RESEARCH

Transgenic Brassica juncea Plants Expressing MsrA1, a SyntheticCationic Antimicrobial Peptide, Exhibit Resistance to FungalPhytopathogens

Anjana Rustagi • Deepak Kumar • Shashi Shekhar •

Mohd Aslam Yusuf • Santosh Misra •

Neera Bhalla Sarin

� Springer Science+Business Media New York 2014

Abstract Cationic antimicrobial peptides (CAPs) have

shown potential against broad spectrum of phytopathogens.

Synthetic versions with desirable properties have been

modeled on these natural peptides. MsrA1 is a synthetic

chimera of cecropin A and melittin CAPs with antimicro-

bial properties. We generated transgenic Brassica juncea

plants expressing the msrA1 gene aimed at conferring

fungal resistance. Five independent transgenic lines were

evaluated for resistance to Alternaria brassicae and Scle-

rotinia sclerotiorum, two of the most devastating patho-

gens of B. juncea crops. In vitro assays showed inhibition

by MsrA1 of Alternaria hyphae growth by 44–62 %. As

assessed by the number and size of lesions and time taken

for complete leaf necrosis, the Alternaria infection was

delayed and restricted in the transgenic plants with the

protection varying from 69 to 85 % in different transgenic

lines. In case of S. sclerotiorum infection, the lesions were

more severe and spread profusely in untransformed control

compared with transgenic plants. The sclerotia formed in

the stem of untransformed control plants were significantly

more in number and larger in size than those present in the

transgenic plants where disease protection of 56–71.5 %

was obtained. We discuss the potential of engineering

broad spectrum biotic stress tolerance by transgenic

expression of CAPs in crop plants.

Keywords Biotic stress � Brassica juncea � Cationic

antimicrobial peptides � MsrA1 � Transgenic plants

Introduction

Despite significant advances in agricultural practices, an

estimated 13.6 % of the world population (*925 million

people) remains hungry. The Asia and Pacific regions con-

tribute *63 % to these hunger statistics [1]. Besides the loss

of arable lands to urban developments, stresses imposed by

various abiotic and biotic factors on crops remain the major

impediment for the agricultural productivity to catch up with

the global demand. Bacteria, fungi, and viruses are the main

phytopathogens affecting crops leading to significant losses

and have been responsible for scripting some of the most

devastating famines in the human history [2, 3]. Plant disease

control by chemical pesticides, apart from being costly, is

increasingly being scrutinized for health and environmental

concerns [2]. Conventional approaches to breed disease-

resistant crop varieties, though significant, are time con-

suming and have fallen short to tackle the vast array of dis-

eases. Thus, the focus has shifted to developing resistance in

plants against a broad spectrum of pathogens by engineering

plants expressing transgenes that can combat these

microorganisms.

Antimicrobial peptides (AMPs) have increasingly

gained attention as candidates for disease protection in

plants. AMPs are usually 12–50-amino acid-long peptides

Anjana Rustagi and Deepak Kumar contributed equally to this work.

A. Rustagi � D. Kumar � S. Shekhar � M. A. Yusuf �N. B. Sarin (&)

School of Life Sciences, Jawaharlal Nehru University,

New Delhi 110067, India

e-mail: neerasarin@rediffmail.com

Present Address:

A. Rustagi

Department of Botany, Ramjas College, University of Delhi,

Delhi 110007, India

S. Misra

Department of Biochemistry and Microbiology, University of

Victoria, Victoria, BC V8W3P6, Canada

123

Mol Biotechnol

DOI 10.1007/s12033-013-9727-8

which are components of innate defense mechanisms in

organisms ranging from microbes to plants and animals [4].

More than 900 AMPs have been reported, which have

either been identified from natural sources or have been

artificially synthesized. The cationic AMPs (CAPs) con-

stitute the largest group, and the term is often inter-

changeably used for the AMPs in the scientific literature.

The CAPs usually acquire an a-helical or a b-sheet struc-

ture and have been found to be active against Gram-posi-

tive and -negative bacteria, fungi, protozoa, and viruses [5].

Most of the AMPs have a common mechanism of action

that targets the differences between host and target cell

membranes and lead to lethal membrane dysfunction by

making pores through it [6]. Besides, they have also been

reported to interfere with cell division, macromolecular

synthesis, and cell wall formation [7]. The transgenic

expression of AMPs has delivered encouraging results in

conferring specific or broad spectrum disease resistance in

plants such as tobacco [8–11], rice [12], potato [13, 14],

banana [15], tomato [16], hybrid poplar [17], and grapevine

[18].

Cecropin A, exhibiting antibacterial and antifungal

activities (isolated from Hyalophora cecropia), and melit-

tin (the major lytic component in the venom of honeybee)

are well-known CAPs. A chimeric peptide, CEMA, com-

bining the bioactive portions of cecropin A and melittin

was engineered by Hancock et al. [19]. In a previous study

by Osusky et al. [13], an MsrA1 peptide was designed by

adding a hexapeptide at the N-terminus of CEMA. The

MsrA1 peptide was predicted, by molecular modeling, to

acquire an a-helical structure and maintain a positively

charged N-terminus. The msrA1 gene when expressed in

transgenic tobacco and potato plants conferred broad-

spectrum resistance to phytopathogens [8, 13].

Brassica juncea (Indian mustard) is an important oilseed

crop grown in many countries around the world and has

been suggested as an alternative crop to canola because of

its higher heat and water-stress tolerance [20, 21]. In India,

mustard contributes 28.6 % to the total oilseed production

and has been projected to provide for 41 % (14 million

tons) of the country’s demand by the year 2020 [22].

However, the crop productivity over the years has been

seriously affected by fungal pathogens, especially, Alter-

naria brassicae and Sclerotinia sclerotiorum [23, 24]. The

fungicide application to counter these pathogens has not

been a very effective and economical proposition, and

therefore, better combat strategies relying on generation of

transgenic plants armored with defense proteins are being

explored [23].

In view of the protective action of MsrA1 in tobacco and

potato, we assessed the efficacy of MsrA1 in conferring

disease-resistance traits to the economically important

oilseed crop of B. juncea. In the present work, we describe

the generation of transgenic B. juncea plants expressing the

msrA1 gene and evaluation of their resistance to A.

brassicae and S. sclerotiorum. The results suggest a high

degree of protection of B. juncea plants against these

fungal pathogens. We discuss our results in the light of

current efforts to generate biotic stress-tolerant transgenic

plants and the economic and environmental impacts that

these approaches could have.

Materials and Methods

Binary Vector for Expression of msrA1 Gene

The construction of pSAI4 plasmid used for the expression

of msrA1 gene has been described by Osusky et al. [13].

The T-DNA portion of this expression construct is shown

in Fig. 1a. The pSAI4 plasmid was transformed into the

Agrobacterium tumefacienes strain GV3101 following the

standard freeze–thaw procedure [25].

Plant Transformation and Regeneration

The B. juncea cv. Varuna seeds used for transformation

were procured from the Indian Agricultural Research

Institute, New Delhi. The seeds were surface sterilized by

treatment with a detergent (Extran) for 5 min, 70 % etha-

nol for 1 min, and HgCl2 (0.05 %) for 10 min followed by

three washes with autoclaved distilled water under sterile

conditions. The sterilized seeds were germinated on semi-

solid Murashige and Skoog’s (MS) basal medium [26].

One-cm-long hypocotyl explants were chopped from

5-day-old in vitro grown seedlings and used for Agrobac-

terium-mediated transformation [27]. In brief, the hypo-

cotyl explants were precultured in liquid MS B1N1 (MS

medium supplemented with 1 mg/l each of BAP and NAA)

at 25 �C for 18–24 h with gentle shaking followed by

coinfection with pSAI4 harboring Agrobacterium at a

bacterial density of 0.5 OD for 30 min. After cocultivation

for 18–24 h with gentle shaking (100 rpm), the explants

were washed twice with MS B1N1 containing augmentin

(200 mg/l) and subsequently plated on semi-solid MS

B1N1 containing AgNO3 (3.4 mg/l), augmentin (200 mg/l),

and kanamycin (50 mg/l). The plates were kept in culture

room maintained at 22 ± 2 �C and 14-h light/10-h dark

photoperiod. The shoots obtained (after 15–20 days) from

the explants were subcultured on the above medium and

finally rooted on MS I2 (MS medium supplemented with

2 mg/l IBA). The well-established control and putative

transgenic plantlets obtained in vitro were hardened and

transferred to the glass house to grow under 25 ± 2 �C

temperature, 16-h light/8-h dark photoperiod, and 70 %

relative humidity. The seeds obtained from the T0 plants

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123

were grown to get the T1 plants for performing the fungal

infection experiments.

DNA Extraction and PCR Analysis of the Transgenic

Plants

Genomic DNA was isolated from the leaves of untrans-

formed control and transgenic plants following the protocol

of Murray and Thompson [28]. The preliminary screening

of the transgenic plants for successful transformation was

performed by polymerase chain reaction (PCR) analysis

using the neomycin phosphotranferase (npt II) and msrA1

gene-specific primers mentioned below:

npt II

Forward primer: 50-GGAGCGGCGATACCGTAAAGC-30

Reverse primer: 50-GAGGCTATTCGGCTATGACTG-30

msrA1

Forward primer: 50-TTACTTAGTTAGCTTCAGCGC-30

Reverse primer: 50-ATGGCTCTAGAGCATATGAAA-30

The PCR conditions for both the set of primers were

identical and included an initial denaturation at 94 �C for

a

b

d

e

c

Fig. 1 Generation and molecular analysis of transgenic Brassica

juncea plants expressing msrA1 gene. (a) T-DNA portion of the

pSAI4 vector construct used for plant transformation. The position of

XbaI restriction endonuclease site used for Southern analysis of the

plants is shown. The amino acid and nucleotide sequence of MsrA1 is

also shown. PCR analysis to screen the transgenic plants for the

presence of transgene was done using msrA1 gene specific (b) and

nptII primers (c). The Southern blot analysis of the transgenic plants

was done after digestion of genomic DNA with XbaI (d) and the

expression of the transgene was confirmed by RT-PCR using the

msrA1 specific primers (e). M DNA molecular weight marker, UT

untransformed control plant, M1–M8 independent transgenic lines

expressing msrA1 gene

Mol Biotechnol

123

5 min, followed by 35 cycles of 94 �C for 30 s, 58 �C for

30 s, 72 �C for 30 s, and a final extension at 72 �C for

5 min. An aliquot of the PCR product was electrophoresed

on a 1.5 % agarose gel and visualized by ethidium bromide

staining.

Southern Blot Analysis

Ten micrograms of genomic DNA from untransformed

control and transgenic plants was digested with XbaI

restriction endonuclease, electrophoresed on a 0.8 % aga-

rose gel and transferred to a Hybond-N? membrane

(Amersham Biosciences, UK) using the capillary transfer

method [29]. The membrane was prehybridized for 2 h at

65 �C in a buffer containing 0.5 M sodium phosphate

buffer, pH 7.2, 1 mM EDTA, and 7 % SDS. Thereafter,

denatured radiolabeled probe (100 bp msrA1 amplicon

obtained by PCR amplification of pSAI4 plasmid and

labeled with [a-32P] dCTP using Random primer DNA

labeling kit (Amersham Biosciences, UK) as per the

manufacturer’s instructions) was added to the prehybrid-

ization buffer and incubated overnight at 65 �C. The

membrane was washed (10 min per wash) sequentially in

39 SSC, 0.1 % SDS; 0.59 SSC, 0.1 % SDS; 0.29 SSC;

0.1 % SDS with constant agitation at 65 �C. The hybrid-

ization signals were captured using phosphorimaging (FLA

5000 imaging system, Fujiflim).

RNA Extraction and Reverse Transcriptase-PCR

Analysis

Total RNA was isolated from the leaves using TRIzol

reagent (Invitrogen, CA, USA) and treated with RNase-free

DNase I to remove any contaminating genomic DNA. RT-

PCR amplification was carried out using a kit (AccuScript,

Stratagene, USA) as per the manufacturer’s recommenda-

tions. Total RNA (1 lg) was reverse transcribed using

MMLV reverse transcriptase. The primers for amplification

of the msrA1 gene were as described above. The primers

for actin (used as an internal control) were 50-AG

TAAGGTGACCTTGCAATTACTTTAGACTTCACCG-30

and 50-AAAGGCTAGCGTTGAAGATGCCTCTGCCGA

C-30. The RT-PCR products were visualized by electro-

phoresis on a 1.5 % agarose gel.

Evaluation of Resistance Against Alternaria brassicae

In Vitro Antifungal Assay

The antifungal activity of MsrA1 protein was assessed

using a plate assay. Alternaria brassicae (Accession No.

ITCC 5097) procured from the Division of Plant Pathol-

ogy, Indian Agricultural Research Institute, New Delhi was

cultured on potato dextrose agar (PDA) medium at

22 ± 1 �C, and the spore suspension was prepared as

described by Sharma et al. [30]. Total protein was extracted

from the leaves of the untransformed control and msrA1-

expressing transgenic plants using protein extraction buffer

(50 mM sodium phosphate buffer, pH 7.5, containing plant

protease inhibitor cocktail from G-Biosciences, USA).

Wells were punched out in agar plates and filled with 50 ll

of A. brassicae spore suspension (106 spores/ml) and

incubated overnight at 30 �C. After the incubation, the

wells were filled with 50 ll (200 lg protein) of one of the

following: untransformed control leaf extract, untrans-

formed control leaf extract treated with proteinase K

(Promega; 100 lg/ml for 2 h at 37 �C), transgenic leaf

extract, or transgenic leaf extract treated with proteinase K

(likewise as in the case of untransformed control). The

plates were further incubated, and the radial growth of A.

brassicae was recorded every 24 h for 5 days. The per-

centage inhibition of hyphal growth was calculated using

the method of Mondal et al. [23].

Inhibition of hyphal growth %ð Þ¼ Diameter of the fungal colony around the wellð

treated with the transgenic leaf extract/diameter

of the fungal colony around the well treated

with the control leaf extract�100

The antifungal activity against A. brassicae was also

checked using the synthetic MsrA1 peptide. The peptide

(amino acid sequence shown in Fig. 1a) was custom syn-

thesized by a commercial source (Link Biotech, India). A

small agar plug containing the fungus was placed in the

center of a petri dish filled with PDA. When the fungal

growth reached to about 45 mm in diameter, sterile filter

paper disks were placed along the circumference of the

growing mycelia. Specific amounts of the peptide were

pipetted onto the disks in 20 ll volumes (sterile water

served as the control). Further growth of the fungus was

monitored by incubating for another 24 h, and the plates

were photographed.

In Vivo Plant Bioassay

The A. brassicae spore suspension was filtered through

cheesecloth to remove the mycelium debris. The spores

were washed twice with sterile distilled water and resus-

pended to a count of 100 spores/ml. For in vivo plant

bioassay, the fifth leaf (from the top) of the 45-day-old

plants was painted with the spore suspension using a

painting brush. The inoculated plants were covered with

transparent polythene bag to maintain artificial epiphytotic

and humid conditions conducive to infection. The data on

lesions per leaf and individual lesion diameter were

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recorded up to 15 days of inoculation. Percentage disease

protection was calculated as described by Mondal et al.

[23].

Disease protection %ð Þ¼ No: of lesions per leaf in untransformed control plantð½�No: of lesions per leaf in transgenicÞ=No: of lesions per leaf in untransformed control plantÞ��100

Evaluation of Resistance Against Sclerotinia

sclerotiorum

Fungal culture of S. sclerotiorum (Accession No. ITCC

6583) obtained from the Division of Plant Pathology,

Indian Agricultural Research Institute, New Delhi was

maintained in the lab following the protocol described by

Jensen et al. [31]. The 45-day-old plants were inoculated

with the fungus by placing a 20-mm mycelial disk at the

third internode (axillary position) of the main stem. The

site of inoculation was covered with parafilm, and the

plants were enclosed under transparent polythene bags to

provide high humid conditions. Disease incidence was

assessed by recording the average size of the lesion on

stem, days to stem breaking, and the number of sclerotia

formed inside the main stem. The percentage disease pro-

tection was calculated as

Disease protection %ð Þ¼ Average size of lesions in untransformed control plantð½� Average size of lesions in transgenicÞ=Average size of lesions in untransformed control plantÞ��100

Statistical Analysis

The infection experiments were carried out three inde-

pendent times, in triplicates with three plants used for each

of the untransformed control and transgenic line. The data

are presented as average ± standard deviation. Results

were analyzed by the Student’s t test. Significance was

defined as P \ 0.05.

Results

Generation and Molecular Analysis of Transgenic

Brassica juncea Plants Expressing msrA1 Gene

Transgenic B. juncea plants expressing the msrA1 gene

were generated by Agrobacterium-mediated transforma-

tion. The plasmid, pSAI4, contains msrA1 gene cloned

between an enhanced CaMV 35S promoter with an AMV

RNA4 translation-enhancing element and NOS termination

sequence (Fig. 1a). The neomycin phosphotransferase II

(npt II) gene present in the plasmid was used for kana-

mycin screening of the transgenic plants. Preliminary

screening of the T0 transgenic B. juncea plants selected on

kanamycin was done by PCR amplification of the msrA1

and npt II genes. In eight independent T0 transgenic lines,

an amplicon of *100 bp was found with the msrA1 gene-

specific primers and of *700 bp with npt II-sspecific

primers (Fig. 1b, c). These fragments were not amplified in

the case of untransformed control plants. Out of these

seven surviving PCR positive lines, five lines (M1, M3,

M5, M6, and M7) were found to be Southern positive.

Figure 1d shows the signals obtained in Southern blot

analysis in the T1 generation of these lines. As the genomic

DNA was digested with XbaI endonuclease—which has a

single restriction site in the T-DNA of the construct—it is

evident from Fig. 1d that a single copy of msrA1 was

integrated in M6 and M7, while two copies of the transgene

were present in each of the lines M1, M3, and M5. The

expression level of the msrA1 gene in the transgenic lines

was assessed by RT-PCR analysis and deduced densito-

metrically after normalizing for the actin levels. Figure 1e

shows the presence of *100 bp band corresponding to the

amplified msrA1 cDNA in all the confirmed T1 transgenic

lines. All the transgenic plants were morphologically

similar to the untransformed control plants and showed no

visible signs of impaired growth or physiology. The plants

from these five T1 transgenic lines as well as the untrans-

formed control plants were used for protection assays

against the fungal pathogens.

Resistance of Transgenic B. juncea Plants Expressing

msrA1 Gene to A. brassicae

In Vitro Antifungal Assay

The efficacy of MsrA1 against A. brassicae was assayed

using an in vitro assay for inhibition of hyphal growth as

described in the materials and methods. The growth of

hyphae around the wells treated with the protein extract

from the transgenic leaves was greatly inhibited compared

to those treated with protein extract from the untrans-

formed control leaves or either of the extracts after pro-

teinase K treatment (Fig. 2a). The hyphal growth inhibition

observed with the extract from different transgenic lines

was calculated with reference to the growth around the

wells treated with untransformed control extract and is

represented as percentage inhibition in Fig. 2b. The per-

centage inhibition in the different transgenic lines varied

between 44 and 62 % over the untransformed control. To

further ascertain the antifungal activity of MsrA1 peptide

against A. brassicae, another in vitro assay was done using

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123

the synthetic peptide. In this assay, the minimum amount of

the synthetic peptide required to inhibit the growth of

mycelia was assessed (kindly refer to ‘‘Materials and

methods’’ for experimental details). The results obtained

24 h after the application of peptide showed marked inhi-

bition of mycelial growth toward filter disks applied with

10 and 20 lg of the peptide (Fig. 2c). This was in contrast

to the flourish of mycelia in the vicinity of disks soaked

with lower (5 lg) amount of the peptide or sterile water

(0 lg). Thus, 10 lg of the synthetic peptide could signifi-

cantly inhibit the fungal growth.

In Vivo Plant Bioassay

The leaves of untransformed control and plants from the

different transgenic lines were inoculated with A. brasicae

a b

II

IV III

c

0

5

10

20

I

Fig. 2 In vitro assay for assessment of antifungal activity of msrA1

expressing Brassica juncea extract and synthetic MsrA1 peptide

against Alternaria brassicae. a Protein extract from different trans-

genic and untransformed control plants was filled in the wells on the

agar plate after 24 h of inoculation with Alternaria spores and the

hyphal growth was monitored. Wells I and II were treated with extract

from M6 transgenic line and untransformed control, respectively. In

the wells III and IV the proteinase K treated extract from M6 and

untransformed control plant, respectively, was used. b Histogram

showing percentage inhibition of hyphal growth in the experiment

described in (a) using protein extract from the different transgenic

lines. c Different amounts (in microgram) of the synthetic MsrA1

peptide made in a total volume of 20 ll sterile water were applied on

sterile filter paper disks placed on the periphery of a growing

(*45 mm diameter) A. brassicae culture. The mycelia growth was

subsequently monitored for 24 h after which the plates were

photographed

Table 1 Evaluation of Alternaria brassicae infection on B. juncea

after 15 days of inoculation

Plants Number of

lesions

(Avg ± SD)

Diameter

range of

spots (cm)

Disease

protection

(% ± SD)

UT 16.0 ± 2.2 0.6–1.4 –

M1 5.0 ± 0.7 0.4–0.8 68.8 ± 3.6

M3 3.6 ± 0.9 0.3–0.7 77.6 ± 3.9

M5 4.4 ± 0.6 0.4–0.7 72.6 ± 3.5

M6 2.3 ± 1.1 0.2–0.6 85.4 ± 5.3

M7 2.8 ± 1.3 0.3–0.8 82.3 ± 6.4

Number of fungal lesions and the diameter range of lesions were

determined. The percentage disease protection in the transgenic plants

with respect to the untransformed control plants was determined as

described in materials and methods. The data presented are the

average ± SD values of three replicate experiments

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123

spores, and the initiation and progression of the disease

lesions were followed in the subsequent days. After

15 days, the number of characteristic lesions on the leaves

of transgenic plants was much less (\5 lesions/leaf) in

comparison to that on the leaves of the untransformed

control plants ([14 lesions/leaf) (Table 1). The size of the

lesions ranged between 0.6 and 1.4 cm in diameter in the

case of untransformed control plants as against 0.2–0.8 cm

in the different transgenic lines (Table 1). In the untrans-

formed control plants, the lesions characteristically mani-

fested as concentric rings that kept on increasing in size

until they merged with each other forming an enlarged

necrotic zone which eventually covered the entire leaf and

dried it up within 30 days of inoculation (Fig. 3a, c). This

process of the spread of infection took more than 50 days

in the case of transgenic plants (Fig. 3b, d). The percentage

disease protection in the different transgenic lines com-

pared with the untransformed control plants varied between

68.8 and 85 % (Table 1).

Resistance of Transgenic B. juncea Plants Expressing

msrA1 Gene to S. sclerotiorum

The untransformed control and transgenic B. juncea plants

were infected with S. sclerotiorum as described in the

materials and methods section. The relative severity of the

infection in the plants was compared by determining the

average size of lesion, days to stem breaking, and the

number of sclerotia formed per main stem at the end of

season. The stages in the progression of Sclerotinia

ba

dc

Fig. 3 Phenotype of Brassica

juncea leaves after infection

with Alternaria brassicae. Leaf

from untransformed control

(a) and representative M6

transgenic line (b) infected with

the fungus after 15 days of

inoculation. Leaf from

untransformed control (c) and

M6 transgenic line (d) after

30 days of fungal inoculation

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infection in the plants are shown in Fig. 4a–h. Whitish

mycelia were observed, which resulted in soft rot lesions

on the stem. The lesions were significantly larger (average

size 7.4 cm) on the stem of untransformed control plants in

comparison to those on the transgenic plants (average sizes

ranging between 2.1 and 3.3 cm for different lines) as seen

in Fig. 4c, d (Table 2). The mycelial expansion toward

noninfected portions of stem was slower in the transgenic

compared with the untransformed control plants (Fig. 4e,

f). The days taken to stem breaking were much less

(31.4 days) in the untransformed control plants, while the

breaking was delayed (until 41.7–65.3 days) in the differ-

ent transgenic lines (Table 2). The number of sclerotia

formed inside the main stem of the untransformed control

plants was also significantly higher (21.7) than in the

transgenic plants (3.1–8.4) signifying the resistance of the

transgenic plants to the fungus (Table 2; Fig. 4g, h). The

percentage disease protection in the transgenic plants with

respect to the untransformed control varied from 56 to

71.5 %. for the different lines (Table 2).

Discussion

Significant yield losses because of varied stress factors

dampen the productivity potential of agriculture, the world

over. Deteriorating environmental conditions, rendering of

large tracts of lands infertile over the years, losses caused

by pest infestation, and development of resistance in dis-

ease causing pathogens are the major challenges that need

to be surmounted by the agriculture community. Fungal

diseases are rated as the most or the second-most important

ba c d

e f g h

UT UT

UT UT

M6

M6

M6

M6

Fig. 4 Stages in the development and progression of Sclerotinia

sclerotiorum infection on Brassica juncea stem. a, c, e Different

stages in the infection of untransformed (UT) control plants. b, d, f

Different stages in the infection of plants of M6 transgenic line (M6)

expressing the msrA1 gene. g, h Sclerotia bodies formed inside the

main stem of UT and M6 plants

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123

factor contributing to yield loss in some of the economi-

cally important crops [32]. B. juncea is an important oil-

seed crop in many countries of the world. It ranks second,

next to ground nut, in contributing to the Indian oilseed

economy and is expected to supply for the increasing

edible oil demands in the decades to come [22]. Yield

losses due to fungal diseases alternaria leaf spot caused by

A. brassicae and soft stem rot caused by S. sclerotiorum are

a serious problem in the cultivation of this crop [23, 33].

These diseases are difficult to control once they set in and

have been reported to lead to complete crop failure in their

most devastating forms [34, 35]. The conventional strate-

gies of tackling the diseases with fungicides and selecting

for the disease-resistant genotypes through breeding are

fraught with concerns of development of resistant fungal

strains, cost, time, and uncertainty.

In view of the above, transgenic approaches for gener-

ation of fungus-resistant plants is increasingly being

explored as a viable alternative. The strategies for confer-

ring fungal resistance either involve the production of

transgenic plants with antifungal molecules like proteins

and toxins or generation of a hypersensitive response

through R (resistance) genes or by manipulating genes of

the systemic acquired resistance pathway. Some of the

proteins used have been pathogenesis-related proteins,

ribosome-inactivating proteins, small cystein-rich proteins,

lipid transfer proteins, storage albumins, polygalacturonase

inhibitor proteins, chitinases, antiviral proteins, and non-

plant antifungal proteins [32].

In the present article, we explored the prospects of

engineering fungal resistance in B. juncea plants by

transgenic expression of MsrA1, a synthetic cationic anti-

microbial peptide. CAPs from microbial sources often have

significant phytotoxicity that limits their direct use for plant

protection. MsrA1 was previously designed by modifying

CEMA which is a chimeric CAP with bioactive portions of

the much investigated cecropin A and melittin proteins.

The change introduced in the MsrA1 was aimed at cur-

tailing the high antimicrobial activity of CEMA to ward off

the unwarranted toxicity effects on the expressing host

plants [13]. We generated five independent transgenic lines

with 1–2 copies of the integrated transgenes. The integrated

gene was transcriptionally active in all these lines. The

expressions of the gene in the lines M1, M3, and M5 (with

two copies of the transgene) were lower than those in M6

and M7 lines which had single transgene copy. This could

be because copy number might have a negative effect on

the transgene expression due to silencing effects [36]. It is,

therefore, mostly desired to have single-copy transgenic

lines. The observation that despite having two copies of the

gene the M5 line showed high number of days to stem

breaking for S. sclerotiorum infection (in comparison with

the single copy carrying M7 line) which could be due to the

reasons that we cannot authoritatively comment upon

based on the present data. The variation in the expression

(and the consequent biologic effect) in independent trans-

formants could be due to several factors including site of

integration on the chromosome, promoter methylation,

posttranscriptional gene silencing mediated by the pro-

duction of aberrant transcripts, etc. [37]. No unintended

deleterious effects on morphology or physiology of the

transgenic plants were evident. In the previous evaluation

of this gene, Osusky et al. [13] found the disease ‘‘’lesion-

mimick’’ phenotype in a particular potato cultivar, while

this abnormality was not seen in another variety used in

their study as well as in tobacco plants expressing the gene

under the control of pathogen-responsive win3.12T pro-

moter in a subsequent study reported from the same group

[8]. This underlines the importance of assessing the effects

of transgenesis in different species and cultivars on a case-

to-case basis.

The transgenic B. juncea plants were found to be

resistant to both the fungal pathogens tested. In the in vitro

assay, protein extract from the transgenic plants inhibited

the hyphal growth of A. brassicae, while the extract from

the untransformed control or the proteinase K treated

transgenic extract could not do so, suggesting that the

MsrA1 protein present in the transgenic plants was active

against the pathogen. Although, the spread of this fungus in

the untransformed control plants was much faster (the

leaves necrosed in 30 days post infection) as seen in the

in vivo bioassay, it was restricted in the transgenic plants

and the infected leaves got necrosed not before 50 days

after infection. Similarly, protection was also observed

against S. sclerotiorum. The spread of fungus from the site

of infection toward uninfected parts was faster and prolific

in case of the untransformed control in comparison with the

transgenic plants. Also, the number of sclerotia formed

Table 2 Evaluation of Sclerotinia sclerotiorum infection on B.

juncea

Plants Size of

lesion (cm)

(Avg ± SD)

Days to stem

breaking

(Avg ± SD)

No. of Sclerotia

in main stem

(Avg ± SD)

Disease

protection

(% ± SD)

UT 7.4 ± 0.9 31.4 ± 3.6 21.7 ± 1.5 –

M1 3.3 ± 1.1 44.5 ± 1.7 7.6 ± 1.5 56.0 ± 3.5

M3 2.7 ± 1.1 41.7 ± 1.5 8.4 ± 2.5 63.0 ± 2.9

M5 2.4 ± 1.4 58.6 ± 2.9 6.7 ± 1.7 68.1 ± 3.2

M6 2.1 ± 1.4 65.3 ± 2.0 3.1 ± 1.5 71.5 ± 1.6

M7 2.8 ± 1.3 53.4 ± 2.0 5.9 ± 2.0 62.2 ± 2.3

The average size of lesion, days to stem breaking, and the number of

sclerotia formed in the main stem were determined. The percentage

disease protection in the transgenic plants with respect to the

untransformed control plants was determined as described in mate-

rials and methods. The data presented are the average ± SD values of

three replicate experiments

Mol Biotechnol

123

inside the main stem was much less in the transgenic

plants. The restricted spread of these two fungi is expected

to translate into higher yields in the transgenic B. juncea

harvest under field conditions.

In conclusion, our work demonstrates the generation of

an economically important oilseed crop having broad

spectrum resistance against fungal pathogens by transgenic

expression of an antimicrobial peptide. This could poten-

tially reduce the yield loss caused by these pathogens and

also decrease the dependence on fungicides that are

increasingly being wished-off for environmental, health,

and economic reasons.

Acknowledgments This work was funded by Grant (No. BT/

PR9616/AGR/02/458/2007) from the Department of Biotechnology,

India to N.B.S. A.R. and D.K. were recipients of Senior Research

Fellowship from the Council of Science and Industrial Research,

India. M.A.Y. is a U.G.C. Dr. D.S. Kothari Postdoctoral Fellow. The

authors thank Dr. Pratibha Sharma, Division of Plant Pathology,

Indian Agricultural Research Institute, New Delhi, for her suggestions

in fungal infection experiments.

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