genes
had significantly higher resistance to BPH (P = 0.0007, P = 0.0251
and P = 0.0001, respectively, by Welch’s ANOVA) than the nontrans-
formed control plants, whereas transgenic plants carrying OsLecRK4
had a mortality rate not significantly different from the nontrans-
genic control (Fig. 3a,b). Though plants transformed with a gene
stack of OsLecRK1 and OsLecRK2 displayed a similar level of resist-
ance as the OsLecRK1, OsLecRK2 or OsLecRK3 single-gene trans-
formants, transgenic plants co-expressing OsLecRK1, OsLecRK2 and
OsLecRK3 exhibited higher resistance to BPH than the OsLecRK1
or OsLecRK2 single-gene transformants (P = 0.0281 and P = 0.0001,
respectively, by Welch’s ANOVA), despite no significant differences
from the OsLecRK3 single-gene transformants (Fig. 3a,b). Real-
time reverse transcriptase (RT)-PCR analysis and seedling mortality
assay of representative transgenic lines showed varying degrees of
correlation between BPH resistance and the transcript levels of the
OsLecRK genes (Supplementary Fig. 5). These observations suggest
that OsLecRK1–OsLecRK3 act together in conferring BPH resistance,
but OsLecRK4 does not play a major role in BPH resistance. Notably,
none of the transformants displayed the degree of resistance observed
in the Rathu Heenati plants, supporting the previous proposition
that besides Bph3, other minor loci may also contribute to the BPH
resistance in Rathu Heenati9.
To further confirm the role of OsLecRK1, OsLecRK2 and OsLecRK3
in BPH resistance, we knocked down the expression of these OsLecRK
genes in a near isogenic line (NIL) carrying the Rathu Heenati Bph3
locus in 02428 background. The reduction of BPH resistance in
the RNA interference (RNAi) transgenic lines was approximately
correlated with the reduction of the transcript levels of these OsLecRK
genes (Fig. 3c–e). Notably, expression of Os06g41560 and Os08g13420
(two paralogous genes sharing the highest sequence similarities to
OsLecRK1–OsLecRK4) was not significantly affected in a RNAi line
(#1) with significantly (P < 0.05, t-test) reduced BPH resistance,
suggesting that these two paralogous genes are probably not involved
in BPH resistance (Supplementary Fig. 6).
Furthermore, we also observed differential BPH resistance in
several recombinant lines with breakpoints in this OsLecRK gene

 advance online publication  nature biotechnology

 letters

a RM8213 A4 RH7845 RM5953 RM307 RM401

F2
n = 2013
RH7845 RH784 RH078 W4 RM16533 RM5,953

BC2F2
RH078 RHD3 RHD9 RH7 RHC10 W1 W4 n = 6,514

57 kb 22 kb
56 6 4 1 2 4 BC3F2
n = 13,256

Os04g020100 Os04g0201200 Os04g0201500 Os04g0201800 OsLecRK1 OsLecRK2 OsLecRK3 OsLecRK4

b OsLecRK1 OsLecRK2
Lectin PAN-AP TM S-TKC Lectin PAN-AP TM S-TKC
246 D/G

591 R/G
35 G/S
72 D/N

122 G/S
129 C/R
178 D/N

556 H/D

134 N/D

424 N/D

752 G/S
357 E/D
398 E/D

680 K/N
90 K/E

151 P/S
445 F/S

612 T/S
289 L/V

632 F/L

631 L/F

758 L/F
492 I/A
554 I/K
Rice
varieties Phenotype
RH
© 2014 Nature America, Inc. All rights reserved.

Ptb33
BP348-e-4-2
Lv-4 R
Lv-7
Sarinah
IR72
02428
9311 S
Nipponbare

OsLecRK3 OsLecRK4
Lectin PAN-AP TM S-TKC Lectin PAN-AP TM S-TKC

441 S-/RK
48 D/G

89 G/D

218 D/G

276 H/Q

302 M/K

539 Q/R
88 Q/K

169 T/M

337 K/Q

428 P/Q

72 H/D
391 R/S

407 A/D

481 M/L

503 N/K
3 H/F

166 S/P

189 A/V
201 A/S

279 P/S
298 S/A

388 E/K

544 F/C

658 V/A
54 R/L

92 M/I
19 K/-
9 I/F

234 T/I

395 I/T

495 I/T

546 T/I

Rice
varieties Phenotype
RH
Ptb33
BP348-e-4-2
Lv-4 R
Lv-7
Sarinah
IR72
02428
9311 S
Nipponbare

Figure 2  Mapping of Bph3 and polymorphisms between resistant and susceptible varieties. (a) Fine mapping of Bph3 to a 79-kb genomic region.
The interval contains seven predicted ORFs. Number of recombinants is shown below the DNA markers closely linked to Bph3. BC2F2, second filial
generation after second backcross. Os04g020100, hypothetical protein; Os04g0201200, armadillo-like helical domain containing protein;
Os04g0201500, amino acid/polyamine transporter II family protein; Os04g0201800, hypothetical protein. (b) Identical amino acids for OsLecRK1,
OsLecRK2 and OsLecRK3 in BPH-resistant varieties. Polymorphic sites in the susceptible varieties are positioned relative to the first amino acid in RH.
An additional amino acid (K) in OsLecRK4 in Nipponbare and 9311 is highlighted in red. Lectin, a type of carbohydrate-binding protein domain; PAN-AP,
Plant PAN/APPLE-like domain; TM, transmembrane domain; S-TKC, serine/threonine kinase catalytic domain; −, deleted or reading frame shifted. The
sequence information for Nipponbare and 9311 is from the GenBank database (http://www.ncbi.nlm.nih.gov/; http://rise.genomics.org.cn/rice/index2.jsp).

cluster. Lines 0027-04, 0200-01 and 0200-06, each of which carry all three
genes (OsLecRK1–OsLecRK3), were highly resistant to BPH; line 0027-15
carrying OsLecRK2-OsLecRK3, but lacking OsLecRK1, conferred
~50% of the resistance; line 2648-07 carrying OsLecRK3 only, but
lacking both OsLecRK1 and OsLecRK2, had only ~25% of the
resistance; whereas line 0200-01 lacking only OsLecRK4 showed
no significant reduction in BPH resistance (Fig. 3f). Taken together,
these results demonstrate that the OsLecRK1-OsLecRK3 gene cluster
represents Bph3 and suggest that members of this cluster act together
in conferring high levels of BPH resistance. These observations
are consistent with previous findings suggesting that clustering
of highly homologous resistance genes or copy number variation
of dissimilar genes at a locus can confer enhanced biotic or abiotic
stress resistance20,21.
Real-time RT-PCR analysis showed that OsLecRK1–OsLecRK3 dis-
played differential expression patterns in response to BPH infestation
between Rathu Heenati and 02428. Upon BPH infestation, expres-
sion of OsLecRK1 was markedly induced in Rathu Heenati and the
NIL line (02428 background carrying the Bph3 locus from Rathu
Heenati), but not in 02428; OsLecRK2 was induced in Rathu Heenati,
02428 and the NIL line, whereas OsLecRK3 showed the most dra-
matic induction by BPH infestation in Rathu Heenati and the NIL
line, but its expression was not detectable in 02428 (Supplementary
Fig. 7). Consistent with a role for OsLecRK1–OsLecRK3 in BPH
resistance, β-glucuronidase (GUS) reporter genes driven by the
promoters of OsLecRK1–OsLecRK3 showed enhanced staining in
the vascular bundles of leaf sheaths, and the GUS activities were
significantly induced upon BPH infestation (Supplementary Fig. 8).
GFP fusions of OsLecRK1–OsLecRK3 proteins were all localized to
the plasma membrane when transiently expressed in rice leaf sheath
protoplasts (Supplementary Fig. 9), consistent with their predicted
membrane localization.

nature biotechnology  advance online publication 

 letters

Figure 3  OsLecRK genes contribute additively a b

to BPH resistance. (a) Representative image a
a
100 b

Seedling mortality rate (%)

showing acquired partial BPH resistance of bc c
b
Kitaake plants transformed with OsLecRK1– 80 bc
OsLecRK4 genomic sequences (gL1, gL2, 60
gL3 and gL4 respectively). Photo was
taken at 10 dpi. RH, Rathu Heenati. 40
(b) Measurement of seedling mortality 20
rates in Kitaake plants transformed d
0
with various OsLecRK genes. Dots represent
the mean of two replicates (about 25 seedlings

Ki H
gL e

gL 4)

gL )

gL L2 )
gL 0)

3)
3
(7

1+ +g (4
ak
R

(1

(1

2+ (1

(1
H

ke

3
gL

gL

gL

gL

gL

gL

gL L1 L4
ta
R

3
each) for an individual transgenic line. The

ta

1+

2+

g
Ki

gL

gL
horizontal bars represent the grand mean of
c d

1+

g
gL
all lines for a given transgene. The numbers c c
100
in parentheses indicate the number of

Seedling mortality rate (%)

independent transgenic lines. Groups that 80 b
b
share the same letters are not significantly b
60
different. Different letters at the top of each
column indicate a significant difference at 40
P < 0.05, by Welch’s ANOVA and Bonferroni
a
correction for multiple tests. (c) Representative 20
a
image of seven representative RNAi seedlings a
damaged by BPH infestation. (d) Seedling 0
© 2014 Nature America, Inc. All rights reserved.

NIL #1 #2 #3 #4 #5 #6 #7 NIL #1 #2 #3 #4 #5 #6 #7
mortality rates of the RNAi transgenic lines.
Error bars, mean of 3 replicates ± s.e.m. e2.5
RNAi lines
f
RNAi lines

(about 25 seedlings each). Groups with same OsLecRK1 OsLecRK2 Seedling mortality rate (%)
OsLecRK3 OsLecRK4 OsLecRK
letters indicate no significant differences.

100
2.0 Lines

20
40
60
80
Relative expression

0
1 2 3 4
Different letters at the top of each column 02428 – – – – c
indicate a significant difference at 1.5
RH + + + + 0 a
P < 0.05, by Welch’s ANOVA and Bonferroni
1.0 0027-04 + + + + 0 a
correction for multiple tests. (e) qPCR
analysis of OsLecRK gene expression in 0.5
0027-15 – + + + b
the RNAi transgenic lines. The expression 2648-07 – – + + b
values were presented relative to those in 0
0200-06 + + + + 0 a
the NIL (near isogenic line). (f) Seeding NIL #1 #2 #3 #4 #5 #6 #7
RNAi lines 0200-01 + + + – a
mortality rates in a set of Bph3 recombinant
lines upon BPH infestation. Plus and minus
denote presence or absence of specific OsLecRK gene, respectively. Error bars, mean of 3 replicates ±s.e.m. (about 25 seedlings each). Different
letters at the top of each column indicate a significant difference at P < 0.05 (n = 3), by Student’s t-test.

Wild rice species are believed to provide a useful source for insect
resistance22. Comparison of genomic sequences for each of the three
OsLecRK genes among 50 rice collections including both wild and
cultivated rice23 (Supplementary Table 1) revealed that these genes
in Rathu Heenati share the highest similarities to those in O. nivara
(Fig. 4a), the progenitor of cultivated rice in Asia and an important
donor of BPH resistance23,24. Notably, the Bph3-containing varieties
identified in our screen (Supplementary Fig. 4) are all indica from
South or Southeast Asia where BPH populations exist year round.
To test the potential utility of Bph3 in breeding BPH resistance vari-
eties, we introduced the Bph3 locus from Rathu Heenati into a suscep-
tible commercial japonica variety, Ningjing 3, using marker-assisted
selection. A Bph3-containing near-isogenic line (R1266) of ~98.9%
Ningjing 3 genetic background was highly resistant to BPH under
both greenhouse and the field conditions (Fig. 4b and Supplementary
Fig. 10). Similar to Rathu Heenati25, R1266 also displayed significantly
enhanced resistance (P < 0.05, t-test) against white back planthopper,
another destructive rice phloem-sucking herbivore (Supplementary
Fig. 11). These results demonstrate that Bph3 is functional in confer-
ring broad resistance against planthoppers.
Plants utilize both cell surface–localized, pattern-recognition
receptors and cytoplasmic R proteins (mostly nucleotide-binding,
leucine-rich repeat proteins) to build a two-tiered immune system
(pattern-triggered immunity and effector-triggered immunity) for

Figure 4  Origin of Bph3 and marker assisted

selection-based breeding of BPH-resistant a OsLecRK1 OsLecRK2
b
cultivars. (a) The OsLecRK cluster from Rathu
Heenati (RH) is most closely related to its
counterpart in the wild rice O. nivara. O. rufipogon
is also a wild rice. The genealogical trees of
50 samples for the OsLecRK1 to OsLecRK3 were
constructed by PAUP* version 4.0b10, using OsLecRK3
the neighbor-joining method. (b) Acquired BPH
resistance in R1266 at the seedling stage. The
O. nivara
Bph3 locus was introgressed into the commercial O. rufipogon
japonica variety Ningjing 3 from RH. R1266, a Cultivar
near-isogenic line whose genome was ~98.9% RH

similar to the genome of Ningjing 3, was selected. Ningjing 3 R1266

 advance online publication  nature biotechnology


effective defense against diverse pathogenic microbes26. It is known
that R gene–mediated resistance is easily overcome by pathogens that
mutate or produce new effectors to counteract effector-triggered immu-
nity27. In contrast, pattern-triggered immunity is generally believed to
confer broad-spectrum and durable resistance to pathogenic microbes,
owing to the conserved and essential nature of pathogen- or microbe-
associated molecular patterns (PAMP or MAMP)28–30, although there
have been rare reports that variations in PAMPs could evade recogni-
tion by host pattern-recognition receptors31. The previously reported
Bph14 and Bph26, which encode cytoplasmic R proteins6,7, may induce
effector-triggered immunity by perceiving the effectors derived from
insect-feeding. In conjugation with the recent findings that LecRK
proteins can function as extracellular ATP receptor or potential cell
surface receptors for insect- or plant pathogen-derived elicitors and
prime pattern-triggered immunity responses32–35, the cell surface
localization of OsLecRK proteins suggests that these proteins may
play a critical role in priming the pattern-triggered immunity response
to BPH infestation by perceiving herbivore-associated molecular
patterns (HAMPs) or damage-associated molecular patterns (DAMP),
or mediating the downstream signaling events, leading to broad-
© 2014 Nature America, Inc. All rights reserved.
spectrum and durable resistance against planthoppers. Further
analyses of the biochemical function of these OsLecRK proteins will
advance our understanding of the molecular mechanisms governing
plant-insect interactions. The cloning of Bph3 and its functional char-
acterization provides target genes and germplasm for breeding rice
cultivars with broad-spectrum and durable insect resistance through
marker-assisted selection or resistance gene pyramiding.
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. Entrez Nucleotide: KF748957–KF748987
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
This work was supported by Jiangsu 333 Program (BRA2012126), NSFC
(31471470), 863 Program of China (2012AA101101, 2011AA10A101 and
2011BAD35B02-02), National Science and Technology Supporting Program
(2011BAD35B02-02), National Key Transformation Program (2014ZX08001-001),
Jiangsu Science and Technology Development Program (BE2012303, BK2010016),
and the Jiangsu PAPD program. We thank R. Guan (Nanjing Agricultural
University) and J. Wang (Chinese Academy of Agricultural Sciences) for statistical
analysis, and J. Zhou (Chinese Academy of Sciences), W. Terzaghi (Wilkes
University) and C. Lin (UC-LA) for proofreading the manuscript.
AUTHOR CONTRIBUTIONS
Y.Q.L., H.K., Z.S., G.P. and J.He performed genetic analysis and mapping of Bph3;
X. Zhang, X.G. and L.J. performed the genetic transformation; H. Wu, H.C., Q.W.
and F.Z. performed the construction of vectors; K.Z., Y.R. and Y.W. performed the
subcellular localization; X. Zheng, Z.Z., Q.L. and F.W. performed sequence analysis;
Y.L.L., J. Hu, L.C. and X.C. performed the phenotypic analysis. J.W., H. Wang and
C.W. directed the project and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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 ONLINE METHODS
Plant materials. The rice cultivar Rathu Heenati (RH), IR72, Ptb33 and other
rice accessions were collected from the International Rice Research Institute
(Los Banos, the Philippines) and have been described previously12,22. Other
rice germplasms used in this work were obtained from the Chinese Crop
Germplasm Resources Center (Beijing, China). Four F2 populations, includ-
ing BP348-e-4-2/02428 F2, Sarinah/02428 F2, Lv-4/02428 F2 and Lv-7/C418 F2
populations were generated to map the BPH resistance loci. The near isogenic
lines (NIL) or R1266 containing Bph3 was selected from the backcross progeny
(backcrossed 4 or 6 times, respectively) between RH with 02428 or Ningjing
3, using 02428 or Ningjing 3 as the recurrent parent. The genotypes of each
NIL at Bph3 locus were determined using molecular markers for fine mapping
(Supplementary Table 2).
Brown planthopper maintenance. A colony of the brown planthopper
(BPH) was collected from rice fields in Nanjing (where the BPHs are mixed
biotypes and mainly biotype 2) (refs. 36,37), and maintained on the susceptible
cultivar Taichung Native 1 (TN1) under greenhouse conditions at Nanjing
Agricultural University.
Evaluation of BPH resistance. BPH resistance was scored according to
the standard evaluation systems of International Rice Research Institution
© 2014 Nature America, Inc. All rights reserved.
(IRRI)38. A seedling bulk test was conducted to phenotype plant reaction to
BPH feeding. To ensure all seedlings were at the same growth stage for BPH
infestation, seeds were germinated in Petri dishes. About 30 seeds from each
individual plant were sown in a 10 cm-diameter plastic pot with a hole at the
bottom. Seven days after sowing, seedlings were thinned to 25 plants at least
per pot. At the second-leaf stage, the seedlings were infested with 2nd to 3rd
instar BPH nymphs at ten insects per seedling. When all the TN1 plants were
dead, the seedling mortality of other cultivars or lines was recorded. At least
two replicates were used for each cultivar or line.
Fine mapping of the Bph3 locus. Three genetic populations segregating for
resistance to BPH were used for fine mapping. These include an F2 (2,103
lines), a BC2F2 (6,514 lines) and a BC3F2 (13,256 lines) population generated
from the cross between RH and 02428, which were self-fertilized to produce F3
lines. The evaluation experiments were repeated three times, and the resistance
score of each F2 individual was inferred from the weighted average of the scores
for the seedlings in the corresponding F3 families. To confirm the phenotypes
of the recombinants, we generated ten F4 lines for each recombinant individual
and evaluated their BPH resistance. Bph3 was initially mapped to a region on
chromosome 4 flanked by the SSR markers RM8213 and RM5953 (ref. 12).
Additional DNA markers were designed based on published sequences of
Nipponbare and 93-11 in the GenBank database (http://www.ncbi.nlm.nih.
gov/; http://rise.genomics.org.cn/rice/index2.jsp) (Supplementary Table 2).
Two newly developed SSR markers RH7845, RM5953 and two Indel markers
RH078 and W4 were used to screen the recombinants from the BC2F2 and
BC3F2 populations, respectively.
Feeding behavior of BPH on RH and 02428. Two methods were used to assess
the preference of BPH for different rice cultivars. Seeds of RH and 02428 were
sown in 10-cm-diameter pots, with one seed per pot. The plants were trimmed
to one tiller 35 days after sowing. In the first assay, two pots were arranged
in a 2 m × 2 m × 1.8 m sealed cage together with a BPH-heavily infested TN1
plant as the BPH source. The number of insects on each variety was recorded
1, 3, 6, 12, 24, 48 and 72 h after the infestation. In the second assay, the plants
were arranged as above except that each plant was infested with 90 BPH. The
number of BPH on the plant was recorded at 3, 6, 12, 24, 48, 72, 96 and 120 h
after infestation. The experiments were repeated three times. To study nymph
survival on seedlings, about 20 pre-germinated seedlings of each cultivar were
grown in a 10 cm-diameter plastic pot. The pots were randomly placed in a
68 cm × 42 cm × 16 cm plastic seed-box supplied with water at the bottom
of the tray. Seedlings were thinned to ten plants per pot 7 days after sowing,
and at the third-leaf stage, the seedlings were infested with 2nd-instar BPH
nymphs at ten insects per plant. The potted plants were then covered with
cylindrical mylar cages (13 × 50 cm). The number of nymphs per plant was
counted 4 days after infestation. The experiments were repeated three times.
nature biotechnology
Honeydew produced by adult insects was estimated as described previously39.
Briefly, newly emerged brachypterous females (12–24 h old) were starved,
but provided with water by a moist filter paper in a Petri dish, for 4 h. A sin-
gle insect was then enclosed in a Parafilm sachet attached to the lower part
(0–5 cm from the base) of a main tiller of a 6-week old rice plant grown in a
pot. A similar sachet (transpiration control) without insect infestation was
attached to the base of a main tiller of a second plant to estimate the transpired
water at the same position on the plant. The insect was removed after 24 h
of feeding by making an incision through the upper part of the sachet. The
sachets were removed from the plants and weighed before and after blotting
on filter paper. The quantity of transpired water accumulated in the control
sachet was used as a correction factor to determine the quantity of honeydew
excreted by the female BPH and the weight of honeydew was calculated by
the following formula:
Honeydew (mg) = (W1–W2) – (T1–T2)
Where W1 = weight (mg) of sachet (originally with an insect) before
blotting, W2 = weight (mg) of sachet (originally with an insect) after blotting,
T1 = weight (mg) of sachet (control) before blotting, and T2 = weight (mg) of
sachet (control) after blotting. Five replicates were used for each cultivar.
Plasmid construction. To construct the plasmids for complementation
test, the genomic DNA fragments (including the promoter regions, the entire
CDS region and the downstream sequence) of OsLecRK1 (~6.8 kb), OsLecRK2
(~5.6 kb), OsLecRK3 (~5.0 kb) and OsLecRK4 (~4.8 kb) were amplified using
gene-specific primers (Supplementary Table 2) from RH seedling DNA,
and the PCR products were inserted into the binary vector pCAMBIA1305.1
to produce pCAMBIA1305-OsLecRK1, pCAMBIA1305-OsLecRK2, pCAM-
BIA1305-OsLecRK3 and pCAMBIA1305-OsLecRK4, respectively. All
constructs were verified by DNA sequencing.
To construct multiple complementation vectors, we added an ApaI site
into the forward primer of 1305-RK2 (KpnI/XbaI); we used primer 1305-RK2
(KpnI/XbaI) to amplify OsLecRK2 from RH DNA, and the PCR product was
inserted into the KpnI and XbaI site of pCAMBIA1305-OsLecRK1 to produce
pCAMBIA1305-OsLecRK1-OsLecRK2. At last, OsLecRK3 was amplified using
primer 1305-RK3(KpnI/ApaI) from RH DNA and the product was inserted
into the KpnI and ApaI site of plasmid pCAMBIA1305-OsLecRK1-OsLecRK2
to produce pCAMBIA1305-OsLecRK1-OsLecRK2- OsLecRK3.
To generate the RNAi construct, two copies of a 719-bp cDNA fragment
sharing 76.4%, 100%, 92.2% and 66.9% homology to OsLecRK1-OsLecRK4,
respectively, were amplified by PCR using primers RNAi1 and RNAi2
(Supplementary Table 2) and inserted as inverted repeats into the PA7 vector
to generate a hairpin RNAi construct, which was then cloned into the binary
vector pCAMBIA1305 with HindIII and EcoRI digestion.
For subcellular localization, the OsLecRK genes coding sequence, amplified
from RH cDNA by PCR using the primers OsLecRK1-GFP, OsLecRK2-GFP
and OsLecRK3-GFP (Supplementary Table 2), was cloned into the vector
PA7, and fused with the N terminus of GFP, resulting in the LecRK–GFP fusion
protein–expressing plasmid PA7-LecRK.
For the β-glucuronidase (GUS) reporter gene constructs, 2,111-bp,
1,681-bp, 2,825-bp and 2,424-bp genomic fragment corresponding to sequence
5′ of the ATG start site of the four OsLecRK genes were amplified using
RH genomic DNA as the template and the primer pairs OsLecRK1-P,
OsLecRK2-P and OsLecRK3-P (Supplementary Table 2), respectively,
and the PCR products were cloned into the pCAM1381 vector to generate
transcriptional fusions with the GUS gene.
Rice transformation. The genetic complementation and RNAi constructs
were transformed into Agrobacterium tumefaciens strain EHA105, respectively,
and subsequently transformed into the rice variety Kitaake and NIL (carrying
Bph3 in a 02428 background), as previously described40. Plants regenerated
from hygromycin-resistant calli (T0 plants) were grown, and T1 seeds were
obtained after self-pollination. The genotypes of each transgenic plant and
their progenies were examined by PCR amplification using gene-specific prim-
ers. For transient assay in rice protoplasts, plasmids for subcellular localization
and a plasma membrane marker aquaporin PIP2a41 were used to co-transform
rice protoplasts, as reported previously42.
doi:10.1038/nbt.3069
 RNA isolation and qPCR analysis. Total RNA was isolated from rice seedlings
using the RNAprep Pure Plant Kit (Tiangen) according to the manufacturer’s
instructions. Quantitative RT–PCRs were performed by a SYBR Premix Ex
Taq RT–PCR Kit (Takara) following the manufacturer’s instructions with the
primers listed in Supplementary Table 2.
Sequence analysis. LecRK family proteins were screened by BLAST searching
of the protein databases of GRAMENE and TAIR (GRAMENE, http://www.
gramene.org/; TAIR, http://www.arabidopsis.org/). The phylogenetic tree of
the LecRK family based on the protein sequences was constructed as previ-
ously reported43. The sequences of OsLecRK1 to OsLecRK3 from 50 accessions
were obtained from the study by Xu et al.23 and used to construct the genea-
logical trees. Genealogical trees were constructed by PAUP* version 4.0b10,
using the Neighbor-Joining (NJ) method44.
GUS histochemical staining. GUS staining was done following the stand-
ard procedures45. Seedlings were collected from at least five independent
lines for each construct, submerged in the staining solution and incubated at
37 °C up to 48 h or until blue color became visible. Then the samples were
incubated with 95% ethanol for 24 h to remove the chlorophylls. Sheath
segments were washed three times and sectioned into about 10-µm-thick
samples for microscopic examination.
© 2014 Nature America, Inc. All rights reserved.
GUS activity was measured by monitoring the cleavage of the β-glucuronidase
substrate 4-methylumbelliferylβ-d-glucuronide (MUG, Sigma)46. Final GUS
activity values were recorded as pmoles MU/min/mg protein. The experiments
were repeated three times.
doi:10.1038/nbt.3069
Microscopy. The GFP fluorescence was visualized with a confocal microscope
(Nikon Eclipse TE2000-U). GUS staining images were captured using a Nikon
80A camera and processed with Photoshop.
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