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.

References

[1] D.E. Groth, E.M. Nowick, Selection for resistance to rice sheath blight

through number of infection cushions and lesion type, Plant Dis. 76

(1992) 721–723.

[2] J.M. Bonman, G.S. Khush, R.J. Nelson, Breeding rice for resistance to

pests, Annu. Rev. Phytopathol. 30 (1992) 507–526.

[3] L.C. Van Loon, E.A. Van Strien, The families of pathogenesis-related

proteins, their activities, and comparative analysis of PR-1 type

proteins, Physiol. Mol. Plant Pathol. 55 (1999) 85–97.

[4] W. Lin, C.S. Anuratha, K. Datta, I. Potrykus, S. Muthukrishnan, S.K.

Datta, Genetic engineering of rice for resistance to sheath blight,

BioTechnology 13 (1995) 686–691.

[5] T. Tabei, S. Kitase, Y. Nishizawa, N. Kikuchi, T. Kayano, T. Hibi, K.

Akutsu, Transgenic cucumber plants harboring a rice chitinase gene

exhibit enhanced resistant to gray mold (Botrytis cinerea), Plant Cell

Rep. 17 (1998) 159–164.

[6] K. Datta, R. Velazhahan, N. Oliva, I. Ona, T. Mew, G.S. Kush, S.

Muthukrishnan, S.K. Datta, Over-expression of the cloned rice thau-

matin-like protein (PR-5) gene in transgenic rice plants enhances

environmental friendly resistance to Rhizoctonia solani causing sheath

blight disease, Theor. Appl. Genet. 98 (1999) 1138–1145.

[7] K. Datta, Z. Koukolıkova-Nicola, N. Baisakh, N. Oliva, S.K. Datta,

Agrobacterium-mediated engineering for sheath blight resistance of

indica rice cultivars from different ecosystems, Theor. Appl. Genet.

100 (2000) 832–839.

[8] K. Datta, J. Tu, N. Oliva, I. Ona, R. Velazhahan, T.W. Mew, S.

Muthukrishnan, S.K. Datta, Enhanced resistance to sheath blight by

constitutive expression of infection-related rice chitinase in transgenic

elite indica rice cultivars, Plant Sci. 60 (2001) 405–414.

[9] B.J. Cornelissen, R.A. Hooft van Huijsduijnen, J.F. Bol, A tobacco

mosaic virus-induced tobacco protein is homologous to the sweet

tasting protein thaumatin, Nature 321 (1986) 531–532.

[10] W.K. Roberts, C.P. Selitrennikoff, Zeamatin, an antifungal protein

from maize with membrane-permeabilizing activity, J. Gen. Micro-

biol. 136 (1990) 1771–1778.

[11] A.J. Vigers, W.K. Roberts, C.P. Selitrennikoff, A new family of plant

antifungal proteins, Mol. Plant Microbe Interact. 4 (1991) 315–323.

[12] L.R. Abad, M.P. D’Urzo, D. Lin, M.L. Narasimhan, M. Renveni, J.K.

Zhu, X. Niu, N.K. Singh, P.M. Hasegawa, R.A. Bressan, Antifungal

activity of tobacco osmotin has specificity and involves plasma

membrane permeabilization, Plant Sci. 118 (1996) 11–23.

[13] D. Liu, K.G. Raghothama, P.M. Hasegawa, R.A. Bressan, Osmotin

overexpression in potato delays development of disease symptoms,

Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 1888–1892.

[14] A.J. Vigers, S. Wiedemann, W.K. Robertz, M. Legrand, C.P. Seli-

trennikoff, B. Fritig, Thaumatin-like pathogenesis-related proteins are

antifungal, Plant Sci. 83 (1992) 155–161.

[15] D.J. Yun, J.I. Ibeas, H. Lee, M.A. Coca, M.L. Narasimhan, Y. Uesono,

P.M. Hasegawa, J.M. Pardo, R.A. Bressan, Osmotin, a plant antifungal

protein, subverts signal transduction to enhance fungal cell suscept-

ibility, Mol. Cell 1 (1998) 807–817.

[16] A. Anand, T. Zhou, H.N. Trick, B.S. Gill, W.W. Bockus, S. Muthuk-

rishnan, Greenhouse and field testing of transgenic wheat plants stably

expressing genes for thaumatin-like protein, chitinase and glucanase

against Fusarium graminearum, J. Exp. Bot. 54 (2003) 1101–1111.

[17] J.M. Neuhans, P. Ahl-Goy, U. Hinz, S. Flores, F.J. Meins, High-level

expression of a tobacco chitinase gene in Nicotiana sylvestris: sus-

ceptibility of transgenic plants to Cercospora nicotianae infection,

Plant Mol. Biol. 16 (1991) 141–151.

[18] E. Jongedijk, H. Tigelaar, J.S.C. van Roakel, S.A. Bres-Vloemans, I.

Dekker, P.J.M. van den Elzen, B.J.C. Cornelissen, L.S. Melchers,

Synergistic activity of chitinase and b-1,3-glucanase enhances fungal

resistance in transgenic tomato plants, Euphytica 85 (1995) 173–180.

[19] Q. Zhu, E.A. Maher, S. Masoud, R.A. Dixon, C.J. Lamb, Enhanced

protection against fungal attack by constitutive co-expression of

chitinase and glucanase genes in transgenic tobacco, BioTechnology

12 (1994) 807–812.

[20] G. Jach, B. Gornhardt, J. Mundy, J. Logemann, E. Pinsdorf, R. Leah, J.

Schell, C. Maas, Enhanced quantitative resistance against fungal

disease by combinatorial expression of different barley antifungal

proteins in transgenic tobacco, Plant J. 8 (1995) 97–109.

[21] R.I.W. Osmond, M. Hrmova, F. Fontaine, A. Imberty, G.B. Fincher,

Barley thaumatin-like proteins: ligand specificity, kinetic and struc-

tural analyses and biological implications, Eur. J. Biochem. 268 (2001)

4190–4199.

[22] J. Grenier, C. Potvin, J. Trudel, A. Asselin, Some thaumatin-like

proteins hydrolyse polymeric b-1,3-glucan, Plant J. 19 (1999) 473–

480.

[23] W.J. Peumans, P. Proost, R.L. Swennen, E.J.M. Van Damme, The

abundant class III chitinase homolog in young developing banana

fruits behaves as a transient vegetative storage protein and most

probably serves as an important supply of amino acids for the synthesis

of ripening-associated proteins, Plant Physiol. 130 (2002) 1063–1072.

K. Kalpana et al. / Plant Science 170 (2006) 203–215 215

[24] R.A. Salzman, I. Tikhonova, B.P. Bordelon, P.M. Hasegawa, R.A.

Bressan, Coordinate accumulation of antifungal proteins and hexoses

constitutes a developmentally controlled defense-response during fruit

ripening in grape, Plant Physiol. 117 (1998) 465–472.

[25] M. Hiilovaara-Teijo, A. Hannukkala, M. Griffith, X.M. Yu, K. Piha-

kaski-Maunsbach, Snow mold-induced apoplastic proteins in winter

rye leaves lack antifreeze activity, Plant Physiol. 121 (1999) 665–674.

[26] A.K. Jacobs, I.B. Dry, S.P. Robinson, Induction of different pathogen-

esis-related cDNAs in grapevine infected with powdery mildew and

treated with ethephon, Plant Pathol. 48 (1999) 325–336.

[27] J. Hejgaard, S. Jacobsen, I. Svendsen, Two antifungal thaumatin-like

proteins from barley grain, FEBS Lett. 291 (1991) 127–131.

[28] K.K. Kumar, K. Poovannan, R. Nandakumar, K. Thamilarasi, C.

Geetha, N. Jayashree, E. Kokiladevi, J.A.J. Raja, R. Samiyappan,

D. Sudhakar, P. Balasubramanian, A high throughput functional

expression assay system for a defence gene conferring transgenic

resistance on rice against the sheath blight pathogen, Rhizoctonia

solani, Plant Sci. 165 (2003) 969–976.

[29] I. Potrykus, C.T. Harms, H. Lorz, Callus formation from cell proto-

plasts of corn (Zea mays L.), Theor. Appl. Genet. 54 (1979) 209–214.

[30] V. Vasil, V. Srivastava, A.M. Castillo, M.E. Fromm, I.K. Vaso, Rapid

production of transgenic wheat plants by direct bombardment of

cultured immature embryos, BioTechnology 11 (1993) 1553–1558.

[31] T. Murashige, F.A. Skoog, A revised medium for rapid growth and

bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473–

497.

[32] S. Zhang, W.Y. Song, L. Chen, D. Ruan, N. Taylor, P.C. Ronald,

R.N. Beachy, C.M. Fauquet, Transgenic elite indica rice varieties,

resistant to Xanthomonas oryzae pv. oryzae, Mol. Breeding 4 (1998)

551–558.

[33] K. Broglie, I. Chet, M. Holliday, R. Cressman, P. Biddle, S. Knowlton,

C.J. Mauvais, R. Broglie, Transgenic plants with enhanced resistance

to the fungal pathogen Rhizoctonia solani, Science 254 (1991) 1194–

1197.

[34] W.P. Chen, P.D. Chen, D.J. Liu, R. Kynast, B. Friebe, R. Velazhahan,

S. Muthukrishnan, B.S. Gill, Development of wheat scab symptom is

delayed in transgenic wheat plants that constitutively express a rice

thaumatin-like protein gene, Theor. Appl. Genet. 99 (1999) 755–760.

[35] S.L. Dellaporta, J. Wood, J.B. Hicks, Isolation of DNA from higher

plants, Plant Mol. Biol. Rep. 4 (1983) 19–21.

[36] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning. A Labora-

tory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY, 1989.

[37] S. Gallagher, S.E. Winston, S.H. Fuller, J.G.R. Hurre, Immunoblotting

and immunodetection, in: F.M. Ausubel, R. Bernt, R.E. Kingston,

D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Short

Protocols in Molecular Biology, John Wiley and Sons, New York,

1995, pp. 1040–1048.

[38] U.K. Laemmli, Cleavage of structural proteins during the assembly of

the head of bacteriophage T4, Nature 227 (1970) 680–683.

[39] S. Sriram, T. Raguchader, P. Vidhyasekaran, S. Muthukrishnan, R.

Samiyappan, Genetic relatedness with special reference to virulence

among the isolates of Rhizoctonia solani causing sheath blight in rice,

J. Plant Dis. Prot. 104 (1997) 260–271.

[40] IRRI, Standard Evaluation System for Rice, INGER Genetic

Resources Centre, International Rice Research Institute, Manila,

1996 , p. 52.

[41] P. Narayanasamy, R. Viswanathan, A new scoring system for rice

sheath rot disease, Madras Agric. J. 77 (1990) 256.

[42] B. Zhu, T.H. Chen, P.H. Li, Analysis of late-blight disease resistance

and freezing tolerance in transgenic potato plants expressing sense and

antisense genes for an osmotin-like protein, Planta 198 (1996) 70–77.

[43] C. Pritsch, G.J. Muehlbauer, W.R. Bushnell, D.A. Somers, C.P. Vance,

Fungal development and induction of defense response genes during

early infection of wheat spikes by Fusarium graminearum, Mol. Plant

Microbe Interact. 13 (2000) 159–169.

[44] R. Vergauwen, F.V. Leuven, A.V. Leare, Purification and character-

ization of strongly chitin-binding chitinases from salicylic acid-treated

leek (Allium porrum), Physiol. Plant. 104 (1998) 175–182.

[45] J. Peng, H. Kononowicz, T.K. Hodges, Transgenic indica rice plants,

Theor. Appl. Genet. 83 (1992) 855–863.

[46] E. Sivamani, P. Shen, N. Opalka, R.N. Beachy, C.M. Fauquet, Selec-

tion of large quantities of embryogenic calli from indica rice seeds for

production of fertile transgenic plants using the biolistic method, Plant

Cell Rep. 15 (1996) 322–327.

[47] P. Christou, T.L. Ford,M. Kofron, Production of transgenic rice (Oryza

sativa L.) plants from agronomically important indica and japonica

varieties via electric discharge particle acceleration of an exogeneous

DNA into immature embryos, BioTechnology 9 (1991) 957–962.

[48] D. Sudhakar, X. Fu, E. Stoger, S. Williams, J. Spence, D.P. Brown, M.

Bharathi, P. Christou, Expression of immunolocalization of the snow-

drop lectin insecticidal protein GNA, in transgenic rice plants, Trans-

genic Res. 7 (1998) 371–378.

[49] C. Fagoaga, I. Rodrigo, V. Conejero, C. Hinarejos, J.J. Tuset, J. Arnau,

J.A. Pina, L. Navarro, L. Pena, Increased tolerance to Phytophthora

citrophthora in transgenic orange plants constitutively expressing a

tomato pathogenesis related protein PR-5, Mol. Breeding 7 (2001)

175–185.

[50] L.C. Van Loon, Induced resistance in plants and role of pathogenesis

related proteins, Eur. J. Plant Pathol. 103 (1997) 735–765.

[51] N. Sakthivel, R. Amudha, S. Muthukrishnan, Production of phytotoxic

metabolites by Sarocladium oryzae, Mycol. Res. 106 (2002) 609–614.