Mol Biotechnol DOI 10.1007/s12033-013-9727-8
RESEARCH
Transgenic Brassica juncea Plants Expressing MsrA1, a Synthetic Cationic Antimicrobial Peptide, Exhibit Resistance to Fungal Phytopathogens Anjana Rustagi • Deepak Kumar • Shashi Shekhar Mohd Aslam Yusuf • Santosh Misra • Neera Bhalla Sarin
•
Ó 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 antimicrobial 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 Sclerotinia sclerotiorum, two of the most devastating pathogens 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
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:
[email protected] 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
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 contribute *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 diseaseresistant crop varieties, though significant, are time consuming and have fallen short to tackle the vast array of diseases. 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
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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) constitute the largest group, and the term is often interchangeably used for the AMPs in the scientific literature. The CAPs usually acquire an a-helical or a b-sheet structure and have been found to be active against Gram-positive 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 melittin (the major lytic component in the venom of honeybee) are well-known CAPs. A chimeric peptide, CEMA, combining 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 broadspectrum 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, Alternaria 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
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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 % ethanol 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 semisolid 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 Agrobacterium-mediated transformation [27]. In brief, the hypocotyl 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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c
b
d e
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
were grown to get the T1 plants for performing the fungal infection experiments.
using the neomycin phosphotranferase (npt II) and msrA1 gene-specific primers mentioned below:
DNA Extraction and PCR Analysis of the Transgenic Plants Genomic DNA was isolated from the leaves of untransformed 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
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
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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 % agarose 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 prehybridization 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 hybridization 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. RTPCR amplification was carried out using a kit (AccuScript, Stratagene, USA) as per the manufacturer’s recommendations. 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 electrophoresis 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 Pathology, Indian Agricultural Research Institute, New Delhi was
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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 msrA1expressing 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, untransformed 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 percentage inhibition of hyphal growth was calculated using the method of Mondal et al. [23]. Inhibition of hyphal growth ð%Þ ¼ðDiameter of the fungal colony around the well treated with the transgenic leaf extract/diameter of the fungal colony around the well treated with the control leaf extractÞ100 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 synthesized 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 resuspended 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% Þ ¼½ðNo: of lesions per leaf in untransformed control plant 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 protection was calculated as Disease protection ð%Þ ¼½ðAverage size of lesions in untransformed control plant Average size of lesions in transgenicÞ= Average size of lesions in untransformed control plantÞ 100
RNA4 translation-enhancing element and NOS termination sequence (Fig. 1a). The neomycin phosphotransferase II (npt II) gene present in the plasmid was used for kanamycin 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 genespecific 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 densitometrically 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 untransformed control plants were used for protection assays against the fungal pathogens. Resistance of Transgenic B. juncea Plants Expressing msrA1 Gene to A. brassicae
Statistical Analysis In Vitro Antifungal Assay The infection experiments were carried out three independent 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 transformation. The plasmid, pSAI4, contains msrA1 gene cloned between an enhanced CaMV 35S promoter with an AMV
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 untransformed control leaves or either of the extracts after proteinase 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 percentage 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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a
b
I
II
IV
III
c 0
5 20
10
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 transgenic 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
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 inhibition 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 significantly inhibit the fungal growth.
Table 1 Evaluation of Alternaria brassicae infection on B. juncea after 15 days of inoculation
In Vivo Plant Bioassay
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
The leaves of untransformed control and plants from the different transgenic lines were inoculated with A. brasicae
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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
Mol Biotechnol 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
a
b
c
d
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 untransformed control plants, the lesions characteristically manifested 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 compared 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
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a
UT
b
M6
c
UT
d
M6
e
UT
f
M6
g
UT
h
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
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 different 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).
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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 disease 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
Mol Biotechnol 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 materials and methods. The data presented are the average ± SD values of three replicate experiments
factor contributing to yield loss in some of the economically important crops [32]. B. juncea is an important oilseed 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 strategies 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 generation of fungus-resistant plants is increasingly being explored as a viable alternative. The strategies for conferring 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 nonplant 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 antimicrobial 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 curtailing 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 transformants could be due to several factors including site of integration on the chromosome, promoter methylation, posttranscriptional gene silencing mediated by the production 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 ‘‘’lesionmimick’’ 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 promoter 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 caseto-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
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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 potentially 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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