Plant Cell Reports

, Volume 22, Issue 2, pp 150–158

Expressed sequence tags from a wheat-rye translocation line (2BS/2RL) infested by larvae of Hessian fly [Mayetiola destructor (Say)]


  • C. S. Jang
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
  • J. Y. Kim
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
  • J. W. Haam
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
  • M. S. Lee
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
  • D. S. Kim
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
  • Y. W. Li
    • State Key Laboratory of Plant Cell Chromosome Engineering, Institute of Genetics and Developmental BiologyThe Chinese Academy of Sciences
    • Department of Crop Science, Division of Biotechnology and Genetic EngineeringKorea University
Genetics and Genomics

DOI: 10.1007/s00299-003-0641-6

Cite this article as:
Jang, C.S., Kim, J.Y., Haam, J.W. et al. Plant Cell Rep (2003) 22: 150. doi:10.1007/s00299-003-0641-6


Of the 16 known biotypes of the Hessian fly [Mayetiola destructor (Say)], biotype L is recognized as being the most virulent. We have previously reported the development of near-isogenic lines (NILs) (BC3F3:4) by backcross introgression (Coker797*4/Hamlet) that differed by the presence or absence of the H21 gene on 2RL chromatin. Florescence in situ hybridization analysis revealed introgressed 2RLs in NILs possessing the H21 gene, but no signal was detected in NILs lacking 2RL. As part of an approach to elucidate molecular interactions between plants and the Hessian fly, a cDNA library from NILs with H21 infested by larvae of biotype L of the Hessian fly was constructed for expressed sequence tag (EST) analysis. Of 1,056 sequenced reactions attempted, 919 ESTs produced some lengths of readable sequences. Based on their putative identification, 730 ESTs that showed significant similarity with amino acid sequences registered in the gene bank were divided into 13 functional categories. Defense- and stress-related genes represented about 16.1%, including protease inhibition, oxidative burst, lignin synthesis, and phenylpropanoid metabolism. EST clones obtained from the cDNA library may provide a clue to the molecular interactions between plant and larva of the Hessian fly larval infestation.


2RLH21Hessian flyESTWheat-rye translocation



Expressed sequence tags


Florescence in situ hybridization


Near-isogenic lines


Rye (Secale cereale L.) chromatins have been used to introduce agronomically useful traits into common wheats (Triticum aestivum L.). The germplasm line known as Hamlet (PI549276) carries the 2BS/2RL chromosome translocation in which Chaupon rye was the donor of the 2RL chromatin (Friebe et al. 1990). Hamlet carries the H21 gene, which confers resistance to biotype L of Hessian fly and is present on 2RL of Chaupon rye (Sear et al. 1992).

We previously reported the development of NILs (BC3F3:4) through backcross introgression (Coker797*4/Hamlet) that differed by the presence or absence of H21 on 2RL (Seo et al. 1997). Molecular markers associated with H21 and 2RL were constructed using these NILs (Seo et al., 2001). ESTs from leaves of young seedlings of NILs carrying 2RL were reported by Jang et al. (1999). Jang et al. (2002) subsequently observed that cDNAs encoding lipid transfer protein termed TaLTP1 and TaLTP2 were differentially expressed in several tissues between NILs but did not elucidate this with their presence on 2RL.

Biotype L of the Hessian fly is recognized as the most virulent form among the 16 biotypes known (Patterson et al. 1992). Friebe et al. (1990) suggested that the 2RL chromatin carried a gene or gene complex that conditioned antibiosis to larvae of the Hessian fly. However, at the time little was known about antibiotic mechanisms related to the interaction of resistance genes with the Hessian fly.

Plants undergo abiotic and/or biotic stresses, such as pathogens, pests, and adverse environments. External stresses cause the induction of numerous genes related to direct and indirect defense mechanisms in plants. Recently, many investigators have focused on interactions between plants and insect herbivores (for review, see Baldwin et al. 2001). Attacks by herbivores have been found to induce jasmonic acid, ethylene, volatiles, secondary metabolites, and defense-related genes in plants (for review, Baldwin and Preston 1999). Korth and Dixon (1997) found that transcripts of proteinase inhibitor II and 3-hydroxyl-3-methylglutaryl-coenzyme A reductase accumulated more rapidly in potato leaves chewed by caterpillars than in leaves damaged mechanically. Defense-related transcripts have been isolated and characterized following herbivore attack using cDNA microarrays (Reymond et al. 2000) and differential display (Hermsmeier et al. 2001). Other investigators have reported that two clones encoding M29b peptidase-like protein and β-glucosidase-like protein were temporally and spatially regulated when nymphal instars were feeding (Van der Ven et al. 2000). Attack by Manduca sexta larvae resulted in an endogenous jasmonic acid amount ninefold above control levels in Nicotiana attenuata (Ziegler et al. 2001).

EST analysis represents one of the most powerful methods used as a rapid complementary approach to biochemical and genetic analysis on a tissue of interest. Recently, many researchers have reported ESTs from diverse tissues in the plant kingdom, and dramatic improvements in the DNA database have assisted in the rapid categorization of EST clones (Covitz et al. 1998; Györgyey et al. 2000; Zhang et al. 2001; Channelière et al. 2002).

We report here the identification of 2RL chromatin introgression in a NIL possessing the H21 gene using FISH. We also performed EST analysis from the cDNA library of wheats infested by the Hessian fly. The EST clones may be expected to provide clues on molecular interaction between plants with the H21 gene and larvae of biotype L of the Hessian fly.

Materials and methods

Plant materials

A NIL line carrying the H21 gene was developed by backcross introgression (Coker797*4/Hamlet) and repeated selection by verification of the resistance to larvae of biotype L of Hessian fly (Seo et al. 1997).

Fluorescence in situ hybridization

Root tips of germinated seeds of NILs were collected in small vials containing distilled water and incubated in ice water for 24 h. The tissues were fixed in Carnoy fixing solution (3:1, EtOH:acetic acid) for 48 h. For chromosome preparation, root tips were squashed with a drop of 45% acetic acid after which the root tips were dyed with 1% acetic carmine for 10 min. Genomic DNA was extracted as described by Sahai-Maroof et al. (1984). Genomic DNA of rye accession Chaupon was labeled with biotin-14-dATP by nick translation. The labeled probe was mixed to a final concentration of 2 ng/μl in a hybridization solution containing 50% formamide, 2× SSC, 10% dextran sulfate, and 100 ng/μl of wheat cv. Chinese Spring genomic DNA as a blocker. The slides with tissues were incubated in 70% formamide in 2× SSC at 70°C for 2 min, subsequently transferred into 75%, 95%, and 100% ethanol at −20°C for 5 min each, and then air-dried. The tissues were hybridized with the labeled probe for 18 h at 37°C in a moist chamber containing 50% formamide in 2× SSC. After hybridization, the tissues were washed four times with 2× SSC and 0.2% Tween 20 at 40°C and once at room temperature and then incubated for 1 h at 37°C in a detection buffer containing 5 μg/ml Avidin-FITC-DN and 5% bovine serum albumin (BSA). After washing, the slides were incubated with 5 μg/ml biotinylated anti-Avidin-FITC in 5% BSA solution. The biotinylated probe was amplified with 5 µg/ml Avidin-FITC-DN and 5% BSA in 2× SSC. The tissues were washed three times with 2× SSC and 0.2% Tween 20 at 37°C for 5 min and then incubated in the detection buffer at 37°C for 1 h. The slides were immersed into distilled water, then air-dried at room temperature and mounted with a thin layer of anti-fade solution containing 1 μg/ml propidium iodide. Fluorescence signals were visualized under an influorescence microscope (Nikon eclipse E800) and the picture taken with Kodak 400 film.

Hessian fly infestation

An NIL possessing the H21 gene was grown for 3 weeks at 25/18°C (day/night) in a growth chamber. Biotype L of the Hessian fly flax seeds was kindly provided by Drs. R.H. Ratcliffel and S.E. Cambron (Purdue University). A bulk population of biotype L of Hessian fly flax seeds was removed from cold storage and held at 24°C for the emergence of adults. Periodic misting was applied to assure adequate moisture for a good emergence of flies. Seedlings with two to three leaves that had been grown apart from the emerging Hessian fly population were transferred to the Hessian fly growth chamber when the insect population reached a maximum. After inspection for eggs on the leaves, the plants were transferred to the Hessian fly-free growth chamber maintained at 24/18°C (day/night) with adequate moisture (over 60%). For construction of the cDNA library, the tissues beneath the leaf sheaths at the base of the plants were harvested at 3, 4, 5, and 6 days after the plants had been transferred to the Hessian fly-free growth chamber.

EST analysis

Total RNA of infested seedling was extracted from tissues beneath the leaf sheaths at the base of the plants using Trizol according to commercial protocols (Gibco/BRL, Gaithersburg, Md.). Poly(A)+ RNA was isolated with the PolyATRact mRNA isolation system (Promega, Madison, Wis.). A cDNA library was constructed using Uni-ZAP XR vector (Stratagene, La Jolla, Calif.). The phage library was excised to pBluescript plasmids as described by the manufacturer (Stratagene). The obtained phagemid of the library was transformed into Escherichia coli strain SOLR. Plasmid DNAs were isolated from the randomly selected clones, which were screened by color selection with IPTG and X-gal, using the Montage Plasmid Miniprep96 kit (Millipore). The inserted cDNAs were amplified with universal T3 sequencing primer using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Foster City, Calif.) according to the manufacturer's instructions. Electrophoresis was performed on an ABI PRISM 3700 Genetic Analyzer (Perkin Elmer). Sequences were edited to remove leading vector and ambiguous sequences at either end. Each edited sequence was translated in all six reading frames and compared with the non-redundant database at the NCBI using the blastx program. Homologies were considered to be significant when the E values were below 10-5 and the similarity scores were greater than 80 (Newman et al. 1994).


Confirmation of 2RL translocation by FISH

Introgression of 2RL chromatin into wheat to form the 2BS/2RL translocation through backcrossing (Coker797*4/Hamlet) was confirmed by FISH. FISH analysis on NILs was carried out with biotin-14-dATP-labeled probes prepared from Chaupon rye, with unlabeled genomic DNA from common wheat cultivar Chinese Spring as blocking. As shown in the FISH analysis, introgressed 2RLs were detected in a NIL possessing H21, but no signal was detected in a NIL lacking 2RL (Fig. 1).
Fig. 1A, B.

Fluorescence in situ hybridization in near-isogenic lines for the H21 gene. A NIL without H21, B NIL with H21 (2BS/2RL). The light chromatin regions in B indicate 2RL introduced from Chaupon rye

EST analysis from a NIL carrying 2RL infested by Hessian fly larva

The primary library contained about 7×106 recombinant phages. Of the 1,056 sequenced reactions attempted in the 5′ side of the inserted cDNA, 919 ESTs produced some lengths of readable sequences. After trimming vector and ambiguous sequences, we determined putative identification using the blastx program in comparison with the protein database.

Among 919 ESTs, 730 clones showed significant similarities with amino acid sequences registered in the database. However, 189 of the other clones did not show high similarities with any sequences of the protein database. A total of 615 clones out of 730 ESTs showed homology to known function genes. Based on their putative identification, 615 ESTS were divided into 13 functional categories, including cell-division cycle (0.8%), cell-wall structure or metabolism (2.9%), chromatin and DNA metabolism (3.4%), cytoskeleton (3.7%), defense- and stress-related (16.1%), gene expression and RNA metabolism (3.1%), membrane transport and intracellular trafficking (6.2%), primary metabolism (35.8%), protein synthesis and processing (16.3%), secondary and hormone metabolisms (3.1%), signal transduction (6.7%), transposon (0.3%), and others (1.6%) (Table 1). Cluster analysis identified 356 groups of sequences among 615 ESTs showing homology to known genes (Table 2). None of the ESTs that were incorporated in this study showed high homology with any of the insect genes registered.
Table 1.

Summary of EST categories


Number of clonesa

Homology to known transcripts


Cell-division cycle

5 (0.8%)

Cell-wall structure or metabolism

18 (2.9%)

Chromatin and DNA metabolism

21 (3.4%)

23 (3.7%)


Defense- and stress-related

99 (16.1%)

Gene expression and RNA metabolism

19 (3.1%)

Membrane transport, intracellular trafficking

38 (6.2%)

Primary metabolism

220 (35.8%)

Protein synthesis and processing

100 (16.3%)

Secondary metabolism and hormone metabolism

19 (3.1%)

Signal transduction

41 (6.7%)


2 (0.3%)


10 (1.6%)

Homology to uncharacterized transcripts


No database match




aValues in parenthesis indicate percentage of ESTs showing homology to known transcripts

Table 2.

ESTs putative identification

Cell-division cycle

Cell-division-cycle protein 48homolog

Cyclin-dependent protein kinase

Kinetochore protein

WD-repeat cell-cycle regulatory protein

Cell-wall structure or metabolism


Cell-wall invertase

Cell-wall invertase 2


Pectin methylesterase

Pectin methylesterase isoform alpha

Proline-rich protein

Xyloglucan endo-1,4-beta-d-glucanase

Chromatin and DNA metabolism

AT-Hook DNA-binding protein

DNA-binding protein


Histone H1

Histone H2A

Histone H2A.2

Histone H2B

Histone H2B.2

Histone H3

Histone H3.3

Histone H4

Histone-like DNA-binding protein

Histidine-containing phosphotransfer protein

Replication protein


Actin 1

Actin-depolymerizing factor 1

Ankyrin-like protein

Ankyrin repeat protein



Tubulin α-chain

Tubulin α-1 chain

Tubulin α-2 chain

Tubulin α-6 chain

Tubulin β-chain

Tubulin β-4 chain

Tubulin β-6 chain

Defense- and stress-related

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase

Anionic peroxidase

Ascorbate peroxidase


Bowman-birk-type protease inhibitor


Class-I chitinase

Chitinase-like protein 1

Cysteine proteinase inhibitor


Disease resistance protein

Disease resistance response protein

DNAJ-like heat shock protein

DNAK-type molecular chaperone

Epoxide hydrolase



Germin F

Germin-like protein 1

Glutathione transferase

Glyoxalase I

Glyoxalase II

Heat shock protein

Heat shock protein 70

Heat shock protein 80

Heat shock protein 82

Herbicide safener binding protein 1


Jasmonate-induced protein 1

Leucine-rich repeat protein


Lipid transfer protein

Oxygen-evolving enhancer protein 3-1

Pathogenesis-related protein

Pathogenisis-related protein 1.2

Patatin-like protein


Phenylalanine ammonia-lyase

Protease inhibitor

Polygalacturonase-inhibiting protein

Safener-induced In2.1-like protein

Superoxide dismutase

Senescence-associated putative protein

Thioredoxin peroxidase

Gene expression and RNA metabolism

Auxin response transcription factor 3

DNA-directed RNA polymerase II

Duplicated domain structure protein

Eukaryotic initiation factor 5A4

Homeodomain leucine zipper protein

Mei2-like protein

Polyadenylate-binding protein

RNA-binding protein

RNA-binding protein cp33

Small nuclear ribonucleoprotein F

Small nuclear ribonucleoprotein polypeptide G

Structure-specific recognition protein

Transcription factor-like protein

Membrane transport, intracellular trafficking

2-Oxoglutarate/malate translocator

ABC transporter protein 1-like

ADP, ATP carrier protein

Amino acid transport protein


Clathrin-associated protein

Coatomer complex subunit

Endomembrane protein

Delta-type tonoplast intrinsic protein

Gamma-type tonoplast intrinsic protein

Membrane protein

Na+/H+ antiporter

Na+ dependent ileal bile acid transporter

Phosphatidylinositol transfer-like protein

Plasma membrane intrinsic protein 1

Proton pump interactor

P-type transporting ATPase

Steroid membrane binding protein

Secretory carrier membrane protein

Sulfate transporter


Transitional endoplasmic reticulum atpase transport protein

Vacuolar ATP synthase catalytic subunit A

Vacuolar ATP synthase subunit B isoform 1

Vacuolar ATP synthase subunit C

Voltage-dependent anion channel protein

H+-transporting APTase

H+-transporting ATP synthase gamma chain

Primary metabolism

1-Deoxy-d-xylulose 5-phosphate reductoisomerase

2-Oxoglutarate dehydrogenase

3-Isopropylmalate dehydrogenase

Acetoacetyl coa thiolase

Aconitate hydratase

Acetyl-CoA carboxylase

AMP deaminase


Acyl-coa synthetase

Adenosine kinase


Aldehyde dehydrogenase

Acid phosphatase

Aldo/keto reductase

Anthranilate synthase alpha 1 subunit



Argininosuccinate synthase

ATP citrate lyase

Carbonyl reductase

Carboxyphosphonoenolpyruvate mutase

Chlorophyll a/b-binding protein

Chlorophyll a/b-binding protein II

Chlorophyll a/b-binding protein CP24

Chlorophyll a/b-binding protein CP29

Cysteine synthase

Cytochrome c1

Cytochrome c oxidase subunit

Cytochrome b5 reductase

Cytosolic 6-phosphogluconate dehydrogenase

Dihydroxyacetone kinase

Diphosphonucleotide phosphatase 1

d-TDP-glucose dehydratase

Dolichyl-di-phosphooligosaccharide-protein glycotransferase


Fructokinase I

Fructokinase II


Fructose-bisphosphate aldolase


Formaldehyde dehydrogenase

GDP-Fuc:Gal-beta-1,3glcnac-R alpha-1,4-fucosyltransferase

Glyceraldehyde 3-phosphate dehydrogenase

Glucose-6-phosphate isomerase


Glutamine synthetase

Glutamate/ornithine acetyltransferase

Glutamate-1-semialdehyde 2,1-aminomutase

Glutaredoxin I

Glutathione-dependent dehydroascorbate reductase

Guanylate kinase

Glycine decarboxylase subunit

Glycine dehydrogenase

Glutamate/aspartate-binding peptide

Glutamine-dependent asparagines synthetase

Inorganic pyrophosphatase

Ketol-acid reductoisomerase


NAD-dependent isocitrate dehydrogenase


Malate dehydrogenase

Methylmalonate-semialdehyde dehydrogenase

Mitochondrial ATP synthase 6 kDa subunit

Mitochondrial F0 ATP synthase d chain

Monodehydroascorbate reductase

NADH dehydrogenase

NADPH-dependent mannose 6-phosphate reductase

Omega-6 fatty acid desaturase

O-Deacetylbaccatin III-10-O-acetyl transferase



Peroxiredoxin Q


Phosphogluconate dehydrogenase

Photosystem I reaction centre subunit III



Protochlorophyllide reductase A

Porphobilinogen deaminase

Purple acid phosphatase

Pyruvate dehydrogenase E1 beta subunit

Pyruvate kinase


Reversibly glycosylated polypeptide

Rubisco subunit binding-protein alpha subunit

Rubisco subunit binding-protein beta subunit

Ribulose-bisphosphate carboxylase activase

Ribulose-bisphosphate carboxylase small chain

Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit

Sucrose:fructan 6-fructosyltransferase

S-Adenosylmethionine decarboxylase

S-Adenosylmethionine synthetase 1

Ubiquinol-cytochrome-c reductase

UDP-glucose:glycoprotein glucosyltransferase


UDP-glucose dehydrogenase

UTP-glucose-1-phosphate uridylyltransferase

Thiazole biosynthetic enzyme 1–2


Triosephosphate isomerase

Xylose isomerase

Protein synthesis and processing

9S ribosomal protein

26S proteasome regulatory subunit

26S proteasome regulatory particle triple-A ATPase subunit4

26S proteasome RPT6a subunit

40S ribosomal protein S1

40S ribosomal protein S3

40S ribosomal protein S4

40S ribosomal protein S5

40S ribosomal protein S9

40S ribosomal protein S10

40S ribosomal protein S11

40S ribosomal protein S18

40S ribosomal protein S25

50S ribosomal protein L4

50S ribosomal protein L31

60S acidic ribosomal protein P0

60S acidic ribosomal protein P3

60S ribosomal protein L3

60S ribosomal protein L6

60S ribosomal protein L7A

60S ribosomal protein L9

60S ribosomal protein L10A

60S ribosomal protein L11

60S ribosomal protein L13A

60S ribosomal protein L18

60S ribosomal protein L25

60S ribosomal protein L35A

60S ribosomal protein L38

Arginyl-tRNA synthetase

Acidic ribosomal protein P0

AAA-type ATPase-like protein

Aspartate aminotransferase

Carboxypeptidase D

Chaperonin 60 beta

Cathepsin B-like cysteine proteinase

Cyclophilin A-1

DNAj-like protein

Elongation factor eEF-1 alpha chain

Elongation factor EF-2

Glycyl-tRNA synthetase


Mitochondrial processing peptidase

Mitochondrial processing peptidase alpha subunit

Mitochondrial processing peptidase beta subunit

Oligopeptidase A

Pentameric polyubiquitin

Peptidylprolyl isomerase

Protein disulfide isomerase 2


Ribosome recycling factor protein

Serine carboxypeptidase

Serine protease


Thylakoid lumen rotamase


Translation elongation factor eEF-1 alpha

Translation elongation factor EF-G

Translation elongation factor Tu

Translation initiation factor

Translation initiation factor eIF-3

Translation initiation factor eIF-4A

Translation initiation factor eIF-5

Translin-like protein

Ubiquitin homolog

Ubiquitin activating enzyme

Ubiquitin-activating enzyme E1

Ubiquitin carboxyl-terminal hydrolase

Ubiquitin-conjugating enzyme

Ubiquitin-conjugating enzyme 9

Ubiquitin extension protein

Ubiquitin-specific protease

Secondary metabolism and hormone metabolism

4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase

β-Amyrin synthase

Auxin-induced protein

Auxin response factor

Auxin-independent growth promoter

Berberine bridge enzyme-like protein

Caffeic acid O-methyltransferase

Cinnamyl alcohol dehydrogenase

Cis-zeatin O-glucosyltransferase 2

Cytochrome P450

Cytochrome P450 monooxygenase

Dihydroflavonol 4-reductase

I ndole-3-glycerol phosphate synthase


Phytochelatin synthetase-like protein

Signal transduction

Argonaute protein

ADP-ribosylation factor

Calcium-dependent protein kinase


Calmodulin-binding protein

Calcineurin B-like protein

Casein kinase I

DR1-like protein

GDP dissociation inhibitor protein

Guanine nucleotide-binding protein beta subunit

Human tumor protein-like protein

Kinase-like protein

Ethylene-responsive small GTP-binding protein

Leucine-rich repeat containing protein kinase

Myb-like protein

OCL1 homeobox protein

Phosphoserine phosphatase


Protein kinase SPK-3

Protein phosphatase

Protein phosphatase 2C

Rac gtpase activating protein

Receptor-like protein kinase

Receptor kinase

Receptor protein kinase

Salicylic acid-activated MAP kinase

Scl1 protein

Serine/threonine protein phosphatase

Shaggy-like kinase

Signal recognition particle 54 K protein

Small Ras-related GTP-binding protein

Stress-induced protein kinase

Transcription factor myb4

Zinc-finger protein


Copia-like retroelement pol polyprotein


Adhesion of calyx edges protein

Glucose regulated repressor protein


Hydrophobic polypeptide

Isp4-like protein

Latex-abundant protein

Light-induced protein

PRLI-interacting factor N

Sphingosine-1-phosphate lyase

Tubby protein

While relatively abundant groups of the mRNA in the cDNA library showed several clusters, most of the clusters were small. Only four clusters contained more than ten EST sequences—the ribulose-1,5-bisphosphate carboxylase/oxygenease small subunit (22 clones), chlorophyll a/b-binding protein (17 clones), lipid transfer protein (LTP) (12 clones), and glyceraldehydes 3-phosphate dehydrogenase (10 clones). In the previous report we characterized two cDNAs encoding lipid transfer proteins termed TaLTP1 and TaLTP2 from 4-week-old leaves of a NIL carrying 2RL that was not infested with Hessian fly (Jang et al. 2002). However, cDNAs encoding lipid transfer protein found in infested plants were not identical with TaLTP1 or TaLTP2 and showed low sequence similarities (data not shown).

Putative pathogen-induced genes

Several investigators have recently reported on molecular and ecological approaches to elucidate interaction between plants and herbivorous insects (Baldwin and Preston 1999; Constabel 1999; Baldwin et al. 2001). In order to prevent and reduce further herbivore damage, plants are known to be active and able to induce defense- and stress-related proteins and phytochemicals. ESTs from Hessian fly-infested wheat also showed relatively high occurrences of genes expected to be related to diverse defense mechanisms (Table 3). Protease inhibitors (PIs) are one type of defensive proteins present in the plant kingdom and are rapidly induced in response to insect attack (For review, see Ryan 1990). In the present study, several types of PIs were represented, including Bowman-birk type PI, cysteine PI, and uncharacterized PI. When infested, the NIL with 2RL produced enzymes involved in the reactive oxygen species pathway and diverse oxidative enzymes, which were required for defense mechanisms, including anionic peroxidase, ascorbate peroxidase, catalase, dioxygenase, lipoxidase, oxygen-evolving enhancer protein, peroxidase, superoxide dismutase, and thioredoxin peroxidase. Cell walls of plants were fortified during pest and pathogen attack. Several clones among the ESTs were correlated to cell wall-fortification, including β-extensin, polygalacturonase-inhibiting proteins, and proline-rich protein. Products of wound-induced phenylprophenoid metabolism are also related to lignin or lignin-like polymers for cell-wall reinforcement. In the ESTs, several clones encoded enzymes involved in phenylprophenoid metabolism; for example, phenylalanine ammonia lyase, caffeic acid O-methyltransferase, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, and cinnamoyl alcohol dehydrogenase.
Table 3.

Genes putatively involved in plant response to infestation of larvae of Hessian fly

Class of target

Putative identification

Accession no.


Number of ESTs

Protease inhibitors

Bowman-brik type PI


Hordeum vulgare


Cysteine PI


Triticum aestivum


Uncharacterized PI


Hordeum vulgare


Reactive oxygen species pathway and oxidative enzyme

Anionic peroxidase


Zea mays


Ascorbate peroxidase


Hordeum vulgare




Hordeum vulgare




Oryza sativa




Oryza sativa


Oxygen-evolving enhancer protein


Zea mays




Zea mays


Superoxidase dismutase


Oryza sativa


Thioredoxin peroxidase


Secale cereale


Cell-wall fortification



Oryza sativa


Polygalacturonase-inhibitor protein


Citrus latipes


Proline-rich protein


Zea mays


Phenylprophenoid metabolism

Phenylalanine ammonia lyase


Oryza sativa


Caffeic acid O-methyltransferase


Lolium perenne



phosphate syntase


Solanum tuberosum


Cinnamoyl alcohol dehydrogenase


Festuca rundinacea



The larvae of the Hessian fly can infest various cereal crops, such as barley, rye, and triticale as well as wheat. A few days following oviposition, susceptible plants become dark-green and show a distinctive dwarfing of leaves caused by the feeding of the larvae. Twenty-eight genes for resistance to the Hessian fly have been reported and two of these are derived from rye. In particular, the H21 gene confers resistance to biotype L of the Hessian fly, which is known to be the most destructive race (Friebe et al. 1990). The development of resistant varieties is the most efficient way to reduce losses. In our previous report, we developed NILs for H21 by backcross introgression (BC3F3:4, Coker797*4/Hamlet) (Seo et al. 1997). Because the H21 gene resides on 2RL chromatin of Chaupon rye (Friebe et al. 1990), a NIL possessing the resistant gene should carry 2RL chromatin. FISH analysis showed that a NIL with the H21 gene possessed rye chromatins, but its NIL counterpart did not. As rye chromatins introduced to wheat are inherited as non-recombination blocks, this finding supports the premise that H21 is located on 2RL chromatin that was translocated from Hamlet (Koebner and Shepherd 1986). Molecular markers described by Seo et al. (1997, 2001) were also expected to tag 2RL translocated chromatin as co-tagging H21.

Mating, oviposition, and larval infestation take 3–5 days and the death of larvae occurred 2–3 days after their establishment in the resistant plants. Therefore, the cDNA library isolated from stems 3–6 days after Hessian fly infestation was expected to be enriched with genes related to interactions between plants and larvae infestation. Zhang et al. (2001) reported 1,016 ESTs from a NaCl-treated Suaeda salsa cDNA library, but defense-related genes showed only 15 clusters in spite of treatment of 400 mM NaCl for 48 h. Among 1,794 ESTs in rose petals, about 15.1% of the clones were associated with defense and stress mechanisms although one cluster of transcripts encoding metallothionein involved 192 clones (Channelière et al. 2002). In our study, defense- and stress-related genes occurred at a high frequency (about 16.1%) and were divided into 44 clusters. This supports the fact that the cDNA library prepared from infested wheat-rye translocations could contain numerous genes associated with interactions between NILs carrying H21 and the larvae of Hessian fly.

The four clusters of abundantly expressed genes in the ESTs were glyceraldehyde 3-phosphate dehydrogenase, rubisco small subunit, chlorophyll a/b-binding protein, and LTP. While three of these groups are involved in primary metabolism and photosynthesis, LTPs have been shown to enhance the in vitro transfer of phospholipids between membranes and to bind acyl chains (for review, see Kader 1996). Plant LTPs can be induced by abiotic and/or biotic stresses, including cold (Pearce et al. 1998), drought (Jang et al. 2002), or pathogen invasion (Molina et al. 2000). In our study, abundant expression of cDNA clones encoding LTP genes might indicate a key role in interactions between plants and larval infestation of Hessian fly.

Against the attack of insect herbivores, plants rapidly induce defense- and stress-related genes and phytochemicals for preventing or reducing further damage (Constabel 1999). For direct defense against herbivores, plants are known to induce defense proteins such as PIs (Korth and Dixon 1997) or polyphenol oxidases (Constabel et al. 2000). PI families are separated by specific criteria for each of the four classes of proteolytic enzymes, including serine, cysteine, aspartic, and metallo-protease (Ryan 1990). Three clusters of PIs were represented in the EST clones, including Bowman-Brik type PI involved in the serine protease inhibitor family, cysteine PI, and an uncharacterized PI. Expression of these PIs might be expected to be included in the defense mechanisms of the plant's response to larval infestation.

The production of a diverse set of stress-related oxidative enzymes may play a key role in plant defense mechanisms against insect herbivores. The oxidative enzymes are able to destroy essential nutrients and then induce anti-nutritive effects in insect diets. Superoxide anions (O2-) are produced in the incompatible interactions of plants and fungi (Doke 1983) and then dismutated either non-enzymatically or via superoxidase dismutase catalysis to hydrogen peroxide (H2O2). Peroxidase uses H2O2 to oxidize a wide variety of biological substrates and is expressed in various stress-related and developmental processes. Several reports suggest that H2O2 and peroxidase play a key role in the defense response of plants to invading pathogens (Mittler et al. 1999; Able et al. 2000). Lipoxygenases, also oxidative enzymes, have several important roles in defense mechanisms. Kolomiets et al. (2000) suggested that transcripts of lipoxygenase were induced during the hypersensitive response development caused by incompatible pathogens in potato and that lipoxygenase might be involved in defense responses against pathogen infection. Our data suggest that the oxidative enzymes, such as several peroxidases and lipoxygenases, were induced by infestation of the Hessian fly and involved in some roles of the defense mechanisms between plants and insects.

The toughness of the cell wall is increased to prevent pathogen ingress, such as insect chewing and other forms of herbivore attack. Increased lignin, one of the structural components of the plant secondary cell wall, represents the mechanical strength and resistance of the plant to biotic and abiotic stresses (Lewis and Yamamoto 1990). Lignin biosynthesis requires the expression of several genes, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, caffeic acid 3-O-methyltransferase, and cinnamyl-alcohol dehydrogenase. Capellades et al. (1996) reported that expression of the caffeic acid O-methyltransferase gene increased and that its promoter responded to wounding and elicitors. Therefore, a NIL carrying 2RL was required to increase expression of lignin synthesis-related genes during larval attack. In EST analysis, the presence of lignin synthesis-related genes suggested lignin synthesis was increased by the attack of insect herbivores.

As far as we know, little is known about the molecular interactions between plants and Hessian fly larval infestation. NILs possessing the H21 gene developed by Seo et al. (1997) have verified that resistance to larva of biotype L of the Hessian fly was derived from 2RL chromatin. Further studies may provide a clue on the molecular interactions between plant and larva of the Hessian fly using NILs and EST clones obtained from the cDNA library.


The authors extend special thanks to Dr. Robert A. Graybosch (USDA-ARS, University of Nebraska-Lincoln) for his thoughtful comments and suggestions on the manuscript and Drs. Rogers H. Ratcliffel and Sue E. Cambron (University of Purdue) for providing Hessian fly stocks. This work was supported by a grant from the BioGreen 21 Program, Rural Development Administration, Republic of Korea. It was also partially funded by a Korea University Grant.

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