Post on 11-Mar-2023
Engineering sheath blight resistance in elite indica rice cultivars using
genes encoding defense proteins
Krishnan Kalpana a,1, Subbiyan Maruthasalam a,1, Thangaswamy Rajesh a,Kandasami Poovannan a, Krish K. Kumar a, Easwaran Kokiladevi a, Joseph A.J. Raja a,Durailagaraja Sudhakar a, Rethinaswamy Velazhahan b, Ramasamy Samiyappan b,
Ponnuswami Balasubramanian a,b,*
a Rice Transformation Laboratory, Department of Plant Molecular Biology and Biotechnology,
Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641003, Indiab Department of Plant Pathology, Centre for Plant Protection Studies,
Tamil Nadu Agricultural University, Coimbatore 641003, India
Received 20 November 2004; received in revised form 25 July 2005; accepted 1 August 2005
Available online 29 August 2005
www.elsevier.com/locate/plantsci
Plant Science 170 (2006) 203–215
Abstract
Genetically transformed lines of the elite indica rice cultivars, ADT38, ASD16, IR50 and Pusa Basmati1, constitutively over-expressing
rice tlp encoding a thaumatin-like protein, have been evolved for management of rice sheath blight disease. The incorporation of tlp transgene
in the genomes of T0 lines was ascertained by polymerase chain reaction and confirmed by Southern hybridization analyses. Expression of tlp
in the putative transformants was confirmed by western blotting analysis. Stable inheritance of the transgene expression was studied up to T2
generation by western blotting analysis. The putative transformants and their progenies expressing tlp showed enhanced resistance against the
sheath blight pathogen, Rhizoctonia solani, when compared to the non-transformed plants. The use of rice chi11, encoding a chitinase, as a co-
transgene along with tlp produced a tlp–chi11 co-transformant that showed enhanced resistance against R. solani than the ones that express tlp
or chi11 transgene alone. In addition to sheath blight resistance, the tlp or chi11 transgenic lines displayed significant levels of protection
against the rice sheath rot pathogen, Sarocladium oryzae.
# 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chitinase; Co-expression; Defense genes; Disease resistance; Pathogenesis-related proteins; Thaumatin-like protein; Rice sheath blight; Rice
sheath rot
1. Introduction
Rice sheath blight (ShB), caused by Rhizoctonia solani
Kuhn, is a destructive disease in most rice-growing areas of
the world. R. solani is soil-borne; the sclerotia or mycelia
present in the plant debris float to water surface during
Abbreviations: BDL, bioassay using detached leaves; BLSI, bioassay
using leaf sheaths intact; PB1, Pusa Basmati1; PR-proteins, pathogenesis-
related proteins; Rs7, Rhizoctonia solani isolate 7; ShB, sheath blight
* Corresponding author. Tel.: +91 422 5511353/453;
fax: +91 422 2431672.
E-mail address: balasubrap@hotmail.com (P. Balasubramanian).1 These two authors have contributed to this article equally.
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved
doi:10.1016/j.plantsci.2005.08.002
irrigation and flood and infect rice plants [1]. The diseasemay
also spread from one hill to another through leaf-to-leaf and
leaf-to-sheath contacts. Infection often leads to extensive
necrosis of leaf sheaths mostly in improved, semi dwarf and
nitrogen-responsive rice cultivars. Genetic resistance to R.
solani has not been recorded in both cultivars and wild
relatives of rice [2]. Control of ShB by fungicides is neither
practical nor sustainable. Apart from being less effective, the
indiscriminate use of fungicides is deleterious to the
ecosystem and health of human and livestock. The undesir-
able environmental and health impacts of fungicides
necessitate alternative strategies for fungal disease manage-
ment. Genetic engineering of crops with genes conferring
.
K. Kalpana et al. / Plant Science 170 (2006) 203–215204
fungal resistance is a promising and long-lasting approach for
themanagement of fungal diseases. This preventive strategy is
not only an alternative approach, but also complementary to
the conventional ones.
Plants perceive signals (elicitors) from pathogens that
initiate parasitic interactions. Host recognition of pathogen’s
elicitors triggers multiple defence responses in hosts,
including the accumulation of defence compounds called
phytoalexins, pathogenesis-related (PR) proteins, evolution
of reactive oxygen species and hypersensitive cell death [3].
It has been demonstrated that a constitutive and higher level
expression of PR-proteins in transgenic plants enhanced
resistance to a variety of pathogens [4–8].
One of the objectives of the present programme was to
evolve rice cultivars with enhanced resistance to ShB by
genetically transforming the high yielding indica rice
cultivars, ADT38, ASD16, IR50 and Pusa Basmati1 (PB1),
with rice tlp gene, encoding a 23 kDa thaumatin-like
protein. Earlier, Datta et al. [6] transformed the indica rice
cultivars, Chinsurah Boro II, IR72 and IR51500, with rice
tlp gene to enhance their resistance against R. solani. Of
the presently transformed high yielding indica rice
cultivars, ADT38 and ASD16, owing to their short
duration, have become farmer-preferred in south India,
where the constant availability of irrigation water is
unpredictable. However, the high fungal disease suscept-
ibility of these cultivars causes significant yield reduction.
Thaumatin-like proteins are members of the thaumatin
protein family comprising its prototype, Thaumatococcus
daniellii thaumatin [9], Nicotiana tabacum osmotin and
Zea mays zeamatin and cereal seed permatin [10,11].
Several members of this protein family are antifungal
[12,13] and they are categorized under PR-protein group 5
(PR5). A possible mechanism of antifungal action of PR5
proteins is by binding to fungal cell surface receptors and
subverting signalling pathways. The antifungal action may
involve plasma membrane permeabilization of the target
fungal cell [10–12,14,15].
Disease resistance is a complex trait controlled by several
groups of genes. Hence, constitutive expression of a single
PR-protein transgene like tlp could not be expected to confer
sufficient level of disease resistance on transformed crops.
The marginal and narrow spectrum disease resistance
conferred by single PR-protein transgene is one of the
major reasons for the reported failures, ranging from poor
performance of the green-house-proven transformants in
field conditions [16] to serious failures as reported by
Neuhans et al. [17]. However, the co-expression of more
than one PR-protein genes, such as chitinase (EC 3.2.1.14)
and b-1,3-glucanase (EC 3.2.1.39), was shown to be much
more effective against development of several fungal
diseases than expression of a single gene [18–20].
The existing literature on TLPs implies that the
antifungal activity of pathogen cell membrane-permeabiliz-
ing TLPs is in concert with those of other antifungal
proteins, such as chitinases and b-1,3-glucanases capable of
hydrolyzing fungal cell wall carbohydrates. A subgroup of
TLPs has been shown to be b-1,3-glucan binding proteins
[21]. Moreover, Grenier et al. [22] showed the hydrolysis of
b-1,3-glucans by some TLPs. Thaumatin-like proteins,
chitinases and b-1,3-glucanases are co-regulated or co-
expressed developmentally [23,24] and when the plants are
subjected to abiotic stress [25] and challenged by pathogens
[26]. Hejgaard et al. [27] reported synergistic activity of
barley TLP and chitinase against the fungi, Trichoderma
viride and Candida albicans. Earlier, rice chitinase genes
(chi11 and RC7) were employed to transform the indica rice
cultivars, Chinsurah Boro II, Basmati122, Tulsi, Vaidehi,
IR64, IR72, IR68899B and MH63, in order to enhance their
resistance against R. solani [7,8]. Hence, in the present
study, apart from tlp transformations of the indica cultivars,
ADT38, ASD16, IR50 and PB1, we also co-transformed the
cultivars, ADT38 and IR50 with tlp and chi11 (encoding a
35 kDa rice chitinase) and compared the level of ShB
resistance of the tlp–chi11 co-transformants with that of tlp
transformants. The transformants were assessed for their
enhanced ShB resistance by ‘bioassay using leaf sheaths
intact’ (BLSI) and ‘bioassay using detached leaves’ (BDL)
[28], which are drastic and non-laborious and the BDL, non-
sacrificial, unlike other methods of bioassay. Moreover, the
transformants were assayed also for their enhanced
resistance against the rice sheath rot pathogen, Sarocladium
oryzae (Sawada) Gams and Hawksworth causing severe rice
yield loss in Asia.
2. Materials and methods
2.1. Transformation vectors employed
The plant transformation vectors, pGL2-ubi-tlp, harbor-
ing a rice thaumatin-like protein gene, tlp, under the control
of a ubiquitin promoter [6], and pMKU-RF2, harboring a
rice chitinase gene, chi11, under the control of ubiquitin
promoter [28], were used to transform mature and immature
embryos of elite indica rice cultivars.
2.2. Preparation of immature embryos
Immature seeds of the rice cultivars, ASD16, ADT38,
IR50 and Taipei309, collected 12–14 days after pollina-
tion, were manually de-husked and surface-sterilized with
1.5% (v/v) sodium hypochlorite for 10–15 min and
washed three times with sterile distilled water. Immature
embryos were isolated under a stereomicroscope (Leica,
Switzerland) using a pair of sterile forceps. The embryos
were incubated for 2 days in the dark at 25 � 2 8C on CC
proliferation medium [29], in such a way that their
scutella were facing up. About 60–70 embryos were
arranged at the center of a 90 mm dia Petri plate
containing CC proliferation medium supplemented with
0.4 M osmoticum [30].
K. Kalpana et al. / Plant Science 170 (2006) 203–215 205
2.3. Callus induction from mature seeds
Manually de-husked seeds of PB1 were surface
sterilized successively with 70% ethanol for 3 min and
1.5% (v/v) sodium hypochlorite for 10–15 min, and
subsequently washed three times with sterile distilled
water. The sterilized seeds were placed onto Murashige–
Skoog (MS) callus induction medium [31] with 2.5 mg/l
2,4-dicholorophenoxy acetic acid (2,4-D) [30], and
incubated in the dark at 27 8C for 21 days. Embryogenic
calli were subcultured onto fresh callus induction medium,
twice, at 18-day interval. The hard friable calli were
arranged at the center of a 90 mm dia Petri plate containing
callus induction medium with 0.4 M osmoticum, 4 h prior
to bombardment.
2.4. Particle bombardment
Biolistic transformation was performed, as described by
Zhang et al. [32]. The helium-driven Biolistic PDS-1000/
He-Particle Delivery system (BioRad, Richmond, USA)
was used in all the experiments. The manufacturer’s
instructions were followed for coating gold particles
(10 mg) with plasmid DNA (20 mg). Two bombardments
per plate were performed at 4 h interval. After bombard-
ment, the immature embryos were transferred to 2,4-D-
supplemented (2.0 mg/l) CC proliferation medium with
50 mg/l hygromycin B. Germinating shoots of the
bombarded embryos were removed after 2–3 days. After
2 weeks, the developing calli were carefully separated from
the rest of the explants, broken into smaller pieces and
planted onto fresh proliferation medium containing 50 mg/l
hygromycin B. Surviving embryogenic calli were sub-
cultured onto the same medium, at 15-day intervals. The
calli that survived three rounds of selection on hygromycin
B were cultured on regeneration medium (CC proliferation
medium without 2,4-D) supplemented with 30 mg/l
hygromycin B, at 25 8C, under 16 h light (110–130 mE/
(m2 s)) and 8 h dark photoperiod, till shoots established
(3–4 weeks). The regenerated plantlets were transferred to
rooting medium, i.e., half strengthMSmediumwith 30 mg/
l hygromycin B.
Likewise, the bombarded mature seed-derived calli
obtained after three rounds of selection on hygromycin B
were transferred to MS regeneration medium [(MS basal
salts supplemented with 3.0 mg/l 6-benzylaminopurine
(BAP), 0.5 mg/l a-naphthalene acetic acid (NAA)]. The
calli were incubated at 25 � 2 8C, in the dark, for 10 days
and subsequently under 16 h light (110–130 mE/(m2 s)) and
8 h dark photoperiod. The emerging shoot buds were
transferred to MS rooting medium (half strength MS basal
salts) with 30 mg/l hygromycin B. Plantlets of 8–10 cm
height were transferred to Yoshida’s culture solution for
better root development [33]. The well-rooted plantlets were
hardened by transferring them to Hoagland’s medium,
before they were potted [34].
2.5. Regeneration and transformation efficiency
Regeneration efficiency (RE) is defined as the ratio of the
number of regenerations to the total number of explants
bombarded. Mean regeneration efficiency (MRE) is defined
as the sum of RE obtained in individual experiments divided
by the total number of experiments conducted. The term
transformation efficiency (TE) refers to the ratio of the
number of putative transgenic lines harboring the transgene(s)
[as assessed by polymerase chain reaction (PCR) or Southern
hybridization analysis] or expressing the same [as assessed by
enzyme-linked immunosorbent assay (ELISA) or by western
blotting analysis] to the total number of explants bombarded.
Mean transformation efficiency (MTE) is defined as the sum
of TE obtained in the individual experiments divided by the
total number of experiments conducted.
2.6. Genomic DNA isolation
Genomic DNA was isolated from the leaves of control
and transgenic plants following the method described by
Dellaporta et al. [35], with required modifications.
2.7. Polymerase chain reaction
Incorporation of transgenes in the putative transformants
was ascertained by PCR of hph gene. Amplifications were
performed in 25 ml reactions containing 10 mM Tris–HCl
(pH 9.0), 50 mM potassium chloride, 1.5 mM magnesium
chloride, 0.001% (w/v) gelatin, 200 mmeach of dNTPs, 75 ng
each of upstream (50-CGTCTGTCGAGAAGTTTC-30) anddownstream (50-TACTTCTACACAGCCATC-30) primers,
25 ng of template DNA and 2 units of Taq DNA polymerase
(Bangalore Genei Pvt. Ltd., India), following standard
protocols [36]. Reactions were carried out in a PTC-100
Minicycler (MJ Research, USA), setting the following
temperature profile: pre-incubation at 94 8C for 3 min,
leading to 35 cycles of melting at 94 8C for 1 min, annealing
at 55 8C for 1 min and synthesis at 72 8C for 1 min, followed
by an extension at 72 8C for 5 min.After the reaction, 10 ml of
the amplified product was analyzed on a 0.8% agarose gel.
2.8. Southern hybridization analysis
Southern hybridization was performed as described by
Sambrook et al. [36] with required modifications. Genomic
DNA from the control and transformed plants was restricted
with HindIII (Bangalore Genei Pvt. Ltd., India). Five
micrograms each of the HindIII-restricted genomic DNA
were resolved on a 1% agarose gel. After partial
depurination with 0.25N hydrochloric acid (20 min) and
denaturation with 0.4N sodium hydroxide, the gel was
equilibrated in 20� saline-sodium citrate (SSC) and the
DNA profile was transferred to a Hybond N+ nylon
membrane (Boehringer and Mannheim, UK) in 20� SSC,
by the conventional upward passive diffusion method.
K. Kalpana et al. / Plant Science 170 (2006) 203–215206
Pre-hybridization was done in 5� saline sodium phosphate
ethylenediaminetetraacetic acid (SSPE), 0.5% sodium dode-
cyl sulfate (SDS), 5� Denhardt’s reagent and 100 mg/ml
denatured sheared salmon sperm DNA, for 2 h at 65 8C.Hybridizationwas done in 5� SSC, 0.1%SDS and100 mg/ml
denatured sheared salmon sperm DNAwith 1 � 106 cpm/ml
[32P]-dCTP labeled probe, for 12 h, at 65 8C. After
hybridization, the filters were washed successively with
0.5� SSC, 2% SDS for 15 min at room temperature, twice,
and 0.2� SSC, 0.1% SDS for 15 min at 65 8C, twice.
The filters were dried and exposed for autoradiography.
2.9. Western blotting analysis
The soluble fraction of leaf proteins was prepared both
from the putative transformants and non-transformant
controls. One gram of leaf material was homogenized in
5 ml of 1 M sodium phosphate buffer (pH 7.0), centrifuged
at 12000 � g for 15 min at 4 8C and the supernatant
collected. Western blotting was carried out following the
method described by Gallagher et al. [37]. The soluble
proteins, resolved by 12% sodium dodecyl sulphate-
polyacrylamide gel electrophoresis [38], were transferred
onto a nitrocellulose membrane (Protran BAS5 celluloseni-
trat, Schleicher and Schuell, Germany), on a Transblot semi-
dry transfer apparatus (Bio-Rad laboratories, USA). The
membrane was blocked with 10% (v/v) Tween-20 and 5%
(w/v) milk powder (Sagar, India) in PBS, for 3 h, at room
temperature. The membrane was then treated with either
anti-tobacco TLP antiserum (a gift from Dr. Legrand,
Strasbourg, Cedex, France) or anti-barley chitinase anti-
serum (a gift from Dr. S. Muthukrishnan, Kansas State
University, USA), for 3 h at room temperature, with gentle
shaking. After the removal of unbound antibody by washing
thrice in PBST, the membrane was incubated for 3 h at room
temperature, in anti-rabbit IgG-alkaline phosphatase con-
jugate (Bangalore Genei Pvt. Ltd., India), diluted to 1:7000
in PBST. The unbound secondary antibody was removed by
three washes with PBST. The antigen band detected either
by the anti-tobacco TLP antibody or anti-barley chitinase
antibody was visualized by treating the membrane with 3-
bromo-4 chloro-3 indolyl phosphate/nitro blue tetrazolium
(BCIP/NBT) (Bangalore Genei Pvt. Ltd., India).
2.10. Progeny analyses
The T1 and T2 progenies of the putative transgenic lines
were checked for the presence of transgene(s) and its
expression, respectively, by PCR and western blotting
analyses. The plants expressing the transgenes were screened
for resistance against the R. solani virulent isolate 7 (Rs7).
2.11. Sheath blight bioassay
The T0 and T1 generation of transformants were screened
for ShB resistance, 40 days after planting. All the R. solani
inoculationswere carried outwithmycelial discs of 5 mmdm,
obtained from 3-day-old cultures of Rs7, grown on potato
dextrose agar, at 28 8C. Each selected leaf sheath was
inoculated with a single mycelial disc. The mycelial disc,
placed on leaf sheath, was covered with absorbent cotton and
securedwith parafilm. The cottonwasmoistened periodically
with sterile distilled water, to maintain high humidity.
Development of symptoms was recorded 7 days after
inoculation (DAI) and the disease intensity was graded using
a 0–5 scale [39]. Based on the grades, the disease intensitywas
expressed as ‘percent disease index’ (PDI), i.e., sum of all the
grades/number of tillers graded � 100/maximum grade.
The T2 generation tlp transformants and T1 and T2
generation tlp–chi11 co-transformants were screened for
ShB resistance, by BDL and BLSI, using a 5-mm mycelial
disc of Rs7 [28]. In BDL, number of infection cushions on
leaves of transgenic and non-transgenic plants were
recorded, 72 h after inoculation (HAI). By BLSI, functional
resistance/susceptibility index (FRI) was calculated up to
168 h, at 24 h intervals. Apart from FRI, highest relative
lesion height percentage (HRLH%) [40] was calculated both
in transgenic and non-transgenic plants, 7 DAI.
2.12. Sheath rot bioassay
Thirty grams of chaffy paddy grains soaked overnight in
100 ml distilled water in a 250-ml Erlenmeyer flask was
sterilized at 15 psi for 1 h. The chaffy paddy grain
suspension was inoculated with 6-day-old mycelial mat of
the sheath rot pathogen, S. oryzae and incubated at room
temperature for 2 weeks. Selected chaffy grains harboring
well-developed mycelia of S. oryzae were used for infecting
50–55-day-old plants of the tlp or chi11 transgenic ASD16
and ADT38 lines and respective non-transformed controls.
The infective grains were placed in between boot leaf sheath
and panicle and secured by an encircled cotton pad wrapped
externally with parafilm. The cotton pad was moistened with
sterile water, at regular intervals, in order to prevent mycelial
desiccation [41]. The disease development was monitored
and recorded 3, 6, 9, 12 and 15 DAI. Disease intensity was
expressed as per cent sheath infection, i.e., length of lesion/
total length of sheath enclosing panicle � 100.
3. Results and discussion
It has been demonstrated that a constitutive and higher
level expression of PR-proteins in transgenic plants
enhances resistance to a variety of fungal pathogens [4–
8]. Thaumatin-like proteins (TLPs), categorized under
Group 5 PR-proteins, are antifungal owing to their
membrane permeabilizing activity [10,11,14,15]. Trans-
genic plants constitutively over-expressing TLPs showed an
enhanced fungal resistance [6,42]. In the present study,
certain high-yielding indica rice cultivars, recalcitrant to
genetic transformation have been transformed with a rice tlp
K. Kalpana et al. / Plant Science 170 (2006) 203–215 207
Table 1
Transformation events using pGL2-ubi-tlp
Genotype No. of embryos
bombarded
No. of lines
regenerated
Regeneration
efficiency (%)
No. of lines
positive to hph
No. of lines
positive to TLP (Western)
Transformation
efficiency (%)
ASD16 887 57 6.43 14 11 1.20
ADT38 1480 110 7.40 14 10 0.80
IR50 1081 57 5.30 20 12 1.10
Pusa Basmati1 163 19 11.70 12 12 7.40
Taipei 309 340 47 13.82 45 43 12.70
gene in order to confer enhanced resistance against the ShB
pathogen, R. solani. The membrane permeabilizing TLPs
are known to be co-regulated or co-expressed with other PR-
proteins such as chitinases and glucanases [23–26,43]
capable of degrading fungal cell wall carbohydrates. Barley
grain TLPs inhibited growth of Trichoderma viride and
Candida albicans and act synergistically with barley grain
chitinase C [27]. Their working in concert is also evident
from their responsiveness to salicylic acid and ethylene [44].
Hence, in the present study, co-transformation of indica rice
cultivars with tlp and chi11 (a rice chitinase gene) was also
carried out to produce TLP and chitinase co-expressing lines
with more enhanced resistance than the tlp transformants.
The tlp transformant and tlp–chi11 co-transformant rice
lines were subjected to thorough molecular analyses and
rigorous bioassays.
3.1. Biolistic transformation and regeneration
Genetic transformation of the indica rice cultivars,
ASD16, ADT38, IR50 and PB1 and the japonica rice
cultivar, Taipei309, was done with ubiquitin promoter-
driven rice tlp gene, by particle bombardment method.
Several lines of putative transgenic plants constitutively
over-expressing TLP were obtained by transforming
immature embryos and mature seed-derived calli. Trans-
formation of indica rice cultivars yielded a total of 143
putative transformant lines, with regeneration efficiencies of
6.43, 7.40, 5.30 and 11.70, respectively, for ASD16, ADT38,
IR50 and PB1. However, the transformation efficiencies of
these indica rice cultivars were only 1.20, 0.80, 1.10 and
Fig. 1. Southern blotting analysis in putative (T0) transgenic indica rice lines fo
control). Lane 2: non-transformed ASD16. Lanes 3–8: transformed ASD16 (KL-A
control). Lanes 10 and 11: transformedADT38 (KL-ADT38-4 and 5) lines. Lane 12
23: transformed PB1 (KL-PB1-1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) lines. Lane 24: pGL
(KL-IR50-1, 2 and 3) lines.
7.40%, respectively (Table 1). Transformation of the
japonica rice cultivar, Taipei309, yielded 47 putative
transformant lines, with relatively higher regeneration and
transformation efficiencies of 13.82 and 12.70%, respec-
tively. Such lower transformation efficiency, when com-
pared to japonica rice cultivars, was common among indica
rice cultivars. The lower transformation efficiency of indica
rice cultivars could be attributed to their recalcitrance, as
suggested earlier by Peng et al. [45] and Sivamani et al. [46].
Earlier, Christou et al. [47] obtained a transformation
efficiency of 0.80 and 3.20% in IR72 and IR36, respectively,
using immature embryos as explants and a selectable marker
system as a candidate gene. Likewise, several of the hitherto
reported indica rice transformations also faced similar
problems of low efficiency [46,48].
3.2. Integration of tlp transgene in putative
transformants
Integration of transgenes in the genome of putative
transformants was ascertained by PCR for hph transgene.
The expected amplified product, a 955 bp DNA fragment,
was obtained from 60 lines of the indica cultivars, ASD16,
ADT38, IR50 and PB1 and 30 lines of the japonica cultivar,
Taipei309 (Table 1). Amplification of DNA from control
plants did not yield the expected product (data not shown).
To confirm the integration of tlp transgene, the PCR-
selected transformants were analyzed by Southern hybridi-
zation. The HindIII-digested genomic DNA from four lines
of the cultivar ASD16 (KL-ASD16-1, 2, 3 and 5), two lines
of the cultivar ADT38 (KL-ADT38-4 and 5), four lines of
r the integration of tlp expression cassette. Lane 1: pGL2-ubi-tlp (positive
SD16-1, 2, 3, 4, 5 and 6) lines. Lane 9: non-transformed ADT38 (negative
: pGL2-ubi-tlp (positive control). Lane 13: non-transformed PB1. Lanes 14–
2-ubi-tlp. Lane 25: non-transformed IR50. Lanes 26–28: transformed IR50
K. Kalpana et al. / Plant Science 170 (2006) 203–215208
Fig. 2. Western blotting analysis of T0 transgenic indica rice lines for TLP expression. Lane 1: pearl millet TLP (positive control). Lane 2: non-transformed
ASD16 (negative control). Lanes 3–5: transformed ASD16 (KL-ASD16-1, 2 and 3) lines. Lane 6: non-transformed ADT38 (negative control). Lanes 7–10,
transformed ADT38 (KL-ADT38-4, 5, 6 and 7) lines. Lane 11: non-transformed PB1 (negative control). Lanes 12–16: transformed PB1 (KL-PB1-2, 4, 6, 8 and
11) lines. Lane 17: non-transformed IR50 (negative control). Lanes 18–20, transformed IR50 (KL-IR50-1, 2 and 3).
Fig. 3. Bioassay of putative T0 transgenic PB1 lines for sheath blight
resistancewithRs7. (A)Transgenic PB1 line and (B) non-transgenic PB1 line.
the cultivar PB1 (KL-PB1-2, 4, 6 and 8) and three lines of the
cultivar IR50 (KL-IR50-1, 2 and 3) were hybridized against
radiolabeled probe prepared from the 3.1 kbp tlp expression
cassette released from pGL2-ubi-tlp by HindIII digestion. A
3.1 kbp hybridization signal corresponding to the whole tlp
expression cassette, was conspicuous in the Southern
hybridization profiles of the transgenic individuals analyzed
(Fig. 1), confirming the integration of tlp.
3.3. Expression of tlp transgene in putative
transformants
TLP expression in the T0 transgenic lines of the indica
rice cultivars, ASD16, ADT38, PB1 and IR50 was checked
by ELISA (data not shown) and subsequently confirmed by
western blotting analyses with a tobacco anti-TLP anti-
serum. As evidenced by the results of western blotting
analyses, over-expression of a 23 kDa TLP was observed in
45 lines among the indica cultivar (ASD16, ADT38, IR50
and PB1) transformants and 19 lines among the japonica
cultivar (Taipei309) transformants (Table 1). No expression
of TLP was observed in non-transformed checks. Fig. 2
shows western blotting profiles of the soluble protein
fractions of the leaves of the putative tlp transgenic lines (T0)
of ASD16 (KL-ASD16-1, 2 and 3), ADT38 (KL-ADT38-4,
5, 6 and 7), PB1 (KL-PB1-2, 4, 6, 8 and 11) and IR50 (KL-
IR50-1, 2 and 3), revealing the detection of the 23 kDa TLP
by the tobacco anti-TLP antiserum. Previously, Datta et al.
[6] reported the constitutive over-expression of TLP and
associated enhanced ShB resistance in transgenic indica rice
lines of Chinsurah Boro II, IR72 and IR51500.
3.4. Sheath blight resistance and its inheritance in tlp
transformants
Two T0 tlp transgenic lines of the cultivar PB1, i.e., KL-
PB1-2 and KL-PB1-4, which proved positive in western
blotting analysis, were evaluated for ShB resistance.
Inoculation of these tlp transgenic lines with Rs7 revealed
a reduced blighting level, as compared to that in the non-
transformed control (Fig. 3). Previously, Datta et al. [6]
showed that transgenic indica rice constitutively over-
expressing rice TLP possessed enhanced ShB resistance.
Likewise, Zhu et al. [42] demonstrated that transgenic potato
constitutively over-expressing an osmotin-like protein, a
member of thaumatin protein family and a PR5 protein,
exhibited higher level resistance to the late blight fungus,
Phytophthora infestans. Chen et al. [34] reported an enhanced
resistance towheat scab pathogen,Fusarium graminearum, in
transgenic wheat constitutively over-expressing TLP.
Fagoaga et al. [49] showed that a constitutive over-expression
of tomato P23 (a 23 kDa PR-5 protein similar to osmotin) in
orange, Citrus sinensis L. Obs. cv. Pineapple, resulted in a
consistent and significant reduction in lesion development
caused by the oomycete, Phytophthora citrophthora and
improved the survival ability of the transformants, when
challenged with oomycetes cultures.
Progeny analysis was done in three T1 tlp transgenic lines
of the cultivar ASD16 (KL-T1-ASD16-1, 2 and 3) by
western blotting analysis. The results revealed the expres-
sion of a 23 kDa polypeptide, corresponding to the TLP
antigen, in the lines KL-T1-ASD16-1.1, 1.2, 1.3, 2.1, 2.2, 2.3
K. Kalpana et al. / Plant Science 170 (2006) 203–215 209
Table 2
Bioassay of transgenic ASD16 (T1) lines expressing TLP against rice sheath
blight pathogen (R. solani)
Lines PDI � S.E.
KL-T1-ASD16-1 40.00 � 1.14
KL-T1-ASD16-2 60.00 � 0.92
KL-T1-ASD16-3 35.00 � 1.86
Non-transformed ASD16 70.00 � 2.04
PDI, percent disease index; as seed set was poor in T0 plants, only a single
plant in each putative transgenic line was spared for the bioassays. Indi-
vidual tillers were considered as separate replications.
Table 3
Assessment of sheath blight resistance by detached leaf assay using infec-
tion cushions
Line Number of infection cushionsa
KL-T2-ASD16-2 15.2d
KL-T2-ASD16-3 16.4d
Non-transformed ASD16 47.8b
KL-C-T1-ADT38-6 23.2c
Non-transformed ADT38 55.6a
In a column, means followed by a common letter (a–d) are not significantly
different at the 5% level by DMRT.a Mean of three replications (five leaf bits/replication).
and 3.3 (data not shown). These results confirmed the
inheritance of tlp transgene, in its functional form, to the T1
progenies of the ASD16 lines tested. Moreover, in bioassay,
inoculation of these lines with Rs7 revealed high levels of
resistance of KL-T1-ASD16-1 and 3 against the pathogen, as
evidenced by a low level of ShB incidence. They scored
significantly lower PDIs (40.0 and 35.0, respectively), as
compared to that in non-transgenic control (70.0, Table 2).
Western blotting analyses of T2 individuals of the ASD16
lines, KL-T2-ASD16-2 and 3 revealed stable inheritance and
expression of tlp transgene (data not shown). Their
resistance against ShB was evaluated by BDL and BLSI,
as described by Kumar et al. [28]. BDL revealed a reduction
in the number of infection cushions on the leaves of the
transgenic lines, when compared to that in the non-
transgenic controls. Frequency of formation of infection
cushion was only 15.2 and 16.4 in the said tlp transgenic
lines, while it was as high as 47.8 and 55.6 in ASD16 and
ADT38 non-transgenic controls, respectively (Table 3).
Moreover, in the tlp transgenic lines, the disease lesions
were relatively smaller and surrounded by a conspicuous
defensive browning, while in the non-transgenic controls,
the disease lesions were larger (Fig. 4), leading to morbid
yellowing of leaves. In BLSI, ShB symptom appeared on
leaf sheaths of non-transformed plants within 48 HAI, while
the symptom was noticed only after 72 or 96 h, in the
assayed tlp transgenic lines. In the leaf sheaths of transgenic
lines, the spread of lesion was slower and the lesions were
Fig. 4. Assessment of sheath blight resistance by detached le
surrounded by zones of extensive browning. However, in
non-transformed controls, blanched lesions appeared, the
faster spread of which led to complete drying of infected leaf
sheaths, within 168 h (Fig. 5). Groth and Nowick [1]
suggested that resistance to the spread of R. solani in rice
could be consequent to production of oxidised phenolics
(dark zone around lesions). In BLSI, FRI was calculated up
to 168 h, at 24 h intervals. FRI values increased gradually in
case of ASD16 control plants, up to 168 h, whereas in the
transgenic individuals, there was a sudden increase in FRI
that continued till 168 h (Table 4). Similarly, Kumar et al.
[28] observed a gradual increase in FRI values throughout
the incubation period in control plants, whereas, in chi11
transformants FRI values rose suddenly, after a gradual
increase in the initial stage. Apart from FRI, HRLH% was
also calculated both in transgenic and non-transgenic plants,
7 DAI. A non-transgenic ASD16 control plant recorded a
HRLH% of 9.48, at 168 h, whereas, it was as low as 2.82 and
3.35 in the two transgenic individuals tested (Table 4).
3.5. Co-transformation of elite indica rice cultivars with
tlp and chi11
Co-transformation of elite indica rice cultivars was
attempted with a view to exploring the possibilities of
enhancing resistance to ShB. Two independent experiments
were conducted using the indica cultivars, ADT38 and IR50.
In case of ADT38, a total of 18 lines were obtained, with a
af assay on T2 transgenic ASD16 lines expressing TLP.
K. Kalpana et al. / Plant Science 170 (2006) 203–215210
Fig. 5. Assessment of sheath blight resistance by intact leaf sheath assay on T2 transgenic ASD16 lines expressing TLP.
regeneration efficiency of 3.7% and co-transformation
efficiency of 0.82%. In IR50, the number of lines
regenerated was 9, with a regeneration and co-transforma-
tion efficiencies of 3.0 and 0.33%, respectively (Table 5).
3.6. Integration and co-expression of tlp and chi11
After co-transformation with tlp and chi11, the putative
transformants (T0) were selected by PCR for hph and gusA
Table 4
Assessment of sheath blight resistance by intact leaf sheath assay using function
Line Functional resistance/susceptibility index (FRI)
24 h 48 h 72 h 96 h
KL-T2-ASD16-2 0.00a 0.00c 0.00d 94.17
KL-T2-ASD16-3 0.00a 0.00c 58.71a 87.65
Non-transformed ASD16 0.00a 24.17a 29.42b 44.15
KL-C-T1-ADT38-6 0.00a 0.00c 54.25a 78.64
Non-transformed ADT38 0.00a 18.45b 22.67c 38.45
In a column, means followed by a common letter (a–e) are not significantly diffa Mean of three replications (five plants/replication).
sequences. Eighteen lines of the cultivar, ADT38 and five
lines of the cultivar, IR50 proved positive for hph. However,
only seven lines of ADT38 and two lines of IR50 proved
positive for gusA (Table 5). The expression of transgenic
TLP and chitinase in transformed plants was examined by
western blotting analysis. An anti-barley chitinase antibody
detected the 35 kDa chitinase in leaf protein extracts from
three T0 lines of the cultivar ADT38 (KL-C-ADT38-4, 5 and
6) and one line of the cultivar IR50 (KL-C-IR50-1) (Fig. 6).
al resistance/susceptibility index (FRI) and HRLH%
a HRLH% (7 DAI)a
120 h 144 h 168 h
a 124.16a 131.47a 165.41a 2.82d
b 115.87b 127.05b 158.70b 3.35c
d 71.70c 85.16c 114.26e 9.48b
c 124.38a 131.50a 150.65c 3.85c
e 68.90c 79.21d 105.27d 11.26a
erent at the 5% level by DMRT.
K. Kalpana et al. / Plant Science 170 (2006) 203–215 211
Table 5
Co-transformation of indica rice with pGL2-ubi-tlp and pMKU-RF2 (chi11)
Genotype No. of
embryos bombarded
No. of lines
regenerated
Regeneration
efficiency (%)
No. of PCR
positive lines
No. of western
positive lines
Co-transformation
efficiency (%)
hph gusA Chitinase TLP Chitinase+ TLP
ADT38 482 18 3.7 18 7 2 2 1 0.82
IR50 301 9 3.0 5 2 1 1 � 0.33
Fig. 6. Western blotting analysis for chitinase expression in T0 transgenic ADT38 and IR50 lines. Lane 1: rice chitinase (positive control). Lane 2: non-
transformed ADT38 (negative control). Lanes 3–8: transformed ADT38 (KL-C-ADT38-1, 2, 3, 4, 5 and 6). Lane 9: non-transformed IR50 (negative control).
Lanes 10–12: transformed IR50 (KL-C-IR50-1, 2 and 3).
Table 6
Bioassay of putative transformants of ADT38 (expressing tlp and/or chi11)
for resistance against Rs7
Putative transgenic lines (T0) Transgene(s) expressed PDI � S.E.
KL-C-ADT38-1 tlp 40 � 0.64
KL-C-ADT38-2 tlp 50 � 0.88
KL-C-ADT38-4 chi11 40 � 1.67
KL-C-ADT38-5 chi11 46 � 2.38
KL-C-ADT38-6 tlp + chi11 30 � 0.51
Non-transformed ADT38 None 60 � 1.98
PDI, percent disease index; only a single plant in each putative transgenic
line was spared for the bioassays. Individual tillers were considered as
separate replications.
Likewise, the anti-tobacco TLP antiserum detected the
23 kDa TLP in three lines of ADT38 (KL-C-ADT38-1, 2
and 6) and one line of IR50 (KL-C-IR50-3) (Fig. 7). The
anti-chitinase and anti-TLP antisera did not detect any
such antigens in the leaf protein extracts of non-
transformed control plants. Among the transformants,
only a single line, KL-C-ADT38-6, was able to bring about
the desired co-expression of tlp and chi11 (Figs. 6 and 7).
No report on the transgenic co-expression of these two
candidate genes in crop plants is available in the existing
literature, except the one dealing with an attempt to
manage wheat scab pathogen, F. graminearum, employing
these two genes [34].
3.7. Enhanced ShB resistance conferred by tlp and
chi11 co-expression
The putative transgenic T0 ADT38 lines (KL-C-ADT38-
1, 2, 4, 5 and 6) upon inoculation with Rs7 recorded a lower
PDI (40, 50, 40, 46 and 30, respectively), as compared to that
observed in the control (60.00). Among them, the line KL-C-
ADT38-6, co-expressing tlp and chi11, recorded the lowest
PDI of 30.00 (Table 6). In this tlp and chi11 co-expressing
line, the number and size of the lesions were smaller than in
the lines expressing either tlp or chi11 (Fig. 8). These results
suggested the functional synergy of activity of TLP and
chitinase in transgenic context, conferring higher disease
Fig. 7. Western blotting analysis for TLP expression in T0 transgenic ADT38 a
transformed ADT38 (negative control). Lanes 3–8: transformed ADT38 (KL-C-A
Lanes 10–12: transformed IR50 (KL-C-IR50-1, 2 and 3).
resistance on the transformants. Earlier studies reported
partial and narrow-spectrum disease resistance of single PR-
protein transformants [50]. However, the co-expression of
more than one PR-protein genes, such as chitinase and b-
1,3-glucanase, was shown to be much more effective against
development of several fungal diseases than expression of a
single gene [18–20].
3.8. Progeny analysis (T1 and T2) of tlp–chi11 co-
transformant
The progeny of the transgenic ADT38 line, KL-C-
ADT38-6, co-expressing TLP and chitinase, was analyzed
for the inheritance and stable expression of both the
nd IR50 lines. Lane 1: pearl millet TLP (positive control). Lane 2: non-
DT38-1, 2, 3, 4, 5 and 6). Lane 9: non-transformed IR50 (negative control).
K. Kalpana et al. / Plant Science 170 (2006) 203–215212
Fig. 8. Bioassay of putative T0 transgenic ADT38 lines expressing tpl and/or chi11 for sheath blight resistance with Rs7. (A) Transgenic ADT38 line (KL-C-
ADT38-1) expressing tlp. (B) Transgenic ADT38 line (KL-C-ADT38-4) expressing chi11. (C) Transgenic ADT38 line (KL-C-ADT38-6) expressing both tlp
and chi11. (D) Non-transgenic ADT38 line.
Table 7
Bioassay of transgenic (T2) lines expressing TLP or chitinase for sheath rot
resistance
Transgenic (T2) lines Transgene
expressed
Percent (%) sheath
infection (15 DAI)a
KL-T2-ASD16-2 tlp 2.90c
KL-T2-ASD16-3 tlp 3.67b
Non-transformed ASD16 None 19.86a
KL-C-ADT38-1 tlp 2.95c
KL-C-ADT38-2 tlp 2.75c
KL-C-ADT38-4 chi11 2.48c
KL-C-ADT38-5 chi11 3.53b
Non-transformed ADT38 None 18.56a
In a column, means followed by a common letter (a–c) are not significantly
different at the 5% level by DMRT.a Mean of 5 replications (3 sheaths/replication).
transgenes. In western blotting analyses of T1 progeny, TLP
antigen alone was detected in all the individuals, probably
because of the silencing of the expression of chi11 transgene
(data not shown). Chen et al. [34] encountered the same
problem while attempting to co-express tlp and chi11 in
wheat against F. graminearum, causing wheat scab, and
attributed it to the transgene silencing. However, bioassay of
T1 progeny against Rs7 revealed appreciable resistance
against the pathogen: the results of BDL evidenced a
significant reduction in number of infection cushions
(Table 3) and BLSI, a delayed symptom expression, reduced
lesion spread and precocious browning around the site of
inoculation (Table 4). Likewise, the bioassay of the
individuals of T2 progeny also evidenced similar spectrum
of resistance against Rs7 (data not shown).
K. Kalpana et al. / Plant Science 170 (2006) 203–215 213
3.9. Enhanced resistance of tlp and chi11 transformants
against S. oryzae
The present tlp and chi11 transformants were assayed
also for their resistance against S. oryzae causing sheath rot.
The results showed enhanced transgenic resistance of both
the tlp and chi11 transformants against S. oryzae, as
evidenced by the reduction in ‘percent sheath infection’, in
comparison to non-transformed controls. The T2 generation
tlp and chi11 transformants showed more than six to eight-
fold reduction in percent sheath infection (Table 7). The
common symptom(s) of sheath rot pathogen is characterized
Fig. 9. Assessment of sheath rot resistance in transgenic
by the formation of grayish brown lesions on the upper leaf
sheath enclosing the panicle [51]. In the present study, tiny
grayish lesions surrounded by thin brown border were
observed as early as 3 DAI on the sheaths of non-
transformed control plants. These lesions enlarged into
characteristic oblong grayish lesions 12 DAI. In transgenic
plants, the development of sheath rot symptom was delayed
and small brownish lesions started appearing only 6 DAI.
The delayed occurrence and relatively slow enlargement of
lesions coupled with extensive browning (a host defense
reaction) around the lesions in transgenic plants (Fig. 9)
suggest enhanced transgenic resistance against S. oryzae.
lines constitutively expressing TLP or chitinase.
K. Kalpana et al. / Plant Science 170 (2006) 203–215214
These data suggests a broader spectrum fungal disease
resistance potential of the tlp and chi11 transformants
produced in the present study.
4. Conclusion
In the present study, we were able to produce transgenic
lines of indica rice cultivars with an enhanced ShB resistance
by virtue of their constitutive over-expression of rice tlp
transgene. The tlp transgenic lines inherited stably the
transgene to their offspring. The inheritance of TLP
expression correlatedwith the ShB resistance of the offspring,
as evidenced by the molecular analyses and bioassay up to T2
generation. Hence, the tlp transgenic lines produced in this
study could be used as a source of ShB resistance in breeding
programmes. Moreover, we also attempted to produce
transgenic indica rice lines co-expressing rice tlp and
chi11. The transgenic line that co-expressed tlp and chi11,
in T0 generation, showed an enhanced level of ShB resistance
than the transgenic lines that expressed tlp or chi11 alone.
However, the chi11 transgene was silenced in T1 generation.
Yet, the results of the present study suggest the viable
synergistic activity of TLP and chitinase in a transgenic
context to enhance fungal disease resistance. In addition, the
enhanced resistance of the tlp and chi11 transformants against
S. oryzae suggests their broader spectrum fungal disease
resistance potential. Presently, efforts are under way to
produce indica rice transformants stably co-inheriting
functional tlp and chi11 transgenes.
Acknowledgements
We are grateful to The Rockefeller Foundation, USA [#
99001/225 (PB) and # 99001/227 (DS)] and the Department
of Biotechnology, New Delhi, India, for providing the
financial support. We greatly acknowledge the help rendered
by Dr. Legrand, Strasbourg, Cedex, France, and Dr. S.
Muthukrishnan, Kansas State University, USA.
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