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Isolation and characterisation of a NBS-LRR resistance gene analogue from

pearl millet
P. Ranjinia; S. Shailasreea; K. Ramachandra Kinia; H. Shekar Shettya
a
Plant Molecular Biology Laboratory, Department of Studies in Biotechnology, University of Mysore,
Manasagangotri, Mysore, Karnataka, India

Online publication date: 15 June 2011

To cite this Article Ranjini, P. , Shailasree, S. , Kini, K. Ramachandra and Shetty, H. Shekar(2011) 'Isolation and
characterisation of a NBS-LRR resistance gene analogue from pearl millet', Archives Of Phytopathology And Plant
Protection, 44: 10, 1013 — 1023
To link to this Article: DOI: 10.1080/03235401003633907
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 Archives of Phytopathology and Plant Protection
Vol. 44, No. 10, June 2011, 1013–1023

Isolation and characterisation of a NBS-LRR resistance gene analogue

from pearl millet
P. Ranjini, S. Shailasree, K. Ramachandra Kini* and H. Shekar Shetty

Plant Molecular Biology Laboratory, Department of Studies in Biotechnology, University of

Mysore, Manasagangotri, Mysore 570006, Karnataka, India
(Received 29 October 2009; final version received 27 November 2009)

Plant resistance (R) proteins belonging to nucleotide-binding site–leucine-rich

repeat (NBS–LRR) family are mainly involved in recognition of effectors secreted
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by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most
drought tolerant cereals, staple food crop of the semi-arid tropics but is highly
susceptible to the downy mildew disease caused by oomycetous Sclerospora
graminicola (Sacc) schroet. Earlier studies have identified several resistance gene
analogues (RGAs) in pearl millet which may be involved in resistance against
downy mildew. Of these, a clone RGPM213 was shown to have more than 60%
identity with R-proteins coding for NBS–LRR-like protein kinase. The exact
nature and function of the R-protein encoded by this gene was not known. In the
present study, the cDNA of RGPM213 encompassing NBS–LRR region was
inserted into an expression vector pRSET-A and transformed into BL21 E.coli
cells. The expressed recombinant fusion protein with a His tag was purified using
nickel affinity purification and it had a molecular weight of 35 kDa on SDS-
PAGE. Immunoaffinity purification using antibodies raised against this
recombinant R-protein identified two proteins of molecular weights 55 kDa
and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of
these proteins followed by homology search in database revealed similarity of the
55 kDa protein with a protein kinase from Brassica oleracia containing serine/
threonine kinase domain.
Keywords: leucine rich repeat; nucleotide binding site; Pennisetum glaucum;
resistance gene analogues (RGAs)

Introduction
Plants face a constant onslaught of pathogenic microorganisms in their environment.
Pathogens have evolved sophisticated weapons to attack plants and plants in turn
have developed diversified defence strategies to ward off pathogens. Similar to
animals, plants use two distinct defence systems to recognise and respond to
pathogen challenge. The first one is by the use of extracellular transmembrane
receptors that recognises pathogen-associated molecular patterns (PAMPs) and
elicits basal defence that is equivalent of innate immunity in vertebrates. However,
some specialised pathogens produce virulence factors (effectors) which can suppress
this ‘‘PAMP triggered immunity’’ and lead to successful infection. To combat these

*Corresponding author. Email: krk@appbot.uni-mysore.ac.in

ISSN 0323-5408 print/ISSN 1477-2906 online

Ó 2011 Taylor & Francis
DOI: 10.1080/03235401003633907
http://www.informaworld.com
 1014 P. Ranjini et al.

pathogens, plants have evolved a second layer of defence which is based on the
recognition of these effectors by highly polymorphic resistance (R) proteins and is
termed as ‘‘effector-triggered immunity’’. R-proteins mainly act intracellularly to
restrict biotrophic and hemibiotrophic pathogens which need living cells for their
survival (Tameling and Takken 2008; Takken and Tameling 2009).
Over 70 R genes conferring resistance to the corresponding pathogen effectors or
associated protein(s) have been isolated and cloned (Liu et al. 2007). In many plant
species, R genes have been identified to be present in the form of clusters (Jones et al.
1993; Meyers et al. 1998, 2003). R-genes have been used in transgenic approaches for
disease control, as they confer broad spectrum of disease resistance. Most of the
plant resistant genes are structurally conserved and their products, R-proteins have
almost similar functional motifs. Based on their structural and functional motifs
R-proteins have been categorised into seven different classes (Takken et al. 2006).
Conserved domains of R genes have been extensively exploited to isolate
unknown resistance gene analogues (RGAs) by using polymerase chain reaction
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(PCR)-based cloning approach (Sharma et al. 2009). This approach using

degenerate PCR primers against the conserved domains has been useful for
isolating novel RGAs from large number of plant species such as cereals (Leister
et al. 1996), cassava (Lopez et al. 2003), sugar beet (Tian et al. 2004), chestnut
rose (Xu et al. 2005), maize (Wenkai et al. 2006), wheat (Wang et al. 2006), sweat
potato (Chen et al. 2007) and sugar cane (Glynn et al. 2008). More importantly
this approach has served as promising tool for the isolation of full-length
resistance genes from crop plants such as wheat (Feuillet et al. 1997), common
bean (Ferrier-Cana et al. 2003) and cotton (He et al. 2003).
Pearl millet [Pennisetum glaucum (L) R.Br] is a staple food crop of the semiarid
tropics and its F1 hybrids are highly susceptible to the downy mildew disease caused
by oomycetous pathogen Sclerospora graminicola (Sacc.) Schroet. Although several
cultivars of pearl millet resistant to downy mildew pathogen have been used, the
resistance was not durable and would eventually breakdown. Utilisation of genetic
resistance is the most economical and environment friendly strategy for disease
control. However, a clear understanding of the molecular mechanisms in host
resistance to pathogen is the essential prerequisite for this approach. Earlier studies
from this laboratory have identified 22 RGAs in pearl millet using PCR strategy with
degenerate primers designed to amplify the common motifs in the resistance genes
(Sarosh 2002). These RGAs were grouped into nine contigs using sequencher 4.0.5
software. Of these, the clone RGPM213 (GenBank accession number AF513969)
showed 60% identity to rice R-protein and was predicted to encode NBS–LRR-like
protein kinase. This RGA has also been mapped to linkage groups 1 and 7 (LG1 and
LG7) of the consensus map of pearl millet genome (Qi et al. 2004). Northern blot
analysis using this cDNA as probe indicated up-regulation of RGPM213 transcripts
in pearl millet following inoculation with the pathogen S. graminicola (Ranjini et al.
2006) and also upon treatment with b amino butyric acid (BABA), a known abiotic
inducer of downy mildew resistance in pearl millet (Shailasree et al. 2001) suggesting
role for this gene in resistance of pearl millet to downy mildew. In the present study,
to identify and characterise the protein encoded by RGPM213, the cDNA
encompassing the NBS–LRR region was expressed as a fusion protein with a His
tag in bacterial expression system. This fusion protein was subsequently purified and
polyclonal antibodies were raised against it, which were used to identify the putative
R-protein from the extracts of pearl millet seedlings.
 Archives of Phytopathology and Plant Protection 1015

Materials and methods

Plant material
Pearl millet cv IP18294, (highly resistant) with 0% downy mildew disease incidence
(DMDI), was used. Seeds were surface sterilised in sodium hypochlorite (0.1%) for
15 min, washed thoroughly with sterile distilled water and germinated on moist filter
paper under aseptic conditions at 25+28C in the dark for 2 days. The seedlings were
harvested and immediately wrapped in aluminium foil and stored at –708C till
further use.

RNA isolation
RNA was extracted from 2-day-old pearl millet seedlings by using standard
protocol of Guanidium thiocyante method as described by Chomzynski and Sacchi
(1987).
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Reverse transcription
cDNA was synthesised using AMV-RT PCR kit (Sigma, USA). Reverse transcrip-
tion was carried out at 428C for 1 h in 20-l reaction mixtures containing 10 mg of
total RNA, 16 reverse transcription buffer, 500 mM of each of dNTP, 3.5 mM of
oligo-dT primers, 1U of RNase inhibitor, 2U Avian Myeloblastosis leukaemia Virus
reverse transcriptase enzyme.

Cloning and sequencing

Two microlitres of cDNA were used for PCR experiments. RGPM213 primers (for
50 -GCCACCACACATACGGACAAT-30 and Rev 50 -GGGTGGGGAAGACGA
CTTT-30 ) were designed based on the conserved NBS–LRR peptide motifs. PCR
amplifications were conducted on a Thermo Cycler (UNO II Biometra, USA). PCR
conditions were 948C for 2 min, 948C for 45 s, 588C for 45 s, 728C for 80 s for 35
cycles followed by 7 min final extension at 728C. The PCR product obtained was
purified using AxyPrep gel extraction kit (Axygen, USA) and cloned in pTZ57R/T
vector (Invitrogen, USA). After transformation in the Escherichia coli JM109 strain,
plasmid DNA was extracted using the alkaline lysis method (Sambrook et al. 1989).
Cloned RT-PCR products were sequenced.

Expression of recombinant RGPM213 protein in E. coli and its purification

The pRSET-A expression vector was used for expression of recombinant RGPM213
in E. coli. Plasmid DNA (pTZ57R/T) was digested with restriction enzymes EcoRI
and HinD III. The RGPM213 cDNA digest containing NBS–LRR region was
cloned into HinD III and EcoRI site of pRSET-A to generate plasmid pRPM213.
Nucleotide sequence analysis confirmed that the fragment was cloned in-frame.
BL21 E. coli cells were transformed with pRPM213 for recombinant protein
expression. Expression was induced with 1 mM isopropylthio-b, D-galactoside
(IPTG) and cells were grown for 5 h before harvest. The expressed fusion protein
was affinity purified from crude bacterial lysate by MagneHis Ni-particles (Promega,
USA) according to manufacturer’s instruction.
 1016 P. Ranjini et al.

Preparation of polyclonal antibodies

Antiserum was raised in New Zealand white rabbit against Nickel affinity purified
recombinant protein. For immunisation, the pure protein in phosphate buffered
saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Phosphate Buffer, pH 7) mixed
with Freund’s incomplete adjuvant (1:1) was injected at multiple sites through
intramuscular routes. Three such injections were given at an intermittent interval of
1 week by increasing the concentration of protein (100–150 mg protein); 500 mg of
protein was used in total. A booster dose was given at the fourth week and the rabbit
was test bled after 4 days. The rabbit was further boosted twice up to 6 weeks in a
similar way. The blood was collected 2 days after the last injection and was allowed
to clot at room temperature (RT) for 30 min followed by 2–3 h at 48C. The
antiserum separated from blood serum was centrifuged at 2006 g for 15 min and
stored at 7208C.
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Immunoblotting
Total crude proteins were extracted from 2-day-old pearl millet seedlings in 50 mM
phosphate buffer (pH 7.0). The pearl millet crude proteins were resolved on 10%
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
blotted to polyvinylidene difluoride membrane using Multiphor II (LKB,
Pharmacia) according to manufacturer’s instruction. The blot was blocked overnight
in 5% fat free milk powder in Tris buffered saline (TBS; 10 mM Tris HCl pH
8.0 containing 150 mM NaCl). Then blot was probed with polyclonal antibody
raised against His-tagged NBS–LRR region containing recombinant protein as
primary antibody for 1 h and 30 min at RT. After washing thrice with TBS, the
blot was incubated with antirabbit IgG horse radish peroxidase conjugate for
1 h 30 min at RT. After washing, the blot was stained for peroxidase activity with
3, 30 -diaminobenzedine and hydrogen peroxide.

Immunoaffinity purification
Pearl millet protein extract was prepared by homogenising the 2-day-old pearl millet
seedlings in 50 mM PBS. The polyclonal antibody bound nitrocellulose membrane
was incubated with 750 ml of pearl millet protein extract for 2 h with shaking at RT.
Nitrocellulose membrane with antigen antibody complex was then washed 56 3 min
each with PBS and the proteins were eluted with 100 mM Tris HCl pH 10.2. The
eluted proteins were precipitated using three volumes of ice-cold acetone and
subjected to SDS-PAGE.

Mass spectrometry
Matrix-assisted laser desorption ionisation- time of flight (MALDI-TOF) spectro-
scopy was performed using Ultraflex MALDI-TOF/TOF spectrometer (Bruker,
Germany) with 337 nm nitrogen laser. The mass spectrometer was operated in a
linear high-power positive ionisation mode at a 25 kV accelerating voltage with a
150 ns time delay. The laser fluence was typically 50 Hz. The immunoaffinity purified
proteins from pearl millet seedling extract which were subjected to SDS-PAGE were
digested in-gel with trypsin enzyme and the digests were purified. Peptides (0.5 ml)
 Archives of Phytopathology and Plant Protection 1017

were spotted on MALDI plate followed by 0.5 ml of alpha-cyano-4-hydroxycinnamic

acid matrix (10 mg/mL in 50% acetonitrile, 0.1% TFA). Spots were dried
completely and loaded into Voyager with internal calibration standard of tryptic
peaks of 842.5 and 2211.1 kDa.

Analysis of MALDI data using Mascot

The m/z values obtained after MALDI-TOF analysis were subjected to Mascot
peptide analysis (http://www.matrixscience.com/search_form_select.htm/) to iden-
tify the conserved peptide motifs.

Results
RGPM213 cDNA cloning and sequence analysis
A sharp band of 500bp was amplified by RT-PCR using pearl millet cDNA as
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template and RGPM213 specific primers (Figure 1). Analysis of the deduced amino
acid sequence of RT-PCR product for conserved peptide motif revealed the presence
of NB-ARC region with P-loop (Kinase 1a) and GxP motifs proposed to function as
molecular switch and which is known to regulate the functions of NBS–LRR
proteins. The deduced amino acid sequence was compared with other four
homologous R-proteins containing NBS–LRR region by multialignment analysis
(Figure 2a). It was found that RGPM cDNA shared more than 60% identity with
R-proteins having NBS–LRR region containing protein kinases in the GenBank
from Saccharum officinarum, Elusine coracana, Oryza sativa and Aegilops venticosa
indicating that NBS–LRR region is highly conserved (Figure 2b). GLPLAL motif
containing amino acids involved in ATP binding is conserved in RGPM213 as in
other NBS–LRR proteins. This indicated that RGPM213 cDNA likely encodes a
NBS–LRR region containing protein kinase in pearl millet.

Expression and purification of His tagged recombinant resistant protein

The expression of cDNA fragment containing NB-ARC domain in pRSET-A was
induced in early log phase of BL21 E. coli cultures by addition of IPTG. SDS-PAGE
analysis of the total proteins from the bacterial culture showed the presence of high
levels of *35 kDa recombinant fusion R-protein in pellet after 5 h growth at 288C
compared to supernatant (Figure 3). Recombinant fusion protein was purified by
MagneHis Nickel particles. SDS-PAGE analysis of the eluted product showed single
band of molecular weight 35 kDa (Figure 4).

Figure 1. Reverse transcription polymerase chain reaction (RT-PCR) product amplified by

primers specific to AF513969. M-100bp DNA ladder, 1-cDNA PCR product of AF513969.
 1018 P. Ranjini et al.
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Figure 2. (a) Sequence alignment of RGPM213 deduced amino acid with NBS–LRR region
containing proteins from S. officinarum (ABK57112.1), E. coracana (ABW049831), O. sativa
(ABA95337.1) and A. venticosa (CAC11100.1). The P-loop and GLPL motif characteristic of
NBS–LRR proteins is highlighted. (b) Phylogenetic analysis of RGPM213 with other reported
NBS–LRR plant R-proteins. The rooted phylogenetic tree is based on the amino acid
sequence alignment of the following NBS–LRR region containing plant resistant proteins.
Saccharum officinarum (ABK57112), E. coracana (ABW04983), O. sativa Japonica Group
(ABA95337) and Aegilops ventricosa (CAC 11100) using BLASTP.

Figure 3. SDS-PAGE analysis showing induction of recombinant resistant protein in E. coli.

SDS-PAGE was performed on a 10% polyacrylamide gel, stained using ponceau-S. M –
Molecular weight marker, 1 – uninduced pellet, 2 – uninduced supernatant, 3 – induced pellet
and 4 – induced supernatant.

Immunodetection of pearl millet R-proteins

The polyclonal antibodies raised against the 35 kDa recombinant protein were used
in Western blot analysis. Two major protein bands with apparent molecular weight
 Archives of Phytopathology and Plant Protection 1019

of 55 kDa and 66 kDa were detected from pearl millet seedling extract (Figure 5).
These two proteins were immunopurified using polyclonal antibodies raised against
fusion protein and were subjected to SDS-PAGE analysis. MALDI-TOF analysis
and the analysis of m/z values using Mascot peptide analysis programme revealed the
homology of the 55 kDa protein with a protein kinase from Brassica oleracia
containing a serine/threonine kinase domain. However, the other protein of 66 kDa
did not match any of the known proteins in the database.
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Figure 4. Showing Coomassie brilliant blue stain of gel before immunoblotting. Samples
were resolved on 10% SDS-PAGE and were electroblotted to membrane. Lane 1 – induced
pellet, Lane 2 – Nickel affinity purified fusion protein.

Figure 5. (a) SDS-PAGE analysis showing immunoaffinity purified pearl millet R-proteins.
SDS-PAGE was performed on a 10% polyacrylamide gel, stained using commassie brilliant
blue. M – Molecular weight marker, Lane 1 – immunoaffinity purified pearl millet proteins. (b)
Western blot analysis of purified fusion protein (Lane 1) and pearl millet total extract (Lane 2)
using polyclonal antibody raised against recombinant fusion protein.
 1020 P. Ranjini et al.

Discussion
The degenerate PCR primer approach is a useful technique for isolating full length
RGAs. The isolated RGA may be a functional resistance gene or closely associated
with the resistance gene. RGA sequences amplified following candidate gene
approach have yielded vital information about the organisation, distribution and
evolution of R genes/RGAs (Kanazin et al. 1996; Pan et al. 2000; Meyers et al. 2003).
In this study, an RGA containing the NBS conserved motifs, P-loop, kinase 2 and
the downstream hydrophobic GLPLAL domain was analysed. The vast majority of
R-proteins activated during pathogen infection predominantly belong to NBS–LRR
class. Ha-NTIR11g, an RGA conferring resistance against Plasmospora halstedii,
encodes CC-NBS–LRR type R-protein (Radwan et al. 2004). In rice, Pi9, Pi2 and
Piz-t resistance genes conferring blast resistance, encode NBS–LRR proteins and
belong to multigene family (Qu et al. 2006; Zhou et al. 2006). Similarly L, M, P
resistance gene of Flax, N protein of Tobacco, RPP1, 4, 5 proteins of Arabidopsis,
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BS2 resistance protein from pepper, Rx1a and Rz2 from potato, all have NBS–LRR
region with Toll and interleukin 1 receptor (TIR)/coiled coil (CC) motif at N-
terminal region (Martin et al. 2003).
Transient expression analysis of I-2 and Mi-1 R-protein mutants in Nicotiana
benthamiana supported the current models for R-protein function in which, NB-
ARC acts as a molecular switch. Structural model of I-2 NB-ARC domain revealed
that, methionine–histidine–aspartate (MHD) conserved element plays an important
role and through which R-proteins orchestrate plant defence (Ooijen et al. 2008).
R-proteins functioning as receptor-like protein or receptor-like kinases with
extracytoplasmic leucine rich repeat (eLRR) play an important role in plant defence
and development, mainly in recognition of various pathogen molecules and plant
hormones (Kruijt et al. 2005). Expression analysis of mutant Cf-9 protein in tomato
showed that Trp and Cys pairs in the N-terminal LRR flanking domains are
important for Cf-9 activity and Avr recognition (Hoorn et al. 2005).
In Arabidopsis, PBS1 gene codes a protein of 50.4 kDa with serine/threonine
protein kinase and myristoylation domain. This kinase domain was 50% identical to
Pti and 42% to Pto. Transient expression of PBS1 induced avrPphB-dependent cell
death in Agrobacterium (Swiderski and Innes 2001). In lettuce, random amplification
of cDNA ends-polymerase chain reaction (RACE-PCR) analysis revealed that
RGC2 in DM3 locus codes for NBS and two LRR domain containing proteins.
Transgenic expression of this full-length cDNA showed resistance against all isolates
carrying Avr3 (Shen et al. 2002). These observations indicate that cloning and
interaction studies of R and Avr gene pair provide new insights into the molecular
basis of host–pathogen interaction.
Sarosh (2002) had previously isolated RGPM213 (AF513969) RGA using
oligonucleotide primers. Sequence analysis revealed that it codes for functional
NBS–LRR-like protein kinase with homology to rice protein kinase. Hence in the
present study, to validate information and to identify the nature of protein
encoded by this gene, partial RGPM213 cDNA encompassing NBS–LRR was
cloned to pRSET-A expression vector. Homology searches of deduced amino acid
of RGPM213 cDNA showed the presence of NB-ARC region that, depending on
the nucleotide bound, defines the activation state of the R-protein, with P-loop
(Kinase 1a) and GxP motifs with consensus sequence GxxxxGKS/T and GLPLAL.
This NB-ARC region has three subdomains NB, ARC1 and ARC2. In ARC2
 Archives of Phytopathology and Plant Protection 1021

subdomain, carboxy terminal MHD motif is highly conserved. Expression of

RGPM213 cDNA in E. coli resulted in production of a *35 kDa histidine
conjugated fusion protein after induction with 1 mM IPTG. Polyclonal antibodies
raised to this *35 kDa fusion protein were used to purify pearl millet R-proteins.
SDS-PAGE analysis of immunopurified proteins showed two bands of 55 kDa and
66 kDa. MALDI-TOF MS analysis of these proteins revealed that 55 kDa protein
is 58% identical to protein kinase from B. oleracia with serine/threonine kinase
domain. This domain is present in most of the R-proteins belonging to class 1, 5
and 7. Further studies are underway to gain complete information about these
proteins and their role in defence reaction of pearl millet against S. graminicola
infection.

Acknowledgements
The authors are grateful to the ICAR Government of India, New Delhi for funds. The
authors thank Prof. Shaila, IISc for her valuable suggestions regarding cloning. We also
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thank Ramachandra R, GKVK for kindly gifting pRSET-A vector. RP acknowledges the
research fellowship received from UGC, New Delhi. SS acknowledges the research funding
received from DST, under the SERC Fast Track Young Scientist Programme, New Delhi.
The authors gratefully acknowledge the DANIDA-ENRECA for facilities.

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