(E)-β-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines

Abstract

Aphids are the major concern that significantly reduces the yield of crops. (E)-β-farnesene (Eβf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-β-farnesene (Eβf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eβf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-β-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using Eβf, a non-toxic mode of action.

Abbreviations

Eβf

(E)-β-farnesene

PCR

polymerase chain reaction

RT-PCR

real time polymerase chain reaction

CTAB

cetyltrimethyl ammonium bromide

WT

wild-type

Mustard aphids Lipaphis erysimi (L) Kaltenbach (Homoptera: Aphididae), which belongs to subfamily Aphidinae reduces crop production up to 70%,¹ with annual estimated loss of 100 millions of dollars worldwide.² The alarm pheromone (E)-β-farnesene (Eβf) is an important volatile sesquiterpene compound acting as the main component of aphid alarm pheromones for many pest aphids.^3,4 Several species of aphids such as Aphis fabae (black bean aphid), L. erysimi (Kaltenbach), Myzus persicae (green peach aphid), Brevicoryne brassicae (Cabbage aphid) released Eβf as ‘prime alarm pheromone compound.⁵ As a natural defense response against aphids plants, for instance, wild potato (Solanum berthaultii) repels green peach aphid (M. persicae) using Eβf.^6,7 Following the importance of Eβf as alarm pheromone, efforts had been made to apply this semiochemical compound to repel aphids either by breeding or by genetic engineering. In the present work, we have isolated Eβf from M. arvensis, and transformed into B. juncea plants. The direct defense response of Eβf on aphid colonization was studied; the resulting emission effects demonstrate the reduction of aphid colonization in the presence of Eβf in B. juncea compared to wild type.

The full length cDNA encoding Eβf from M. arvensis (accession number: ADC92564), cDNA library was constructed using a SMART cDNA library kit (BD Biosciences, USA). The Eβf cDNA was cloned into binary plant expression vector pBinAR,⁸ the cDNA encoding Eβf gene was driven by the CaMV-35S promoter, and an octopine synthase (ocs) terminator with the expression cassette of the nptII gene within the T-DNA (Fig. 1a), and was transformed into. Agrobacterium tumefaciens strain GV 3101 by freeze-thaw method.⁹ The Agrobacterium strain harboring the desired chimeric construct was used for transformation of B. juncea (cv. varuna) using hypocotyl as explant.¹⁰

Figure 1.

Development and analyses of Eβf transgenics of B. juncea T-DNA of the chimeric pBin-Eβf (a), PCR detection of transgenic plants using nptII gene (b), and RT-PCR of Eβf gene and its expression in independent transgenic lines, (c). Amplification of β-actin cDNA was used as internal control. NC-designated as non-template control and lane 1–9 are different transgenic lines of B.juncea.

For genomic PCR, DNA was isolated from leaf tissues employing the C-TAB. The putative transgenic plants were analyzed by using Eβf gene specific primers, and with kanamycin resistance marker (nptII gene). Aphids nymphs (without wings) were collected from infected wild type B. juncea plant grown in field condition were used in the experiment. Aphid movements were recorded on transgenic leaves emitting Eβf using detached leaf^11,12 for aphid bioassay. A circular disc from the leaves of WT plants (2 cm in diameter) was removed, and replaced by a similar size disc excised from the leaves of transgenic plants. A circular portion of the transgenic leaf tissue was surrounded by non Eβf leaf tissue of WT plants. Similarly, in control, a 2-cm leaf disc from the leaf of WT plant was replaced by a leaf disc from other WT plant. Ten aphid nymphs were released at the center of the leaf discs. Each leaf disc was pricked¹³ using toothpick for 10–12 times to enhance the emission of volatiles immediately after release of aphids. The behavior response was measured by recording the number of aphids, which moved away from disc at different time intervals of 15 and 30 min. Experiments were conducted in 3 replicates for each of the transgenic lines. Data were statistically analyzed as per completely randomized experimental design and critical difference (CD) value was calculated through the statistical software Sequential Optimum Selection (SOS).¹⁴ The percent repellency was calculated as: Percent repellency = [(number of aphids walk away in case of transgenic leaf disc-number of aphids walk away in case of untransformed leaf disc)/ total number of aphids released] × 100.

Aphid population data were recorded from 9 replicates of randomly selected plants of both wild type and transgenic plants. The data were collected after 60 days of plant transplantation in the field area assigned to grow transgenic plant. Top 5 cm of shoots of each of the transgenic lines was tapped gently with a wooden pencil to dislodge the aphids on a piece of white paper. Aphid from each plants falling on the paper were counted separately. Data were collected from early flowering stage till the maturity, at weekly intervals from the start of infestation. The extent of aphid colonization was compared with transgenic lines and WT plants. The percent (%) relative population of wild type and each transgenic line was calculated. The seasonal mean population (SMP) of each lines and wild types were calculated by dividing the total number of aphids counted during the whole season by the total number of sampling dates. The data were analyzed separately for each line and time point compare to control, analyses of variance were conducted using SAS/STAT. The means were compared using the Tukey's test at P ≤ 0.05. A least significant difference (LSD) was calculated using SAS at 0.05 level of probability for comparison of treatment means.

The suitability for heterologous expression for the synthesis of Eβf in transgenic Arabidopsis thaliana¹⁵ led us to select Eβf as a target gene for genetic engineering of crop genotypes to produce exogenous aphid alarm pheromone Eβf. A cDNA library of M. arvensis young leaves were constructed and used for PCR amplification of a full coding cDNA sequence of Eβf. We constructed plant transformation vector pBin-Eβf by cloning the Eβf gene in pBinAR using CaMV35S constitutive promoter. Several putative transgenic lines (FS1, FS2, FS3, FS4, FS5, FS7, FS11, and FS16) were generated through A. tumefaciens strain GV-3101 mediated transformation. The primary transformants were analyzed through genomic PCR using npt II gene (Fig. 1b), and RT-PCR with Eβf gene specific primers (Fig. 1c).

Behavior of L. erysimi was studied in transgenic and untransformed B. juncea lines. On transgenic leaf discs, the aphid nymphs started showing alarm response, and aphids walk away to a WT tissue area within 15–30 minutes (Fig. 2a). In case of WT leaf disc the released aphids either did not show such walking and migration or their movement was at a much reduced rate. Transgenic line FS3 and FS1 produced maximum alarm response followed by FS 16, FS11, and FS7 at 30 minutes time (Fig. 2b). The results revealed that no significant difference between 15 min and 30 min assays indicated the efficacy of EβF as aphid alarm pheromone to induce repellency through expressing in plants. The aphid walkaway response varies in the transgenic B. juncea expressing Eβf (Fig. 2b), and therefore the FS3 and FS1 lines were found to confer 80% repellence followed by FS16 (75%), FS11 (72.5%), FS7 (70%), FS5 (65%), FS2 (60%), and FS 4 (37.5%).

Figure 2.

Alarm response of L. erysimi on leaf disc of transgenic B. juncea and WT. Ten aphid nymphs were released at the center of the leaf disc and migration of the aphids from transgenic area to WT surrounding area were observed and recorded over a time span of 0–30 minutes, panel a (a), The number of aphids showing alarm response in terms of percentage repellency was showed in the graph after 15 and 30 min of insect release, panel b (b). The data recorded in 3 sets were statistically analyzed and presented as Mean±SD. LSD = 0.41 for 15 min and LSD = 0.40 for 30 min, P < 0.05. Abbreviations-WT, wild type (Untransformed plant), FS are individual transgenic plants of Brassica juncea expressing Eβf gene.

Quantitative estimation of maximum aphid population obtained during the growth period of crop demonstrated the reduced aphid colonization on the transgenic compared to the WT plants over the period of crop maturity (Table 1). During the period of observation, aphid population was significantly reduced in transgenic plants expressing Eβf as compared to control (Table 1).

Table 1.

Aphid population data. Mean number of aphid/ top 5 cm inflorescence of transgenic canola lines expressing (E)-β-farnesene (Eβf) compared to control

No. of days	N	WT	FS1	FS2	FS3	FS4	FS5	FS7	FS11	FS16
1	9	13.88a	1.66b	2.7b	2.4b	4.33b	2.8b	1.5b	2.22b	2.55b
8	9	25.66a	2.55b	1.9b	2.33b	2.22b	4.88b	2.55b	4.11b	3.66b
15	9	39.11a	3.55b	3.11b	3.33b	6.77bc	4.11b	3.44b	4.66b	4.33b
23	9	61.9a	3.88b	3.44b	3.5b	10.8bc	4.33b	455b	4.88b	3.55b
31	9	55.7a	3.11b	4.22b	3.6b	9.77bc	4.22b	4.11b	3.11b	3.66b
39	9	43.33a	3.22b	3.7b	3.1b	6.6b	3.9b	3.33b	3.9b	2.7b
47	9	25.55a	2.9b	3.7b	3.44b	4.22b	3.8b	3.9b	3.7b	2.9b
%SMP		100	7.8	8.5	8.1	16.6	10.5	9	10	8.7

Notes: N represents number of replicates; FS, different transgenic lines, Values followed by same letter do not differ significantly within each row Tukey's test Test (α ≤ 0.05).

The maximum aphid population of mean value (10.8) was observed on fourth week (23 days) in transgenic line FS4, While minimum aphid population of mean value (3.4) was observed in transgenic line FS2 (Table 1). Moreover, the percent seasonal mean population of each line varies from 7.8 % in FS1 to 16.6 % in FS5 (Table 1), compared to wild types. Such response was variable when independent lines were considered. The transgenic lines were found most resistant to aphid colonization, compared to the control (Fig. 3).

Figure 3.

Aphid colonization. Aphid colonization was observed in wild type plants (Right panel) and no aphid colonization was observed in transgenic plant of B. juncea expressing Eβf gene (Left panel)

The current study demonstrated that Eβf isolated from M. arvensis reduced the aphid colonization of L. erysimi and protect the plant B. juncea. Aphid colonization was minimized on transgenic Arabidopsis plants producing Eβf.^16,17 The results supported the applicability of genetic engineering of sesquiterpene like compounds as an alternative approach against aphid colonization in crops.¹⁸ We have tested the direct response of Eβf gene from M. arvensis deployed in B. juncea plant against aphid colonization. Observing the aphid behavior response in transgenic B. juncea within a time interval of 0–30 min, the aphid behavior response assay revealed the impact of Eβf gene as aphid repellent gene. Aphids walk away from the transgenic alarm zone, as compared to WT B. juncea, and the percentage rate of emission of Eβf as the volatile compound was found to vary among plants, even they all are of the same age. In particular, transgenic plants FS1 and FS3 showed maximum repellency (80%, Fig. 2b).

Previously, Jing-Gong¹⁹ demonstrated the aphid behavioral response in field, including Aphididae family and concluded that Eβf was major component that serves as alarm pheromones. Such response indicated that Eβf provides direct defense to the crop against aphid colonization. Relatively, the aphid dynamic data on B. juncea expressing Eβf gene showed reduced the aphid colonization as compared to the WT plant (untransformed) B. juncea (Table 1; Fig. 3). In particular, the SMP value of WT increased (61.9) constantly from first to fourth week of observation, and then gradually decreased (25.5) on seventh week. The same trend was also observed in all the transgenic lines, however the maximum aphid population was observed in FS4 transgenic line which followed the similar repellency pattern showed in aphid leaf disc data. The positive correlation between aphid population data and aphid repellency was observed. For instance, the SMP aphid population of FS1 (7.8) shows maximum aphid repellency (80%) Figure 2b. Transgenic plants grown to maturity, and no sign of deviation was shown for another plant, characteristics including morphology, flowering time, pollen fertility, and seed setting with reference to WT plants. Morphologically, all plants grew normally, and flowering appeared at the same time. Our finding here support that Eβf involved in direct defense against aphids.

Funding

Funding from the ICAR-NATP project, Govt. of India is gratefully acknowledged.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Dr. Vaibhav Singh, for assistance in statistical analysis (EMS, The Netherlands).