Euphytica

DOI 10.1007/s10681-014-1302-2

Cloning of a gene encoding L RR pr otein and its validation

as candidate gall midge r esistance gene, Gm4, in r ice
Dhanasekar Divya K udapa Himabindu
Sur esh Nair Jagadish S. Bentur

Received: 22 June 2014 / Accepted: 8 November 2014

Springer Science+Business Media Dordrecht 2014

Abstr act The Asian rice gall midge, Orseolia

oryzae, is an important pest of rice. Gm4, a major
rice gall midge resistance gene in the indica rice
variety Abhaya has been ne mapped within 0.3 Mb
region with the anking markers RM22551 and
RM22562. Among the 70 putative candidate genes
identied in the reference Nipponbare ricegenome in
this region, two genes coding for leucine rich repeat
(LRR) proteins were shortlisted for further analysis.

Electr onic supplementar y mater ial The online version of

this article (doi:10.1007/s10681-014-1302-2) contains supplementary material, which is available to authorized users.
D. Divya K. Himabindu J. S. Bentur (& )
Directorate of Rice Research, Rajendranagar,
Hyderabad 500030, India
e-mail: jbentur@yahoo.com
Present Address:
K. Himabindu
International Crops Research Institute for the Semi-Arid
Tropics, Pattancheru 502324, India
S. Nair
International Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067,
India
Present Address:
J. S. Bentur
Agri Biotech Foundation, Rajendranagar,
Hyderabad 500030, India

Polymorphisms was observed between the parents

improved samba masuri (ISM) (susceptible parent)
and Abhaya (resistant parent) and the recombinant
inbred line (RIL)-derived near isogenic lines (preNILs) 482R (resistant line) and 489S (susceptible
line) with one pair of the markers targeting the
candidate genes. Polymorphism co-segregated with
the trait in a larger mapping population consisting of
40 resistant and 40 susceptibleRILs (F10 generation).
A major segment of this gene (LOC_Os08g09670.1),
covering one of the two exons, was cloned and
sequenced from both parents as well as from the two
pre-NILs 482R and 489S. Data revealed a large
deletion in the Abhaya fragment as compared to that
of ISM. At least ve amino acid substitutions were
detected, between the alleles, that are likely to
inuence the protein function. Expression analysis
of this putative candidate gene through reverse
transcription real time PCR showed more than two
fold increase in its expression levels in the resistant
parent Abhaya and resistant line 482R at 24 h after
gall midge infestation when compared with the
uninfested controls. These results suggested the
LRR coding gene to be the candidate for Gm4. In
addition, the current work also identied a functional
marker (LRR-del) for the detection of the gene for
use in marker-assisted introgression of Gm4.
K eywor ds Oryza sativa (L.) Gall midge biotype
(GMB) Resistance gene Functional markers
Sequence polymorphism LRR gene

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I ntr oduction
The Asian rice gall midge [Orseolia oryzae (WoodMason)] is a highly destructive pest of rice in the
continent and is ranked third in economic importance
in India (Bentur et al. 2003). Breeding and cultivation
of resistant rice varieties is the main approach to
contain thispest. So far 11 gall midge resistancegenes
have been reported and nine of these have been
mapped (Yasala et al. 2012; Sama et al. 2014). Seven
distinct biotypes of the pest have been characterized
based on the reaction of gene differential rice varieties
(VijayaLakshmi et al. 2006). Noneof thereported gall
midge resistance genes is effective against all the
biotypes. Emergence of new and virulent biotypes in
response to extensive cultivation of resistant rice
varieties carrying a single gene is a cause for concern.
Pyramiding two or more resistance genes into a single
cultivar has been proposed to achieve durable gall
midge resistance (Cohen et al. 2004). Molecular
markers can support classical breeding in achieving
such goals of gene pyramiding. However, the choice
of genecombinationsto providedesired durability can
be better judged with detailed information on the
resistance genes, used in the combination, and their
respectivemodeof action. Gall midgeresistancegenes
differ in their nature of resistance i.e. with or without
the expression of tissue necrosis typical of a hypersensitive reaction (HR) (Bentur and Kalode 1996).
Genes differing in their resistance mechanism i.e.
HR? and HR- types, could be ideal combination for
pyramiding. It is also possible to extend the range of
resistance across biotypes by combining diverse
resistance genes. Gall midge resistance in the rice
variety Abhaya has been introgressed from the land
race Ptb10. The resistance gene Gm4 identied in
Abhaya (Shrivastava et al. 1993) has been tagged and
mapped using RAPD (Nair et al. 1996) and RFLP
(Mohan et al. 1997a) markers on to chromosome 8 of
rice. Gm4 conferred resistance against ve of the
seven biotypes with HR? type of resistance mechanism. The gene has been mapped within 0.33 Mb
region on chromosome 8 between the SSR markers
RM22551 and RM22562 (Himabindu 2009).
The nucleotide-binding site leucine-rich repeat
(NBS-LRR) proteins are large, abundant proteins
involved in the plant defense through detection of
diverse pathogens, including bacteria, viruses, fungi,
nematodes, insects and oomycetes. NBS-LRR

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containstwo distinct domainsof theprotein connected

through linker region. The rst of the NBS-LRR
encoding genes to be cloned was from Arabidopsis
(Bent et al. 1994). While over 150 NBS-LRR encoding genes are reported in Arabidopsis thaliana (Meyers et al. 2003), rice genome has 653 NBS-LRR
genes(Maroneet al. 2013). Riceblast resistancegenes
Pi-ta, Pi9 and Pi54 encode NBS-LRR proteins (Bryan
et al. 2000; Qu et al. 2006; Ramkumar et al. 2011).
Two bacterial blight (BB) resistance genes Xa1 and
Xa26 are also members of the NBS-LRR family (Sun
et al. 2004). The brown planthopper resistance gene
Bph14 encodes coiled coil nucleotide binding leucine
rich repeat (CC-NB-LRR) protein (Du et al. 2009).
NBS-LRR resistance proteins are classied into two
sub groups. The rst subgroup consistsof proteins that
have a distinct N-terminal region (TIR domain) that
resembles the cytoplasmic domains of the Drosophila
protein Toll and themammalian interleukin-1 receptor
protein. The proteins of the second subgroup lack this
region but may have a leucine zipper or coiled-coil
domain in the N-terminal region (Pan et al. 2000).
NBS and TIR domains have a conserved signaling
function but LRR domain is involved in the pathogen
recognition by either direct interaction with avr
protein or indirectly by binding to the protein complex
between avr protein and other host proteins (Kobe and
Diesenhofer 1995). The LRR domain is a common
motif found in more than 2,000 proteins involved in
proteinprotein interactions and ligand binding (Jones
and Jones1997). Many of thecloned R genesbelong to
NBS-LRR class and confer race-specic resistance
against the pathogens (Martin et al. 2003). In the
present study, we identied a LRR gene as the
candidate gene for Gm4 based on physical location,
structural diversity, co-segregation and functional
validation. We also report a functional marker
(LRR-del) for this gene for use in marker-assisted
breeding.
M ater ials and methods
Plant material
Gall midge resistant rice variety Abhaya was crossed
simultaneously with the gall midge susceptible rice
varieties improved samba mahsuri (ISM) and TN1.
Linkage analysis and map construction have been

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performed earlier using the mapping population

consisting of 91 recombinant inbred lines (RILs) in
F10 generation developed from the cross TN1 X
Abhaya and validated in 233 F2:3 population of the
cross ISM x Abhaya (Himabindu 2009). In the present
study, theformer population wasused for validation of
new designed markers. Leaf samples were collected
from the test plants and used for DNA isolation and
genotyping with molecular markers.

fragments were gel eluted and cloned using Topo

TA cloning kit (Invitrogen, USA) as per the manufacturer s instructions and were transformed into
DH5a cells. Plasmid DNA was isolated using QIAprep spin miniprep (Qiagen, USA). The puried
plasmid DNAs were sequenced at Macrogen, South
Korea. All the four sequences have been submitted to
the GenBank and have been assigned accession
numbers (GenBank ID: KM366134KM366137).

DNA isolation and PCR

In silico analysis of LRR protein

Total genomic DNA was isolated from the leaf tissues

of selected parents and their derived RIL population
through method of Zheng et al. (1995) and used for
PCR amplication following the protocol of Chen
et al. (1997). The map locations, SSR primer
sequences and other details of markers used are
available online at http://www.gramene.org. The PCR
products were resolved on 3 % agarose in 0.5 9 TBE
buffer, stained with ethidium bromide (0.5 l g/ml) and
documented under UV light. The size of the amplied
fragments was calculated using Alphaease software
(Alpha Innotech, USA) with 100 bp ladder (Fermentas, Lithuania) as size reference.

Thesequencedataof thePCR amplied fragments(more

than 50 % of the LRR gene) was analyzed using the
software Bioedit (http//www.mbio.ncsu.edu/bioedit/
bioedit.html). Translation of nucleotide sequences to
amino acid sequences was done for each of the PCR
amplied fragments. Thepredicted amino acid sequences
of thePCR amplied fragmentsfrom ISM, Abhaya, 482R
and489SwerealignedwithLRRisolatedfromrice(Oryza
sativa japonicagroup [Accession no.EAZ05904.1], using
clustalW software (http://embnet.vital-it.ch/software/
clustalW.html). The amino acid sequences were also
usedfor predictingthe3D modelsof proteinusingSWISSMODEL (http://swissmodel.expasy.org). The effect of
substitution on a hypothetical protein structure was
examined in silico by protein variation effect analyzer
PROVEAN and SIFT (http://provean.jcvi.org).

Identication of pre-near isogenic lines through

recombinant inbred lines (RILs)
Forty RILseach with R (with nil gall midgedamage)
and S (with 100 % plant damage) phenotype were
used for the study. These lines were genotyped with
320 SSR markers, spread across the genome including-gene linked markers on chromosome 8. The lines
were rst screened for foreground selection and
further shortlisted for background selection. The pair
(482R and 489S) with maximum genotypic similarity
with contrasting phenotype was identied as pre-NILs
through RILs (Himabindu et al. 2010) for further
study.
PCR amplication, cloning and sequencing
of the candidate Gm4 genes
Primer pairs were designed to PCR amplify different
parts of the genes. In view of the polymorphism
detected by one of the primer pairs -LRR1- between
the parental lines ISM and Abhaya and the RIL
derived 482R and 489S pre-NILs, the amplied

Quantitative real time PCR analysis

The rice varieties TN1, Abhaya, 482R and 489S were
grown under standard condition (Rawat et al. 2012). A
set of 15-day-old plants was exposed to gall midge
biotype1 (GMB1 with 25 femalesand 10 males). Two
days after insect release, the trays were transferred to
high humidity chamber and left there for 2 days.
Uninfested plants were treated as control. The tissue
sampled was the apical meristem region on which gall
midge maggots feed. This part is sampled by cutting
the stem part of the plant at the base and 1 cm above.
After egg hatching, the samples were collected at 24
and 120 h after infestation (hai) as per the protocol
described earlier (Rawat et al. 2012). Real-time PCR
was performed using Applied Biosystems 7500 real
time PCR system with the SYBR green chemistry
(Applied Biosystems, USA) according to the manufacturer s instructions. Gene-specic primers for real
time PCR were designed from the unique region of

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Fig. 1 Representation of
similarity and dissimilarity
observed, at different SSR
loci on chromosome 8,
between the two pre-NIL
pairs (482R & 489S and
321R & 368S). Figures on
left indicate physical
position in Mb

LRR gene using the Primer Express software Version

3.0 (Applied Biosystems, USA). Rice ubiquitin gene,
OsUBC (accession no. AK059694) was used as the
endogenouscontrol. PCR reactionswerecarried out in
10 l l reaction containing 2 l l of rst stand cDNA, 1X
PCR buffer, 125 l M dNTPs, 1.5 mM MgCl 2, 0.2 l M
primers and 1U Taq polymerase. The thermal prole
used was: 94 C for 2 min1 cycle followed by
35cycles of 94 C for 20 s, annealing at 60 C for
20 s, 72 C for 30 s and nal extension of 72 C for
5 min. Melt curve analysis was also performed after
completion of PCR cycles to check specicity of the
PCR amplication. To calculate mean relative expression levels, cDNAs from three biological samples in
two technical replications each were used. Minimum
change in mean value of two fold with reference to
respectiveuninfested samplewastherst criterion and

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statistical differences in mean values between the

treatments in Student s t test at P\ 0.05 was the
second criteria noted. Relative transcription levels are
presented graphically.

Results
Development of pre-NILs through RILs
Pre-NILs developed through RILs were utilized for
functional validation of the candidate genes. Initially,
40 resistant and 40 susceptible RILs (F10 generation)
from the cross TN1 X Abhaya were selected based on
contrasting phenotypesgall midge resistance or
susceptibility. Of these, two NIL pairs had the same
allele distribution pattern at most of the 15 SSR loci

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Table 1 Pre-NILs identied from RILs from a cross between
TN1 X Abhaya
S. No Chr. No No. of SSR tested No. of SSRs
monomorphic between
482R
versus
489S
No.

321
versus
368
%

No.

1
2

1
2

21
30

19
27

90
90

18
27

86
90

3
4
5

3
4
5

29
31
30

25
26
29

86
84
97

26
27
29

90
87
97

6
7
8
9
10

6
7
8
9
10

29
22
15
31
27

27
22
9
31
26

93
100
60
100
96

27
21
6
28
26

93
95
40
90
96

11
12
Total

11
12
12

30
25
320

27
24
292

90
96
91

27
21
283

90
84
88

SSR simple sequence repeat marker

screened for on chromosome 8, carrying Gm4, except

at the two anking loci viz., RM22551 and RM22562
(Fig. 1). When subjected to further screening using an
additional 305 SSR markers mapping to other chromosomesaswell, two RILs, 482R and 489S, displayed
an overall genotypic similarity of 91 % but with
contrasting phenotypes (Table 1). These two lines
482R and 489S were considered as pre-NILs developed through RILs and used in functional studies
along with the parents, TN1 and Abhaya.
Identication and cloning of LRR gene
Based on the ne linkage map, genes located between
the SSR markers RM22542 and RM22571 encompassing 0.6 Mb region were analyzed for locating,
Gm4. The exact physical positions of the linked
markers were determined using edit software against
the indica sequence database (http://rice.genomics.
org.cn/rice/index2.jsp). In all, 70 genes were predicted in this genomic region using the software
package FGENESH (http://www.softberry.com). Of
these, 13 genes coded for hypothetical proteins whose
functions are unknown, 9 genes encoded for germin-

like protein, 12 for Cyclin-like F-box domain containing proteins which recruit particular substrates for
ubiquitin. Two genes code for protein kinase-like
domain containing proteins which are kinase enzyme
modifying proteins by adding phosphate group. Two
genes in the region code for LRR-containing proteinswidely implicated in plant defense against pathogens.
One gene is associated with ubiquitin domain containing protein which is known to be involved in
metabolism or housekeeping functions. Further, genes
encoding DNA or RNA binding domain containing
proteins, cytochrome P450 family protein, a mitochondrial enzyme involved in oxidation, WRKY
domain containing transcription factor protein
involved in regulation of various biological processes
were also observed in the region.
One of the two genes coding for LRR was suspected
tobealikely candidategenebasedonthepolymorphism
detected between the parents with a set of primers
designed to amplify different fragments of these two
genes (Table 2). One of the markers was designed in
such a manner that it targeted a part of the gene
approximately 1 kb downstream of the 30untranslated
region(UTR) of LRRgene(LOC_Os08g09670.1) andit
amplied a 1.8 kb fragment in both the susceptible
parent ISM andthesusceptibleline489S, whilea1.5 kb
fragment was amplied in the resistant parent Abhaya
and theresistant line482R (Fig. 2). Thispolymorphism
was also conrmed in all the 40 resistant and 40
susceptible RILs (data not shown). This gene, in the
reference genome, stretch from 5.5858 to 5.5825 Mb
(3.3 kb) region with two exons from 5.585843 to
5.585543 Mb and from 5.584156 to 5.583509 Mb,
probably coding for two components of protein that are
100 and 216 amino acid residueslong, respectively. The
amplied segment of the genestretch from 5.584177 to
5.5825 Mb (* 1.8 kb) thus covering 56 % of the gene
and 68 % of the translated segment of the gene.
Sequence analysis of the cloned amplicons from the
resistant parent Abhayaand resistant line482R revealed
the deletion of 4, 16, 4 and 273 bp regions downstream
of the gene as compared with the amplicons from the
susceptible parent ISM and susceptible line 489S
(Fig. 3). Based on the sequence polymorphism a new
primer pair, LRR-del, was designed that targeted this
region, for genotyping. Thismarker amplied * 620 bp
fragment in TN1 and a 350 bp in Abhaya (Fig. 4). This
marker can, therefore, be considered as functional
marker for detection and introgression of Gm4.

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Table 2 Details of primers used for PCR amplication of two genes (encoding leucine rich repeat proteins in the Gm4-encompassing region) and those used for quantitating relative expression (qRT) of LRRs in the different rice varieties
Primer

Physical position
(Mb)

Forward primer
50-30

Reverse primer
50-30

Fragment size (kb)

ISM

TN1

Abhaya

LRR1
LRR1 .1
LRR1 .2
LRR2

5,584,177
5,583,213
5,583,213
5,628,269

AACTTCTGCTACCATTGCAG
TGCTGTCCTAACCATTGGTG
TGCTGTCCTAACCATTGGTG
TGTTCGACGAAATGCCGCTG

TCTAGGTCGGTTGCCTGTGG
AGTACGTTGTACGCTCCGAG
CACCGGGTATCGGAGGTTTC
ACCGTAGCACACGACGTCTG

1.8
2.1
2.4
2.9

1.8
2.1
2.4
2.9

1.5
2.1
2.4
2.9

LRR del
LRR
qRT

5,583,333
5,583,982

GTGGATCGAGAGAAGACAAG CTTGAGGACGATATTCAAGC
CGCTTCAGACTGAGTCAACA CTTCCAATCCTTCATTGGTG

0.6
0.6
0.35
0.119 0.119 0.119

In silico analysis of LRR protein

Fig. 2 PCR products amplied by leucine rich repeat (LRR)

primers, LRR1 F and LRR1 R, in gall midge susceptible parent
ISM, resistant parent Abhaya and along with those amplied
from pre-NILs, 482R and 489S. L-1 kb ladder. Arrows on the
left indicate molecular weights in kbp

Characterization of LRR gene

The cloned sequence information of about 1.8 kb fragment revealed 100 % homology with Oryza sativa
Japonica PAC clone:P0412D08 at nucleotide level and
42 % homology with LOC_Os08g09670.1 (F-box protein family) at amino acid level. Based on the cloned
sequence information we tried to predict 3D structure of
the LRR protein and any structural difference between
Abhayaand ISM. The3D structureanalysisrevealed that
Abhaya sequences showed homology with mammalian
F-box/LRRprotein whileISM sequencesshowedhomology with uncharacterized protein Q64V53_BACFR (Fig.
S1). However, the amino acid sequences, despite few
substitutions, did not reveal any changes in the 3D
structure between Abhaya and ISM.

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Six frame translationsof the DNA sequence resulted in

14 possible ORFs within the sequence. In the case of
cloned fragments of Abhaya and 482R, 10 ORFs were
observed while 7 ORFs were noted in ISM and 489S.
Further, two of 14 ORFswerethelongest. Sequencesof
one of the longest ORF from all the four samples were
aligned for similarity. Differences based on the referenceLRR sequencefrom thedatabasewerenoted at the
following amino acid substitution between resistance
and susceptible lines: H36Y, N39D, D67E, D88G,
E118G. Also one more amino acid substitution was
noted at G79S only in 482R sequence (Fig. 5). Nearly,
eighty-twoamino acid deletion wasobserved inAbhaya
and 482R sequences in the intronic region as compared
with ISM and 489S sequences. Two pre-miRNAs
sequences (osa_miR7692 and osa_miR1428) were
observed on either side of the four deletion regions in
the ISM and 489S.

Validation of LRR gene through real time reverse

transcription PCR
Relative expression of this LRR gene (LOC_
Os08g09670.1) was measured in Abhaya, TN1 and the
two derived pre-NILs482R and 489Sat two time points
(24 and 120 hai) following gall midge infestation. At
24 hai, 3.75- and 2.22-fold increasein thetranscript level
was noticed in the resistant Abhaya and 482R, when
compared with the respective uninfested controls
(Fig. 6). However, in the susceptible TN1 and 489S
increasewaslessthantwo fold. However, at 120 hai time
point, thetranscript level waslessthan two fold in all the

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Fig. 3 Graphical
representation of the LRR
regions amplied in the
parental lines ISM and
Abhaya by the various PCR
primers (used in this study)
and their location with
reference to the fragment
LOC_Os08g09670.1
(uppermost bar). Indels are
also shown (vertical arrows)

Fig. 4 PCR products amplied by LRR-del primers, LRR-del

F, LRR-del R, in resistant parent Abhaya, the resistant (482R),
susceptible (489S) pre-NILs, susceptible parents TN1 and ISM.
L-100 bp ladder. Arrowson theright indicatemolecular weights
in bp

genotypes when compared with their respective uninfested controls.

Discussion
Discovery of new gene/gene sources would enable
breeders to improve rice varieties with broad range
and durable gall midge resistance. Use of molecular

markerslinked to theresistancegenescan behelpful in

accelerating gene-pyramiding efforts (Mohan et al.
1997b; Katiyar et al. 2001; Bentur et al. 2003). The
crucial question currently being addressed is the
choiceof gene combinations that needs to be deployed
to achieve durable gall midge resistance. The Gm4
gene in Abhaya is a potential candidate for gene
pyramiding since it provides HR? type of induced
resistanceagainst veof theseven gall midgebiotypes
GMB1, GMB2, GMB3, GMB4 and GMB4M (Vijaya
Lakshmi et al. 2006). Though Gm4 has been tagged
and mapped on to Chromosome 8 of rice earlier by
Nair et al. (1996) and Mohan et al. (1997a) using
RAPD and RFLP markers, these are not close enough
to be used reliably in marker assisted selection (MAS)
programs. Using a set of 44 SSR markers on chromosome 8, Himabindu (2009) identied much closer
anking markers, RM22551 and RM22562, and tested
them in alternative mapping populations segregating
for Gm4. Our current analysis of the genomic region
encompassing these markers narrowed down two
genes encoding LRR proteins as likely candidate
genes coding for Gm4. Based on PCR amplicon size
polymorphisms, displayed by one set of the PCR
primers used, between the parents and among 482R
and 489S pre-NILs, and also among 40 R and 40 S

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Fig. 5 Sequence alignment of the predicted amino acid

sequences of the PCR amplied and sequenced LRR regions
from the different rice samples (Abhaya, 482R, ISM and 489S)
using LRR1 primers. Observed differences in the predicted
amino acid sequences of the different fragments are boxed.

Fig. 6 Relative expression proles of LRR in Abhaya, 482R,

TN1 and 489S after gall midge biotype 1 infestation. Error bars
represent Mean S.D. Light bars represent 24 hai and darker
bars represent 120 hai time points. Means that are not
statistically signicant are marked *

RILs, one of the genes, LOC_Os08g09670.1, was

shortlisted as the candidate Gm4 gene. RT-PCR-based
functional validation studiesfurther conrmed therole
of this gene in gall midge resistance. Thus, we
conclude that this LRR gene is the candidate gene
representing Gm4 in Abhaya. More markers were
developed targeting thedeletion in thegeneand oneof
these designated as LRR-del showed distinctly scorable polymorphism between the parents and can be
considered as functional marker for use in MAS as

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Asterisk indicate positions which have fully conserved residue,

colon indicate strongly similar properties- [ 0.5 in the gonnet
PAM 250 matrix, dot indicate weakly similar properties- =\ 0.5
in the gonnet PAM 250 matrix

there was perfect co-segregation of the marker alleles

with the gall midge resistance/susceptible phenotype.
NBS-LRR is a large class of genes associated with
disease resistance in several plants (McHale et al.
2006). NBS-LRR class of proteins is reported to be
involved in recognizing the pathogen/insect derived
elicitorsand initiatethedefenseresponsefollowing pest
attack (DeYoung and Innes 2006). A common feature
of these proteins is the presence of LRR domain which
is associated with hypersensitive response mediated
induced resistance against the biotic stress factors. The
expression of this class of genes, in contrast to
constitutively expressed resistance genes such as
Xa21 (against bacterial blight) (Song et al. 1995) and
Gm1 (against gall midge) (Rawat et al. 2012), arelikely
to be less of a metabolic load on the plant. Seven
nucleotidebinding (NB) and/or LRR genesarereported
in the rice genome anking previously mapped gall
midge resistance genes Gm2, gm3, Gm6, Gm7 and
Gm11 (Yasala et al. 2012) all conferring HR? type
resistance. Of these genes, a NB-ARC coding gene has
been earlier identied and functionally validated as the
candidate gene for gm3 (Sama et al. 2014). The present
report is the second putative gall midge resistance gene
to be cloned and validated.
Sequence analysis of different alleles of NBS-LRR
class of genes often revealed a large deletion in the

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functional allele in contrast to its non-functional

counterpart. The functional allele of the recessive
gene gm3 coding for NB-ARC domain containing
protein, lacked a complete exon of 320 bp in the
resistant rice variety RP2068-18-3-5 when compared
with the allele found in the susceptible rice variety
TN1. In addition, a 92 bp region was also deleted in
RP2068-18-3-5 when compared with thethird allelein
the rice variety Phalguna carrying Gm2 (Sama et al.
2014). Likewise, in the NBS-LRR-domain-containing
protein coding for Pi54 lacked a 144 bp segment in an
exon in the functional allele (Ramkumar et al. 2011).
We report here four separate deletions of 4, 16, 4 and
273 bp in the Abhaya allele at the untranslated region
compared with the allele present in the susceptible
parent, ISM.
In addition, several indel changes in the transcribed
regions resulted in amino acid changes at protein level
as well. Five amino acid changes in the coding region
of LRR domain differentiated the resistant and
susceptible parents and so also the alleles from 482R
and 489S. Though it is yet to be experimentally
validated, theamino acid substitution from histidineto
tyrosine at 36 position, aspartic acid to glycine at 88
position and from glutamic acid to glycine at 118
position, probably affected the protein function. In
contrast, the other two amino acid substitutions from
asparagine to aspartic acid at 39 position and from
aspartic acid to glutamic acid at 67 position may not
affect the function of the protein as predicted by the
softwarePROVEAN and SIFT. Thericeblast genePita encodes a predicted 928 amino acid cytoplasmic
receptor with a centrally localized NB site. A single
amino acid substitution from alanine to serine at 918
position converted the Pi-ta resistance allele to pi-tasusceptible allele (Bryan et al. 2000). Dodds et al.
(2001) reported that six amino acid changes, conned
to the LRR b-strand/b-turn motif, can determine
specicity differences among plant resistance genes.
Further, glycine to glutamic acid change at the
conserved glycine residue in the p2-X151 mutant
allele completely ablated the P2-conferred rust resistance in ax. Bacterial blight resistance gene xa5
showed two amino acid changes in the coding region
sequences between resistance and susceptible alleles.
A changein amino acid from valineto glutamic acid in
resistance allele was suggested to be the cause for
resistance against BB (Iyer and McCouch 2004).
Similarly, in the TIR-NBS-LRR domain of the gene

RAC1 (a dominant gene conferring resistance to white

rust pathogen Albugo candida in Arabidopsis) a single
amino acid substitution in LRR domain, at 74 position,
from isoleucine to lysine, resulted in a mutant allele,
rac1, leading to susceptibility to the pathogen (Borhan
et al. 2004).
Functional signicance of a large deletion of
273 bp in the Abhaya allele at the untranslated region
compared with that of ISM allele can only be
predicted. It is known that the untranslated region
contains both binding site for regulatory proteins as
well asmiRNAswhich inuencethepost-translational
gene expression. These regulatory regions are known
to be involved in functions related to polyadenylation,
translation efciency and stability of mRNA. By
binding to specic sites within the 30-UTR, miRNAs
can initiate or terminate gene expression by either
inhibiting translation or directly causing degradation
of the transcript. Since the deletion in Abhaya
involved binding sites for two of the miRNAs it can
be predicted that the gene in ISM and 489S with
undeleted segment is prone to gene regulation by the
two miRNAs. Further, partial binding (88 and 89 %)
of the miRNAs is suggestive of suppression of NBLRR gene expression and not total inhibition (Martinez and Tuschl 2004). This argument is supported by
our observation that NB-LRR gene did express after
Gminfestation in both TN1 and 489S, though thelevel
of expression was less than two fold (Fig. 6). Also,
osa-miR7692 is reported to be regulated by elicitors
from the blast fungus Magnaporthe oryzae and playsa
negative role in regulating alternatively spliced transcript of OsNramp6 (Natural resistance-associated
macrophage protein 6) processing in plants (Campo
et al. 2013). The other microRNA osa_miR1428 is
reported to be involved in rice grain development and
in translation to cleave target mRNAs (Zhu et al.
2008). Similarly, the location where a deletion of a
327 bp fragment from the 30UTR region of the
resistance allele of xa5 gene, conferring resistance
against bacterial blight, wasused to develop functional
markers (Iyer and McCouch 2004). However, the
exact mode of miRNAs function can only be clearly
elucidated after additional functional studies are
carried out.
Acknowledgments Wethank theProject Director, Directorate
of RiceResearch, Hyderabadfor thefacilitiesandencouragement.
This work was supported by a grant from the Department of

123

Euphytica
Biotechnology (F.No: BT/AB/FG-2(PH-II)(4A)/2009 to JSB and
F.No: BT/AB/FG-2(PH-II)(4B)/2009 to SN), Government of
India.

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