RESEARCH PAPER |
Differential transcript accumulation in Cicer arietinum L. in response to a chewing insect Helicoverpa armigera and defence regulators correlate with reduced insect performance
1National Institute of Plant Genome Research, Aruna Asaf Ali Marg, JNU Campus, New Delhi 110 067, India
2Centre of Advanced Study in Zoology, Department of Zoology, University of Delhi, Delhi 110 007, India
To whom correspondence should be addressed. E-mail: praveenverma{at}india.com
Received 29 January 2008; Revised 12 March 2008 Accepted 17 March 2008
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Abstract |
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Monitoring transcriptional reorganization triggered in response to a particular stress is an essential first step for the functional analysis of genes involved in the process. To characterize Cicer arietinum L. defence responses against Helicoverpa armigera feeding, transcript patterns elicited by both herbivore and mechanical wounding were profiled and compared, and the application of defence regulators was assessed. A combination of approaches was employed to develop transcript profiles, including suppression subtractive hybridization (SSH), macroarray, northern blot, and cluster analysis. Of the 63 unique genes isolated, 29 genes expressed differentially when Helicoverpa feeding and wounding responses were compared. Comparative macroarray analyses revealed that most of the Helicoverpa-induced transcripts were methyl jasmonate (MeJA) and ethylene (ET) regulated. The effects of mild insect infestation and the exogenous application of signalling compounds on larval feeding behaviour were also monitored. Bioassays were performed to measure dispersal percentage and growth of larvae on elicited plants. Larvae released on elicited plants had decreased larval performance, demonstrating the central role of induced plant defence against herbivory. Similarly, wounding and exogenous application of MeJA and ET also affected larval growth and feeding behaviour. Our results demonstrated that Helicoverpa attack up-regulated large transcriptional changes and induced chickpea defence responses. Therefore, the results of this study advance the understanding of non-model plant–insect interactions on a broader scale.
Key words: Chickpea, ET, Helicoverpa, induced plant defence, MeJA, SA, SSH
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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Plants respond to both pathogen and herbivore attack by constitutive and induced defence mechanisms (Karban and Baldwin, 1997; Thomma et al., 1998; Kessler and Baldwin, 2002). The advantage of induced defence depends on the type of attacker and the subsequent cost of defence. Induced defences operate via both direct and indirect modes. Defence-related protein expression, reinforcement of the cell wall, biosynthesis of secondary compounds, and production of reactive oxygen species are examples of direct induced defences. Volatile organic compounds provide indirect defence by attracting enemies of the attacker (Paré and Tumlinson, 1997; Kessler and Baldwin, 2002). Complex cross-talk networks have been uncovered which serve to recruit various signal pathways in the regulation of defence induction (Walling, 2000; Rojo et al., 2003). While methyl jasmonate (MeJA) signalling plays a primary role in chewing insect defence (McConn et al., 1997; Reymond et al., 2004; De Vos et al., 2006), ethylene-mediated expression is also involved (Stotz et al., 2000; Kessler and Baldwin, 2002; von Dahl et al., 2007). In addition, salicylic acid (SA) is an important plant-produced signal. During biotrophic pathogen interactions, SA activates plant defence responses against pathogen attack (McDowell and Dangl, 2000; Glazebrook, 2005).
Plants, like animals, alter their induced defences in response to prior experiences (Baldwin and Schmelz, 1996). A mild insect infestation promotes an adaptive mechanism resulting in deterioration of plant quality as a food source; this reduces insect preference and performance on an induced plant compared with that on an uninduced plant (Agrawal, 1998; Voelckel and Baldwin, 2004). Induced resistance to subsequent attacks is due to plant changes in molecular and biochemical composition, which subsequently modify metabolic processes involved in the adaptive response. Limited reports describe the effect of induced defence on the growth and feeding behaviour of herbivores (Agrawal, 2000; Voelckel and Baldwin, 2004). Therefore, further exploration in other plant systems is warranted.
Plants distinguish between mechanical damage and herbivory. Insect attacks on plants results in wounding, but a plant's molecular response to mechanical damage differs (Korth and Dixon, 1997; Reymond et al., 2000). Several different types of elicitors, including fatty acid conjugates (volicitin; Alborn et al., 1997) and enzymes (glucose oxidase, Felton and Eichenseer; 1999; β-glucosidase; Mattiacci et al., 1995; and alkaline phosphatase; Funk, 2001), are present in the oral secretions and regurgitant of herbivores (Paré and Tumlinson, 1999), which may contribute to the differential response.
Large-scale transcriptional changes accompany insect-induced resistance, and herbivore-specific cues orchestrate the responses (Kessler and Baldwin, 2002). Transcript pattern changes in response to herbivory have been generated in many plant species including Arabidopsis thaliana (Reymond et al., 2000, 2004; Kempema et al., 2007), Nicotiana attenuata (Hermsmeier et al., 2001; Hui et al., 2003), Citrus sinensis (Mozuruk et al., 2006), Picea sitchensis (Ralph et al., 2006b), and poplar (Ralph et al., 2006a; Major and Constabel, 2006). These studies have provided insights into the molecular basis of insect–plant interactions, but little information regarding cultivated crops are available. Moreover, recent studies reveal that differential gene expression is dependent on the type of attacker and in some cases species specific (Zarate et al., 2007). For example, insect-inducible genes identified in N. attenuata had little sequence homology with up-regulated genes in Arabidopsis (Korth, 2003). Moreover, attack from the same lepidopteran herbivore resulted in species-specific transcriptional responses in two species of solanaceous host plants (Schmidt et al., 2005). Therefore, studying each insect–plant interaction is required.
Chickpea (Cicer arietinum L.) is an important legume crop due to its role in the human diet and use in animal feed. One of the major threats to its successful production is the generalist herbivore, Helicoverpa armigera, which damages the aerial parts of the plant, including leaves and pods. Since most studies examining Helicoverpa–chickpea interactions have focused on specific gene or protein dynamics (Johnston et al., 1991; Jongsma et al., 1995; Giri et al., 1998; Peng et al., 2005; Srinivasan et al., 2005), our aim was to identify target genes up-regulated during mild insect infestation which may contribute to the defence response. To isolate Helicoverpa-induced genes, a subtractive cDNA library was constructed from chickpea seedlings under Helicoverpa mild infestation using SSH. In addition to known defence genes, a number of genes and their presumed biochemical functions, that have not been previously associated with defence responses against insects, were identified. Using macroarray, transcript patterns elicited by both herbivore and mechanical wounding were profiled and compared. Comparative expression patterns on exogenous applications of various signalling compounds were obtained to evaluate the dynamics of regulatory pathways. In addition to investigating the effects of elicitation by mild insect infestation, induced plant defences in chickpea were evaluated by examining signal compound elicitation on larval feeding behaviour.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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Plant and insect growth conditions
Chickpea seeds (C. arietinum L.; Pusa-362) procured from the Indian Agricultural Research Institute, New Delhi, India, were sown in pots containing autoclaved potting soil mixture (peat compost and vermiculite; 1:1 v/v). Plants were grown for 4 weeks in a greenhouse with 16/8 h light/dark cycle at 22–25 °C, 50–60% relative humidity (RH), and watered regularly during cultivation.
Larvae of H. armigera were reared in the laboratory at 25 °C and 65–70% relative humidity (RH) on a 14/10 h light/dark cycle. The larvae were fed on an artificial diet as described by Armes et al. (1992). The freshly moulted fifth-instar larvae were starved overnight before releasing them on the plants.
Plant treatments
Insect infestation was achieved by the release of fifth-instar H. armigera larvae on 4-week-old chickpea plants (one larva per plant) and allowed to feed for 3–4 h at 25±2 °C until 
15–20% of the leaf area was consumed. Larvae were then removed, and the entire shoot was harvested and stored at –80 °C after quick freezing in liquid nitrogen. To mimic insect infestation, leaves were wounded with a punch machine (hole diameter=4.5 mm) until 15–20% of leaf area was removed, maintaining time span (3–4 h; continuous wounding with intervals of 1 h) and physical conditions (at 25±2 °C; 65–70% RH) similar to those of insect feeding. Plants were subsequently harvested. For treatments involving exogenous signalling molecules, equal volumes of aqueous solutions of MeJA (100 µM; Aldrich, St Louis, MO, USA), SA (5 mM; Sigma, St Louis, MO, USA), and ethephon (50 µM, 2-chloroethanephosphonic acid, Sigma, St Louis, MO, USA) were sprayed onto chickpea plants according to published procedures (Stotz et al., 2000). Each plant received not more than 500 µl of the aqueous solutions of the signalling compounds. The plants were then maintained in individual enclosures under the same conditions and harvested at different time points. In order to verify the effect of treatments, mRNA levels of marker genes namely PR-5 (Thomma et al., 1998), LOX (Stotz et al., 2000), and β 1,3-glucanase (Felix and Meins, 1987) for SA, MeJA, and ET treatments, respectively, were checked by northern hybridization.
Isolation of RNA and construction of subtracted cDNA library
Total RNA was prepared following treatment using 1 g of tissue (pooled from 20 plants grown at the same time under similar physical conditions) with TRIzol® Reagent (Invitrogen® Life Technologies, Rockville, MD, USA). Poly A+ RNA was purified using an mRNA isolation kit (Roche Applied Science, Manheim, Germany) according to the manufacturer's protocol. A forward suppression subtractive hybridization (SSH) was performed using PCR-SelectTM cDNA Subtraction Kit (BD Biosciences, Palo Alto, CA, USA) following the manufacturer's protocol. The enriched differentially expressed cDNAs were cloned into the pDrive Cloning Vector (Qiagen, Germany). In order to confirm differential expression of the individual clones during mild infestation by Helicoverpa, differential screening was performed with macroarray using subtracted cDNA probes, and unsubtracted probes respectively. The differentially expressed clones were then selected for sequencing. The recombinant plasmids were sequenced via Big Dye TerminatorTM kit version 3.0 (Applied Biosystems, Foster City, CA, USA) and examined with the 3700 ABI Prizm 96 capillary sequence analyser. All sequences were screened for homology in GenBank database using BLASTx (http,//www.ncbi.nlm.nih.gov/BLASTX). Sequences were submitted to GenBank and the assigned accession numbers are provided in Table 1.
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cDNA macroarray and data analysis
Individual clones of the subtracted cDNA library were amplified, purified, and denatured by adding an equal volume of 0.6 M sodium hydroxide. Equal volumes of each denatured PCR product (about 100 ng) were spotted on HybondTM N membranes (Amersham Pharmacia Biotech, NJ, USA) using a 96 well dot-blot apparatus (Bio-Rad Laboratories, CA, USA). In addition, PCR products of chickpea actin cDNA (Accession no. AJ012685 [GenBank] ) using primers (5'-GGTAACATTGTCTTGAGTGG-3' and 5'-CCAGATCCGTAACAATACAC-3') and neomycin phosphotransferase (NPTII) gene from the binary vector pBI121 (Accession no. AF485783.1) using primers (5'-TGCTCGACGTTGTCACTGAAG-3' and 5'-GTCAAGAAGGCGATAGAAGGC-3') were respectively spotted as an internal control and a negative control. The membranes were neutralized with neutralization buffer (0.5 M TRIS–HCl, pH 7.4, 1.5 M NaCl) for 3 min, washed with 2x SSC, and immobilized with UV cross-linker (Stratagene, La Jolla, CA, USA).
Probes were prepared for DNA array hybridization by first-strand reverse transcription (PowerscriptTM RT, BD Biosciences, CA, USA) with 1 µg mRNAs isolated from different samples and labelled with 
32P-dCTP (10 µCi µl–1; 3000 Ci mmol–1). Radiolabelled cDNAs were purified by Sephadex G-50 (Amersham Pharmacia Biotech, NJ, USA), suspended in prehybridization buffer (7% SDS, 0.3 M sodium phosphate pH 7.4, 1 mM EDTA) and hybridized at 60 °C overnight. The membranes were then washed three times with washing buffer (1x SSC, 1% SDS, 20 min each at 60 °C). Autoradiographs were scanned employing a FSMI (Fluor-S-Multiimager, CA, Bio-Rad, USA) to acquire images and signal intensities analysed by subtracting background noise. Actin cDNA was used as the internal control whose subtracted volume value was used for comparison with the control values. Differential screening and expression pattern data were generated as means (±SD) of the three independent experiments to ensure biological and technical replications. A paired Student's t test on log2-transformed data was applied to determine if statistical differences between expression ratios of each treatment and control pair were evident. Genes significantly different from controls in any of the treatments were selected and presented in Table 1. The following two criteria were chosen to demarcate differentially expressing genes based on a previous report (Major and Constabel, 2006): (i) a greater than 2-fold induction level; and (ii) a P <0.05 level of significance as determined by a t test for three independent experiments. Expression profiles of stress-inducible cDNAs were also analysed by clustering performed using SOTA (self organizing tree algorithm) by TIGR Multiple Experiment Viewer version 3.0 using complete linkage (available at http://www.tigr.org/software/tm4/menu/TM4).
Northern hybridization
Twelve micrograms of total RNA were fractionated in 1.2% agarose gel containing formaldehyde and transferred onto positively charged HybondTM N membrane (Amersham Biosciences, NJ, USA) according to Sambrook and Russell (2001). Equal loading and lane transfer was verified by membrane staining with methylene blue (0.02%). PCR-amplified individual cDNA fragments (with primers corresponding to adaptor 1 and 2R, provided in the SSH kit) were purified from agarose gel extraction. In addition, LOX2 (Accession no. AJ276265
[GenBank]
) PR-5 (Accession no. AJ010501
[GenBank]
), and PR-2 (Accession No. CV793598) were amplified (the primers used for amplification 5'-TGAAGCCAGTGGCCATCGAAT-3' and 5'-CGAAGGCCGTGTGGGAAGAT-3'; 5'-TGGTGGACTTCAATGCAC-3' and 5'-GGCATCTCTATATGAGGAGC-3'; and 5'-CGTCTCACGGATCTTTCCGTT-3' and 5'-GCTATTTGACATCTGCCGTG-3' primer sets, respectively,) and purified from agarose gel isolation. Probes were labelled with 32P-dCTP using NEBlot® kit (New England Biolabs, MA, USA) and purified. Northern hybridization was performed and band-intensity was evaluated as described above for cDNA macroarray.
Stay/dispersal experiment
Chickpea plants were subjected to MeJA, SA, and ET treatments and wounded mechanically as described previously. For elicitation by insects, plants were infested with newly moulted fifth-instar larvae for 3 h until 15–20% tissue was consumed. After treatment, plants were incubated for 3 h in individual enclosures. The first-instar larvae were removed from the stock culture on wet filter paper and placed at the bottom of round glass Petri dishes for 15 min. The treatment satiated the larvae with water and achieved identical physiological conditions. Twenty first-instar larvae (20 larvae=1 replicate) were separately released on each of the treated or control plants. In order to trap straying first-instar larvae, a white sheet coated with odourless glue was placed under treated and control plants in the centre of a circular arena (10 inches in diameter). Double-sided tape was fixed on the inner margin of the arena before larvae release. Six h after initial release, the number of trapped larvae was recorded. The experimental procedure included five replications. Water-treated plants served as the control for the above-mentioned experiments. Dispersal percentage was calculated based on the number of larvae dispersed from the plant surface and the total number of larvae released. Five independent experimental data sets were analysed statistically using analysis of variance (ANOVA; Tukey's Test; Sigma Stat 2.0; Jandel Scientific Software, 1995; Jandel corporation, San Rafael CA).
Feeding bioassays
Each freshly moulted fifth-instar larva was individually released on control/treated plants (50 plants for each control/treatment), and covered with wire mesh to restrict movement. The initial weight of larva (IWL) was recorded before release and the final weight of larva (FWL) noted after 24 h of feeding. The relative body weight gain of the larvae was calculated as the difference between IWL and FWL. For conducting bioassays with excised plant tissues, equal amounts of freshly excised control/treated plant tissues were weighed separately, which gave the initial weight of the tissues (IWT) and transferred into the numbered Petri dishes (9 cmx3 cm). The neonate fifth-instar larvae (50 larvae for each control/treatment) were weighed individually which gave the initial weight of the larvae (IWL). Larvae were released individually into the numbered Petri dishes containing the control/treated plant tissues (2000 mg). The same amount of plant tissue was kept in Petri dishes without larvae under the same conditions to estimate the loss of moisture for calculating the corrected final weight of consumed tissues. All the Petri dishes were kept inside the BOD incubator, maintaining the same temperature and humidity as mentioned earlier. Larvae were allowed to feed for 24 h after which larvae were taken out and weighed individually which gave their final weight (FWL). The relative body weight gain of the larvae was calculated as the difference between IWL and FWL. The unconsumed plant tissues were also weighed separately which gave their final weights (FWT). Amount of tissue consumed was calculated by subtracting the corrected FWT from IWT. The data obtained from five independent experiments conducted both on live and excised plants were analysed statistically using ANOVA (Tukey's test).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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Identification of differentially expressed genes
In order to decipher genes up-regulated during mild infestation by Helicoverpa which may lead to defence, a forward subtractive cDNA library was constructed using the suppression subtractive hybridization (SSH) strategy. As a result, 715 recombinant colonies were obtained which were subjected to differential screening and sequencing. After screening for induction during insect infestation and sequencing, 63 unique genes were identified by BLASTX analysis which included transcripts not previously reported to be induced during insect attack and some functionally unknown transcripts. In addition to this, some transcripts already known to be responsive to insect attack in other plants were also obtained which appears to support the validity of the subtracted cDNA library. The library served to elucidate transcriptional changes and subsequent differential responses in chickpea triggered by Helicoverpa mild infestation.
The potential role of elicited transcripts
To gain insights into the function of differentially expressed genes, they were categorized into eight classes based on their putative roles during Helicoverpa infestation (Table 1; Fig. 1). The major functional category corresponded to genes involved in defence, secondary compound synthesis, and cell wall fortification and was classified as defence-related (29%). In addition, another category comprised genes involved in signalling and gene regulation (10%) and a significant fraction of genes were involved in detoxification (8%). Genes were also found to play a role in protein synthesis (6%), abiotic stress (6%), photosynthesis or energy metabolism (6%), and a major fraction (13%) are listed as miscellaneous. Genes, whose function were not ascertained (22%) were categorized as ‘unknown functions’, and considered to be Helicoverpa-responsive. This is also to be mentioned that for some of the genes the functional categorization might be arbitrary and there may be some overlaps.
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Among the genes likely to be directly involved in defence, PR proteins (PR-10 and PR-5), hevein-like protein, and LTP/protease inhibitor were found in the subtractive library. Secondary metabolites such as phytoalexins, radical scavengers, and structural barriers serve a vital role in pathogen and insect defence. Several genes potentially involved in secondary metabolite synthesis were identified, including leucoanthocyanidin dioxygenase, dihydrofolate synthetase, homogentisate 1,2-dioxygenase, cytochrome P450, and hydroxymethyltransferase. Evidence suggested homogentisate 1,2-dioxygenase was involved in phenylpropanoid and lignin biosynthesis (Raes et al., 2003). Furthermore, hydroxymethyltransferase was shown to be up-regulated in response to elicitation of insect oral secretions (Giri et al., 2006). Endo-1, 4-β-D-glucanase, cellulose synthase, and pectinmethylesterase encoding proteins that function in cell wall fortification were also up-regulated. During the induced defence response, an increased accumulation of secondary metabolites, cell-wall reinforcing enzymes, and defensin proteins with toxic, antidigestive, and antinutritive activity has repeatedly been associated with diverse plant–insect interactions which reduce the palatability of the subsequent attackers and serve as a defensive tool for the plants (Kessler and Baldwin, 2002, 2004).
Genes potentially involved in protection of cells from oxidative stress were up-regulated on insect attack namely thioredoxin h, metallothionein-like protein, and RUB 1. Thioredoxins are a group of small proteins functioning in the regulation of redox status of the cell during oxidative stress (Gelhaye et al., 2004). The precise role of metallothionein is not clear, but a dual role has been assigned to this protein; the detoxification of metal ions released during protein breakdown and serving as a metal chelator and to function as metal binding proteins for storage or transport into developing organs (Giritch et al., 1998). The involvement of ubiquitin–proteasome-dependent proteolysis during insect feeding is reflected by the up-regulation of RUB1 and F Box proteins, which are associated with the ubiquitination cascade. The exact role of F Box protein has not been implicated in herbivory but a regulatory role for ubiquitin-dependent proteolysis during senescence has been assigned to this protein (Gepstein et al., 2003). An F box protein, SON1, has been implicated in the regulation of the induced defence response independent of SA (Kim and Delaney, 2002). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also shows 6-fold inductions during insect infestation. This gene has previously been reported to be up-regulated by herbivoral attack in native tobacco (Giri et al., 2006). GAPDH mainly play role in catalytic function of glycolysis, but it may be a part of reactive oxygen species signalling during herbivory. Two of the clones homologous to GTP-binding proteins and ATPase were also induced on Helicoverpa infestation. GTP-binding proteins are known to regulate many cellular responses including signal transduction, cytoskeletal organization, and vesicle trafficking (Haizel et al., 1995). Ran-A1, a GTP-binding protein has previously been reported to be induced on insect attack in Nicotiana attenuata (Hui et al., 2003). ATPases are reported to be up-regulated in poplar on insect attack and the function assigned to them may be actively transporting a range of ions like H+, Ca2+, Na+, etc. into or out of the vacuoles or cells to support many biological functions (Ralph et al., 2006b). Aphid feeding could induce the expression of H+ ATPase in a resistant plant indicating its role in defence (Thompson and Goggin, 2006).
Ethylene and MeJA were induced in response to insect herbivory and wounding in several plant species and therefore considered key regulators in plant defence mechanisms (Arimura et al., 2000; Winz and Baldwin, 2001; De Vos et al., 2005). In addition, ET and MeJA mediate up-regulation of defence-related genes such as protease inhibitors (O'Donnell et al., 1996), defensin (Penninckx et al., 1998) and PR proteins (Díaz et al., 2002). In the present study, Helicoverpa infestation induced a gene probably involved in ethylene biosynthesis (ACC oxidase), suggesting increased ethylene biosynthesis following insect attack. Furthermore, recent studies reported that ACC oxidase (Ralph et al., 2006a, b; von Dahl et al., 2007) were induced in plant–insect interactions. The induced expression of ACC oxidase indicates the pronounced accumulation of ET in the process which may contribute to induced plant defence by regulating expression of defence-related genes or proteins that may affect the infesting larvae. One of the genes regulated by auxin (GH1) were also induced by Helicoverpa infestation, suggesting involvement of this phytohormone during the response. Jang et al. (2003) reported the induction of auxin-induced protein and response factors during the Hessian fly larval attack on wheat–rye plants. Moreover, ethylene and auxin are determined regulators of the octadecanoid pathway (Walling, 2000), suggesting a defensive role during herbivory. A group of genes was identified in this study whose direct or indirect roles in insect defence were not previously known, including HMGB1 (High Mobility Group B1), Pi starvation-induced protein, GH1 protein (auxin-induced), cold-induced protein BnC24B, PPF-1, RAB11A, and among several others. Furthermore, some of these genes were involved in other types of stress, such as abiotic stress. Other genes identified in our study were up-regulated due to a stress response or the facilitation of transcriptional and translational changes during stress. In addition, it is proposed that the genes with unknown function are defence-related genes as most of them are induced on the application of defence regulators.
Cluster analysis revealed distinct responses to H. armigera infestation, mechanical damage, MeJA, ET, and SA
In order to achieve a comprehensive overview of expression profile of genes that were co-expressed during insect infestation, mechanical damage and treatments of signalling compounds, SOTA clustering was performed. The expression ratios obtained by macroarray were log2 transformed in order to reduce the noise level. The analysis yielded 11 clusters and the clusters with n >10 were selected to study the expression patterns for functionally similar genes (Fig. 2). The maximum number of genes were grouped into cluster 11 which comprised genes which showed a very high expression level during Helicoverpa infestation, MeJA and ET treatments (Fig. 2C). In contrast to this, expression of the genes in this cluster was less during mechanical damage and SA treatment. This group was found to be enriched in genes involved in defence, abiotic stress, protein synthesis and destination, and genes of unknown functions. Another major group, cluster 4, consisted of defence-related genes and genes playing a role in signalling and gene regulation and detoxification as well. The genes in this cluster showed similar expression patterns during Helicoverpa infestation, MeJA and ET treatments but their expression was almost nil during mechanical damage. In cluster 1, genes showing higher expression during Helicoverpa infestation and no induction by SA were placed. Almost all functional categories are represented in this cluster. Detailed information on genes within each cluster can be found in Supplementary Fig. S1 at JXB online. The miscellaneous class and genes with unknown functions showed no clear clustering and were present in almost all the clusters, which may be due to the heterogeneous composition of these categories. Characterization of these genes can provide a valuable insight into understanding the chickpea–Helicoverpa interaction better.
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Different transcript signatures for Helicoverpa feeding and mechanical wounding
Of 63 unique genes selected for further analysis, the transcripts of 46 genes were up-regulated upon Helicoverpa infestation, but wounding altered transcript levels of only eight genes above the cut-off value (as described in the Materials and methods) compared with the control (Fig. 3C). For the genes whose mRNA levels were co-induced during both types of stress, the transcript levels were higher on Helicoverpa infestation (Table 1). Helicoverpa infestation and wounding pair expression ratios were compared, and revealed 29 gene ratios were significantly different and are presented as ‘volcano plots’ (Jin et al., 2001) in Fig. 3B. The genes differentially induced during Helicoverpa infestation were probably related to insect-specific elicitors present during infestation but absent during wounding (McCloud and Baldwin, 1997; Korth and Dixon, 1997). Previous reports have demonstrated similar differential gene responses during mechanical damage and insect infestation (Reymond et al., 2000; Schittko et al., 2001; Reymond et al., 2004). Many defence-related genes were placed in this category, including pre-hevein-like protein, LTP/protease inhibitor, PR-10, cysteine protease, and hydrolase, among others. Pre-hevein-like protein is reported to be up-regulated by insect infestation but not by mere mechanical damage (Reymond et al., 2000). A subset of five genes was analysed by northern blot to validate the macroarray dataset (Fig. 2A). In general, the results of RNA gel-blot were consistent with the macroarray expression data analysis, with few differences between the two methods. These results further strengthen the fact that plants distinguish between mechanical damage and insect infestation and insect-elicited transcriptional changes differed from mechanical damage (Reymond and Farmer, 1998; Zhu-Salzman et al., 2005). LOX gene served as a marker for wounding and insect infestation (Hui et al., 2003; Reymond et al., 2004).
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H. armigera-responsive genes are differentially regulated by MeJA, ET, and SA
Among the three defence regulators, MeJA altered more transcripts than SA and ET (Table 1; Fig. 4D). Out of 63 genes, 47 were up-regulated by MeJA (74.6%), 39 by ET (61.9%), and 27 by SA (42.85%). Eighteen genes showed mRNA increases in all three treatments, including three well-known defence-related genes (cellulose synthase, hydroxyisobutryl-coenzyme A hydrolase, homogentisate 1, 2-dioxygenase) and two abiotic stress related genes (dehydrin 1, cold-induced protein). Since none of these genes showed exclusive up-regulation by SA (Fig. 4D), its association in this interaction was either less pronounced or it was involved in the signalling pathway cross-talks. Three plant defence genes (PDF1.2, PR1b, and Osmotin) were identified in Arabidopsis and induced synergistically by MeJA and ET (Xu et al., 1994; Penninkx et al., 1998, Kessler and Baldwin, 2002). In insect- and MeJA-induced responses, studies have shown that a large proportion of genes are commonly induced by both the responses (Reymond et al., 2004; Bodenhausen and Reymond, 2007). To confirm the expression data, the same subset of five selected genes was analysed by northern blots. The results demonstrated congruence between both methods, with the exception of a few minor differences (Fig. 4A, B, C).
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Elicited chickpea plants could defend effectively during subsequent infestation by H. armigera
To indicate induced plant defence in chickpea, stay/dispersal tests were performed which showed that the percentage dispersal from control plants was significantly lower than ET- and MeJA-treated and pre-infested plants. The mean proportion of dispersed larvae from control plants was 5±3.5 (mean ±SD), compared with 35±7.9 for ET-treated plants. The dispersal percentage for MeJA-treated and pre-infested plants was 30±7.9 and 19±4.18, respectively. No significant difference was found between the dispersal percentage of SA-treated, wounded and control plants (14±4.18 and 12±5.7) (Fig. 5A). The negative effect on plant acceptance on phytohormone-treated plants may be attributed to both the elicited defence response and the direct influence of the phytohormone on the insect's behaviour. Significantly less aphid infestation had been observed previously on MeJA-treated plants (Ellis et al., 2002; Zhu-Salzman et al., 2004), suggesting effective plant defence elicited by MeJA. Involvement of JA and ET increases due to chewing insects was shown by the induction of modest but significant increases in ET production and a clear increase in JA production (De Vos et al., 2005; Leitner et al., 2005). The differential behaviour of larvae on pre-infested plants may be attributed to the pronounced accumulation of signalling compounds (MeJA and ET) and allelochemicals, which detract the larvae.
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The effects of induced plant defence were tested by feeding larvae on elicited plants under no choice conditions. The results were consistent with the previous experiment. The lowest mean body mass change was observed for larvae feeding on ET-treated plants (41.73±1.97 mg), followed by pre-infested plants (50.73±1.31 mg). The body mass increment of larvae fed on MeJA-treated plants (59.65±2.01 mg) and wounded plants (53.14±2.77 mg) were significantly different from control plants. In contrast to other treatments, the average body mass change of larvae fed on SA-treated plants (72.10±1.89 mg) was not significantly different from control plants (Fig. 5B). Similarly, in the experiment where tissue consumed was calculated, which allowed us to correlate between the amount of tissue consumed and relative weight gain of the larvae, it was observed that the lowest tissue was consumed for ET-treated (292.6±6.6), followed by pre-infested tissues (349±12.6) The amounts of tissue consumed by the larvae feeding on MeJA-treated tissue (354±8.3) and wounded tissue (360±11.8) were significantly different from control plants. But the consumption was not significantly different when SA-treated and control tissues were compared (Fig. 6A). The results of relative weight gain of the larvae were similar to the previous experiment (Fig. 6B), suggesting that the reduced weight gain of the larvae feeding on ET- and MeJA-treated, mechanically wounded and pre-infested plant tissue are because of less consumption of the treated tissue as compared to control. The reduced growth of larvae fed on MeJA-elicited plants corresponded with previous reports. For example, JA elicitation of N. attenuata conferred induced resistance in both field (Baldwin, 1998) and laboratory (van Dam et al., 2000) trials. N. attenuata increased production of secondary metabolites following MeJA elicitation, which diminished the plant's palatability for Manduca sexta (Kessler and Baldwin, 2004). In both the experiments performed in this study, the ET-elicited induced response was also effective. MeJA-induced ethylene production is reported to be responsible for defence responses (Dicke and van Poecke, 2002; Hudgins et al., 2004). The results of Bi et al. (1997) suggested that exogenous application of SA on cotton plants did not affect the growth of Helicoverpa zea, congruent with our study.
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Higher defence status was also maintained by induced plants (with mild insect infestation) than by uninduced plants, which may be attributed either to the induction of defence-related anti-nutritive and anti-digestive proteins (Kessler and Baldwin, 2002, 2004) or to the much earlier events occurring before gene expression, such as detection of defence regulators. Gene activation and subsequent metabolic changes can be detected even after approximately 1 h of infestation (Maffei et al., 2007) although it might take few more hours to cause induced defence. Moreover, events occurring before gene expression (such as the pronounced accumulation of signalling compounds) can affect growth and feeding behaviour of the larvae. There is evidence to suggest that H. zea can intercept the plant defence signals elicited by its own feeding activity and can detect plant signal molecules and the allelochemical end-products (Li et al., 2002). Therefore, we can say that even if the toxic concentrations of anti-feed compounds may not be available in the induced plants, H. armigera could detect a higher defence status by tasting the signals.
In conclusion, this study shows that Helicoverpa attack triggers changes in transcript levels that are distinct from mechanical damage and are controlled mainly by MeJA and ET. Directly or indirectly, the majority of the genes identified as being Helicoverpa activated, may have a significant effect on insects performance, as it was depicted that elicitation with mild insect infestation, MeJA, and ET affected larval feeding behaviour. It is expected that further functional characterization of these novel Helicoverpa-responsive genes which are regulated by MeJA and ET will extend our understanding about defence responses against insects and in developing new strategies for crop protection.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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An online supplementary figure, S1 is available at JXB online. This figure provides detailed information on the genes within each cluster which are given in Fig. 2.
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Acknowledgements |
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This research was supported by the National Institute of Plant Genome Research, New Delhi. The authors acknowledge Professor Paula Levin Mitchell, Winthrop University, SC for critically editing the manuscript. We thank Mr Mohan Gidwani for photography. One of the authors, AS, acknowledges the Council of Scientific and Industrial Research, Government of India for providing a fellowship.
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* These authors contributed equally to the paper.
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