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Originally published In Press as doi:10.1074/jbc.M401405200 on March 17, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23782-23789, May 28, 2004
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The Interactions of Allium sativum Leaf Agglutinin with a Chaperonin Group of Unique Receptor Protein Isolated from a Bacterial Endosymbiont of the Mustard Aphid*

Santanu Banerjee{ddagger}§, Daniel Hess¶, Pralay Majumder{ddagger}||, Debjani Roy**, and Sampa Das{ddagger}{ddagger}{ddagger}

From the {ddagger}Plant Molecular and Cellular Genetics, Bose Institute, P-1/12, C.I.T. Scheme, VII-M, Calcutta 700054, India, the Protein and Peptide Analytics Laboratory, Friedrich Miescher Institut for Biomedical Research, P.O. Box 2543, CH-4002 Basel, Switzerland, and **Bioinformatics Centre, Bose Institute, P1/12, C.I.T. Scheme, VII-M, Calcutta 700054, India

Received for publication, February 9, 2004 , and in revised form, March 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The homopteran pest Lipaphis erysimi, commonly known as the mustard aphid, causes major damage to oil seed crops. These aphids affect the growth of the plant by sucking on the plant sap, and while feeding, they also transmit disease-causing luteoviruses to the host plant in a non-replicating and circulative manner. The aphid species is capable of transmitting viruses such as barley yellow dwarf virus, potato leaf roll virus, and bean leaf roll virus, which are commonly known as plant luteoviruses and belong to the family Luteoviridae. They are phloem-limited, single-stranded, positive-sense RNA viruses that are strictly dependent on aphids as vectors for their transmission from plant to plant (1-4). The plant-aphid-virus interaction is highly specific for all three components (plant, aphid, and virus). Control of aphids using transgenic plants has been suggested as an alternative to pesticide spray. To date, the only commercial insect-resistant transgenic plants are those expressing genes from the soil bacterium Bacillus thuringiensis encoding insecticidal crystal proteins (Bt-toxins) (5-10). Aphids, including L. erysimi, are not affected by Bt toxins. However, some carbohydrate binding plant lectins, e.g. Galanthus nivalis agglutinin, Phaseolus hemagglutinin, and wheat germ agglutinin have been reported to be able to control homopteran pests (11-15). We demonstrated previously (16, 17) the detrimental effects of the mannose-binding Allium sativum leaf (ASAL) and bulb (ASA) lectins on the growth and development of L. erysimi when provided in an artificial diet bioassay. Additionally, the brush border membrane vesicle (BBMV1) of L. erysimi gut epithelial cells responsible for its binding to the lectin have been purified by ligand blot analysis (16). Furthermore, the specific ligand-positive ~56 kDa receptor protein of ASAL, purified from L. erysimi, was characterized and identified as a symbionin protein (SymL) belonging to the Cpn 60 class of insect chaperonins, which is evolutionarily related to the Escherichia coli GroEL protein. Such SymL protein has been reported previously to be coded by a mutualistic intracellular symbiotic bacteria, Buchnera aphidicola. It resides in a specialized tissue of the insect gut, which supplies several essential amino acids to aphids that they do not get from plant sap (18). This SymL also binds to the read-through domain (RTD) of the coat protein of the single-stranded virus particles located in the hemocoel and escorts them to the salivary gland and finally helps the viruses to be transmitted to different plants while the aphids feed on them (1-4).

The L. erysimi symbionin described here establishes a unique oligosaccharide-mediated binding affinity with ASAL. The bioinformatics approach further indicates that the above binding may elicit changes in surface accessibility of symbionin leading to its inactivation of the luteovirus RTD binding ability thus reducing the incidence of viral transmission from one plant to another.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mustard aphids (L. erysimi) were obtained from our institutional experimental farm and maintained on live mustard plants or artificial diets. Mannose-binding garlic leaf lectin (ASAL) has been purified from the leaves of garlic grown in the same experimental farm. All reagents and chemicals used were of analytical grade.

Isolation of Aphid Brush Border Membrane Vesicle—Approximately 1000 aphids fed with a sublethal dose of ASAL were dissected, and the excised midguts were suspended in insect ringer solution (0.65% NaCl, 0.25% KCl, 0.03% CaCl2). The BBMV was isolated from the excised midgut suspension by differential precipitation of the luminal epithelium of the insect midgut with the addition of 12 mM MgCl2 salt as reported earlier (16).

Ligand Blot Assay of Total BBMV Protein-—The total BBMV protein was dissolved in 1% sodium deoxycholate and ~5 µg each were separated onto duplicate lanes using 12% SDS-PAGE. After electrophoresis, one of the lanes was stained with Coomassie Brilliant Blue, whereas the other was transferred electrophoretically to a Hybond-C (Amersham Biosciences) membrane in a Hoeffer submerged electroblot apparatus. The membrane was transiently stained with Ponceau-S (Sigma). After blocking with 5% bovine serum albumin, the membrane was incubated with 2 nM ASAL solution for 1 h and subjected to ligand blot analysis challenging with ASAL polyclonal antibody.

Purification of the Major Receptor Protein—Approximately 100 µl (~ 0.8-1 mg) of total aphid BBMV protein extract was separated using 12% SDS-PAGE along with protein molecular weight standards in a Bio-Rad Mini-Protean III electrophoresis module. The marker lane, together with 3-4 mm of the preparative lane, was excised and stained with Coomassie Brilliant Blue. A doublet of two bands, resolving closely in the range of 56 kDa was excised after aligning with the Coomassie Brilliant Blue-stained molecular weight standard. The proteins were electroeluted from the gel piece using an Atto electroelution apparatus (Atto Corp.). The electroeluted sample was dialyzed against 50 mM ammonium bicarbonate buffer, pH 8.0, and the resulting SDS-free protein sample was lyophilized to dryness and subjected to ligand blot assay as described earlier.

The proteins were further separated using 10% preparative SDS-PAGE with a longer run time for better resolution of the two protein bands of the doublet. The upper band, with a higher sensitivity to ASAL, was again purified by electroelution and analyzed in an TSK G3000SW HPLC gel filtration column with 20 mM Tris-Cl, pH 7.5, as the running buffer.

Two-dimensional PAGE Analysis of the Receptor Protein—The electroeluted proteins were additionally separated by two-dimensional gel electrophoresis in a Bio-Rad Mini-Protean II Isoelectric focusing unit using carrier ampholytes (3-10) according to the protocol given by the manufacturer. For the second dimension, gels were run in a Mini-Protean III SDS-PAGE module. The profiles were monitored by staining the gels with Coomassie Brilliant Blue as well as by ligand blot analysis.

Identification of the Receptor Protein by Liquid Chromatography-Tandem Mass Spectrometry—The protein spot was excised from the two-dimensional gel, reduced with 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide, and cleaved with porcine trypsin (Promega, sequencing grade) in 50 mM ammonium bicarbonate buffer, pH 8.0, at 37 °C overnight (19). The extracted peptides were analyzed by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a magic C-18 100-µm x 10-cm HPLC column (Spectronex) connected online to an ion trap Finnigan DecaXP (Thermo Finnigan). A linear gradient from 5 to 50% buffer B (0.1% formic acid, 80% acetonitrile in H2O) in buffer A (0.1% formic acid, 2% acetonitrile in H2O) was delivered with a Rheos 2000 HPLC system (Flux) at 100 µl/min for 30 min. A precolumn flow splitter reduced the flow to ~300 nl/min, and the peptides were manually loaded with a 10-µl Hamilton syringe on a captrap (Michrom BioResources) mounted in the injection loop of the mass spectrometer. The eluting peptides were ionized by electrospray ionization and detected, and the individual ions were automatically selected and fragmented in the ion trap. Ten peptide fragments have been sequenced. Individual MS/MS spectra and derived sequence information of peptide fragments were compared using the program Turbo-Sequest (20) against the combined Swissprot (40.28) and Trembl (21.7) protein sequence data bases.

Glycoprotein-specific Staining of BBMV Protein—The proteins extracted from the total mustard aphid BBMV by dissolving in 1% sodium deoxycholate were separated using 12% SDS-PAGE and stained specifically for covalently bound oligosaccharides according to the methods of Moller and Poulsen (21). This method involves periodic acid oxidation of the fixed proteins in SDS-PAGE, staining with Alcian blue and subsequent silver enhancement staining at high temperatures to specifically stain the glycoproteins. Finally the stain was developed with 2.5% sodium carbonate for 30-60 s. The staining profile was recorded, and the gel was further stained with Coomassie Brilliant Blue according to standard procedures to stain the total proteins including the non-glycosylated ones.

Competitive Inhibition of ASAL-receptor Interaction in Presence of Mannose and Ligand Blot Analysis after Deglycosylation of the Receptor—Total aphid BBMV proteins, dissolved in 1% sodium deoxycholate and dialyzed, were run on two separate 12% SDS-polyacrylamide gels, and both of these were transferred onto Hybond-C membranes and marked as "A" and "B." The ligand blot analysis mentioned above was performed on two membranes, one modification was introduced for membrane B wherein 1 nM ASAL pre-incubated with 1 M {alpha}-D-mannose was used instead of "only ASAL." After this point, the ligand blot analysis was carried out on both the membranes as mentioned earlier (16). Likewise, ligand blot assays both in the presence and absence of {alpha}-D-mannose were carried out with the purified ~56-kDa receptor protein also. In another experiment, 5 µg of total BBMV proteins of L. erysimi was deglycosylated, using the N-glycosidase F deglycosylation kit (Roche Diagnostics) according to the protocol described in the kit manual and analyzed using ligand blot to check the response of deglycosylated receptor to ASAL.

Bioinformatic Study of the ASAL Lectin: SymL Receptor Association
Building of the Model of ASAL and SymL—The nucleotide sequence of ASAL was obtained from the NCBI data base (Accession number U58947 [GenBank] ) and translated in silico using the translation tool at the Swiss Bioinformatics Server (ExPASy.ch). The 110 amino acid sequence thus obtained, represented a single subunit of the protein and was used for building the model of ASAL. The three-dimensional coordinates of the template structure, 1KJ1 [PDB] (22), were obtained from the Protein Data Bank (rscb.org/PDB/) (23). The coordinates of chain A were used for the purpose. The amino acid sequence of SymL was obtained from the NCBI Entrez data base (Accession number AAK52957 [GenBank] B. aphidicola (Myzus persicae)), and the three-dimensional coordinates of its template structure, 1GRL [PDB] (24), were obtained from the Protein Data Bank. The comparative homology modeling was done using the program Modeller (25). Energy minimizations were carried out by 4000 cycles of conjugate gradient using the consistent valence force field in a Silicon Graphics R4600 work station using the program Discover (Biopsy Technologies, San Diego, CA). The integrity and quality of the models were assessed using the program Procheck (26), which also generated the Ramachandran plot of the models.

Addition of the Oligosaccharide Unit to the SymL Model—On closer inspection of the amino acid sequence of the symbionin protein, two asparagine-linked glycosylation signatures, Asn133-Lys134-Ser135 and Asn534-Ser535-Ser536, were found. However, our modeling studies showed that the second signature remains buried into the quaternary structure of the tetradecameric SymL; hence, the first signature sequence was chosen as the possible glycosylation site. A consensus oligosaccharide moiety, (GlcNac)2(Man)7, was obtained from the NMR solution structure of human Cd2 protein available in the Protein Data Bank (23) under the code name 1GYA [PDB] (27). This oligosaccharide moiety was covalently added to the Asn133 position of the modeled SymL using the program Insight-II (Biosym Technologies) on a silicon graphics R4600 work station. The resultant Protein Data Bank coordinate file was named sm.pdb

Docking of Glycosylated SymL with ASAL—The modeled structure of ASAL (ligand) was docked into the active site of its receptor SymL using the program Gramm (28, 29) on a PC-LINUX work station. The experiment generated 4-5 good models, and the best model was chosen based on its energy rankings. The quality of the docked model was verified using the program Procheck, and Ramachandran plots were generated. The values of the accessible surface area for both the native protein (sm.pdb; SymL) and the ligand-bound protein were calculated using the homology module of the Insight-II program. The differences in accessible surface area between native SymL (sm.pdb) and the docked complex (smgl-asal) was calculated for every residue using Insight-II. The schematics were rendered using the program MolScript (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of the BBMV Receptors by Ligand Binding Assay—Aphid BBMV protein was separated by SDS-PAGE as described under "Experimental Procedures." The total protein profile was monitored by Coomassie Brilliant Blue staining (Fig. 1A). The ligand binding receptor was identified on Hybond-C membrane as an ~56-kDa protein, as shown in Fig. 1B.



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  		FIG. 1.
BBMV protein profile in SDS-PAGE and its ligand blot. A, lane 1, molecular weight marker; lane 2, Coomassie-stained total BBMV protein. B, ligand blot analysis of the total BBMV protein, arrowhead indicates ~56-kDa receptor protein.

 
Separation of Individual Receptors by Two-dimensional PAGE—On further analysis using 10% SDS-PAGE (Fig. 2A) and isoelectric focusing (Fig. 2B), the protein was split into a doublet with two bands interacting with ASAL. The higher molecular weight protein was further analyzed using two-dimensional PAGE (Fig. 2C) and subsequent ligand blot analyses were repeated (Fig. 2D). The receptor was further verified by HPLC as a single peak shown in Fig. 2E.



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  		FIG. 2.
Purification and biochemical characterization of the Lipaphis BBMV receptor protein through various methods. A, purified receptor analyzed in 10% SDS-PAGE with overrun-generated doublet. B, ligand blot analysis of the purified receptor doublet separated in two-dimensional PAGE. Arrows show the positions of the two spots. The higher molecular weight receptor protein separated at a pH range of 8.5-8.8 and was targeted for further purification. Isoelectric focusing was run with 3-10 pH range carrier ampholytes in the first dimension. C, two-dimensional PAGE analysis of the pure receptor protein. Ioelectric focusing was run with 3-10 pH range carrier ampholytes in the first dimension and 12% SDS-PAGE in the second dimension. D, ligand blot analysis from a replica of the two-dimensional gel in C. E, HPLC gel permeation chromatography of the receptor protein run in an TSK G3000SW column (LKB Bromma) to verify the purity.

 
Identification of the Purified Receptor Protein—The tryptic peptides of the two-dimensional PAGE-isolated receptor protein were analyzed by LC-MS/MS. Fig. 3A shows the base peak chromatogram of the LC-MS/MS analysis of the tryptic digest of the protein. The derived sequence information of the peptide fragments was compared using Turboseqest 36, and the receptor protein had been identified as 57,885-Da E. coli GroEL-related molecular chaperonin symbionin from Buchnera aphidicola (Swissprot identifier Q93T48). The peptide sequences are depicted in Fig. 3B.



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  		FIG. 3.
Identification of receptor protein. A, base peak chromatogram of the LC-MS/MS analysis of the tryptic peptides of SymL. The peptides are numbered according to their occurrence in the protein sequence from the N to C terminus. They have been identified from the MS/MS spectra using Turbosequest and are labeled in the chromatogram. B, sequence of the identified protein. The sequenced peptides are shown in red.

 
Characterization of the Receptor as Glycoprotein—The glycoprotein-specific staining revealed that the active receptor is a glycoprotein. In the staining protocol using 2.5% Na2CO3, glycoproteins lit up within 30 s and the non-glycosylated proteins remained unstained within this brief exposure time. Further Coomassie Brilliant Blue staining of the gel could detect all glycosylated and non-glycosylated proteins (Fig. 4A showing only the stained glycoproteins in lane 1 compared with Coomassie Brilliant Blue-stained total BBMV proteins, lane 2). From the results, it is evident that the ligand-positive ~56-kDa receptor protein is glycosylated.



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  		FIG. 4.
Monitoring of the glycosylated nature of the BBMV receptor. A, depicts the glycoprotein-specific staining of mustard aphid BBMV proteins. Lane 1, stained glycoproteins; lane 2, Coomassie-stained total proteins. Arrowhead shows the ~56-kDa ASAL receptor. B, shows deglycosylation and subsequent ligand blot analysis of aphid BBMV Proteins. Lane 1, crude BBMV fraction; lane 2, deglycosylated BBMV fraction. Arrowhead shows the major ASAL receptor. C, the inhibition of ligand blot analysis in the presence of mannose. Lane 1, ligand blot analysis of aphid BBMV; lane 2, ligand interaction inhibited by mannose. Here, the membrane was incubated with ASAL in the presence of 1 M {alpha}-D-mannose. D, ligand blot analysis of the pure receptor in the presence of mannose. Lane 1, ligand blot analysis of ~56-kDa receptor; lane 2, ligand interaction inhibited by mannose.

 
Ligand Blot Assay of the Receptor after Deglycosylation and Mannose Inhibition—The results of the ligand binding experiments carried out with total BBMV and deglycosylated BBMV established that the ligand binding ability of the receptor is abolished after deglycosylation (Fig. 4B, lane 2). Subsequent mannose-inhibition assays of the ligand blot analysis of total L. erysimi BBMV proteins (Fig. 4C) and purified receptor protein (Fig. 4D) were carried out where the ASAL probe was presaturated with excess {alpha}-D-mannose prior to the ligand blot analysis. In fact, under these conditions ASAL did not bind to the receptors (Fig. 4, C and D, lane 2 in each), although it bound to the receptors in the absence of {alpha}-D-mannose (Fig. 4, C and D, lane 1 in each). This result clearly indicates that the binding of ASAL to the receptor in the insect midgut takes place at N-linked mannosylated oligosaccharide and is dependent upon the mannose-binding capacity of the lectin. Deglycosylation of the BBMV protein was seen to diminish the interaction of the two proteins to a large extent, thereby reiterating a lectin-glycoprotein interaction in the case of the ASAL and BBMV receptors. However, no mobility shift was apparent in SDS-PAGE analysis of the ~56-kDa receptor band even after the deglycosylation reaction.

Molecular Interaction between ASAL and SymL Models—The docked smgl-asal (SymL-ASAL) complex is shown in Fig. 5. The docked model, when analyzed critically, showed the interaction between the mannose molecules Man554 and Man557 of SymL with respective ASAL residues Gln90, Asp92, Asn94, Val96 Asp60, Asn62, and Val64 as shown in Table I, the schematic representation of which is depicted in Fig. 6A. The ASAL residues interacting here satisfy the mannose binding domain of the garlic lectins as reported earlier in the case of garlic bulb lectin, ASA (20). Most interestingly, however, we also found that a few main-chain residues, Gln432, Gln436, Gln436, and Arg118 of SymL, formed hydrogen bonds with Tyr97, Ser99, Asp100, and Ile101 residues of ASAL (Table I and Fig. 6B). Table II summarizes the two types of interactions mentioned above.



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  		FIG. 5.
Model of ASAL (red) docked with SymL (blue) through the oligosaccharide moiety. The oligosaccharide moiety was taken from the Protein Data Bank structure 1GYA [PDB] and covalently added to Asn133 of SymL in silico. Interactions in terms of hydrogen bonds were mapped and represented in Tables I and II.

 


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  		TABLE I
Table shows the interaction of the mannose residues with the ASAL residues (left two columns) and the main-chain interaction between the residues of ASAL and SymL (right two colums)

 



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  		FIG. 6.
Molecular interactions of ASAL and SymL. A, shows the interaction of the two mannose residues with the ASAL amino acid residues. The oligosaccharide chain is depicted in the ball-and-stick conformation. ASAL is shown as a schematic and the yellow regions are the residues interacting with mannose. Asn133 of SymL is shown in the ball-and-stick conformation and is magenta. The rest of the receptor is in the wire frame conformation. B, schematic representation of the main-chain interactions of ASAL and SymL. ASAL is depicted as a schematic and its interacting residues are shown in yellow. The interacting SymL residues are shown in ball-and-stick conformation. The rest of the SymL molecule is shown in the wire frame conformation.

 


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  		TABLE II
Table shows hydrogen bond formation between ASAL and SymL

 
Surface Accessibility Profile of the Free SymL Molecule and the ASAL-SymL Complex—The surface accessibility calculations done on the ASAL-SymL complex model determined the reduced surface accessibility in comparison to the free SymL molecule. The reduced accessibility was envisaged in the residues of SymL that are responsible for the possible interaction with other potential molecules (RTD domain of virions) depicted in Fig. 7. In other words, the binding of ASAL to the SymL receptor covers up a certain area of the surface of the receptor, making that area sterically inaccessible to solvents or other molecules e.g. viral RTD.



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  		FIG. 7.
Changes in the molecular surface accessibility of symbionin. Surface accessibility before (blue) and after (green) binding of ASAL. The arrows show the region of maximum accessibility variations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
L. erysimi, commonly known as the mustard aphid, affects plants by transmitting luteoviruses while sucking on the phloem sap and causing interference with the growth and development of plants. After determining the efficacy of ASAL on our target aphid, L. erysimi, we extended our efforts to investigate the binding affinity of ASAL to the insect gut. The total BBMV was isolated from the L. erysimi gut, and the specific ligand-positive receptor of ASAL was identified. The receptor was further analyzed and, interestingly, identified as a symbionin (SymL) protein. The SymL protein is coded by a bacterial endosymbiont of the aphid. It is known from earlier reports that various aphid species, e.g. M. persicae, carry a prokaryote called B. aphidicola within their body, which is known to have had a symbiotic association with aphids for the last 160-280 million years (1-4, 18, 31, 32). The aphids carry these primary symbionts and there are various attributes associated with their long association. The prolonged association of Buchnera and the aphids has resulted in a massive reduction in the genome size of the former. The Buchnera genome is only 641 kb, in comparison to its closest non-symbiotic relative E. coli (4.6 Mb) (33). Buchnera is localized in the aphid hemocoel in a specialized organelle called mycetocytes. The benefits of the association are strictly nutritional, i.e. aphids obtain certain essential amino acids from the bacterium, which over-expresses them in the plasmids recruited for the purpose, and in return the bacterium obtains most of its housekeeping requirements from the aphid hosts (18, 31)

The importance of the study of symbionin (SymL) in aphids has increased in the last two decades, because it was found to be involved in virus transmission. Symbionin is the protein that escorts the virion to the aphid salivary gland. In fact, symbionin is recognized by the luteovirus particle, which has a minor capsid protein, the RTD, exposed on the virion capsid. The bound virion-SymL complex passes through the midgut epithelial membrane into the aphid hemocoel by a mechanism as yet unknown and enters the accessory salivary gland of the aphid, which is speculated to be a receptor-mediated endoexocytosis. The virion is known to concentrate itself in the accessory salivary gland of the host until it is released into the phloem of a healthy plant during the next infestation cycle of the aphid (3, 4, 34) The symbionin has been found to have extensive sequence homology (varies from aphid to aphid in the range of 80-92%) to the E. coli GroEL chaperonin. Both of them are also identical in terms of their quaternary association (35).

The present symbionin protein extracted from mustard aphid BBMV had been characterized as a glycoprotein using glyco-specific staining. Its binding affinity for ASAL had already been established using ligand blot analyses. Because ASAL is a mannose-binding protein, ASAL-SymL binding should be mannose-mediated. In agreement with this theory, deglycosylation of the BBMV proteins was seen to diminish the interaction of the two proteins considerably, thereby reiterating a lectin-glycoprotein interaction in the case of the ASAL and SymL molecules. However, no mobility shift was apparent in SDS-PAGE analysis of the ~56-kDa receptor band even after the deglycosylation reaction. Moreover, SymL responded negatively to the ligand reaction when ASAL presaturated with {alpha}-D-mannose was used as the ligand. From these results, it can be predicted that the binding of ASAL to the receptor in the insect midgut is probably dependent on its capacity to bind to the exposed mannose unit(s) of the receptor.

Additionally, the molecular aspect of the binding of the present SymL protein with luteovirus particles for its transmission onto the host plants was investigated using a bioinformatic approach. From the sequence information, it has been established that each monomer of SymL may be divided into three major domains: the equatorial, the intermediate, and the apical domain. The equatorial domain extends from the amino acid residues 1 to 135 and from 410 to the C-terminal end of the protein. Studies on the potato leaf roll virus have shown that in the SymL monomer, amino acid residues 1-121 and 409-474 come together to form a single potato leaf roll virus-RTD binding site (36). The apical and intermediate domains, on the other hand, remain partially or fully buried into the bucket-like quaternary structure of the protein (36).

The independent docking experiment with the SymL-RTD and the ASAL-SymL complex RTD gave new insight into the virus transmission ability of SymL when it remains bound with ASAL. The in silico model structure suggested that ASAL binds to the oligosaccharide unit attached to Asn133, which is situated on the equatorial domain of the SymL monomer. The mannose residues of the oligosaccharide moiety were found to interact with Gln90-Asp92-Asn94-Val96 and Asp60-Asn62-Val64 of two mannose binding domains of ASAL. This result corroborates the mannose binding domain defined earlier in the case of garlic bulb lectin, ASA (22). ASAL also establishes contact involving the residues, Tyr97, Ser99, Asp100, and Ile101 with the side chains of SymL amino acid residues Gln432, Gln436, Gln436, and Arg118. Additionally, the interacting Arg residue Arg118 and Gln residues Gln432 and Gln436 of SymL fall at the defined RTD binding domain of the same (residue 1-121 and 409-474 coming together serves as RTD binding domain). Thus, the SymL-ASAL docked model probably fails to recognize the virion particle because of the unavailability of the amino acid residues essential for RTD binding, as reflected in the surface-accessibility calculation profiles of the amino acids of free SymL and the ASAL-SymL complex as shown in Fig. 7.

The amino acid sequence analysis of the model structure of the SymL protein purified by us from the L. erysimi BBMV tissue envisaged the presence of a significant number of putative myristoylation signature sequences as well as exposing a hydrophobic patch between residues 502 and 522, which also supports the possibility of the SymL-virion complex coming into close association with the membrane and then passing on to the hemocoel for further transmission into plants. The detrimental effects of the lectins we have studied on the aphids and other homopteran insects (16, 17) have been absolute in moderately high doses (50 or 100 µg/ml). However, at lower doses, where mortality reaches 50%, the antagonistic effects of lectin in terms of a reduction of luteovirus transmission clearly would become significant.

On the basis of these observations, it is envisaged that the developing transgenic crops, which would express this monocot mannose-binding lectin (ASAL), can efficiently control aphids and as a result, reduce the viral attack. In cases where the aphids may not be eliminated completely and the plants may encounter very low levels of aphid infestation upon consumption of the expressed lectin, it is predicted that they will lose their SymL-mediated virus transmission ability. Thus, lectins with such dual utility could serve as a powerful tool in an integrated pest management program. It remains elusive whether, indeed, the spread of the virus is reduced when aphids are exposed to ASAL expressed in transgenic plants.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Provided a Fellowship and infrastructural facilities by Bose Institute, Calcutta, India. Back

|| Provided a Fellowship by the Council for Scientific and Industrial Research (CSIR). Back

{ddagger}{ddagger} Funded by the Department of Atomic Energy, Government of India. To whom correspondence should be addressed. Tel.: 91-033-23379544; Fax: 91-033-23343886; E-mail: sampa{at}bic.boseinst.ernet.in.

1 The abbreviations used are: BBMV, brush border membrane vesicle; RTD, read-through domain; HPLC, high pressure liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry/mass spectrometry. Back


   ACKNOWLEDGMENTS
 
We thank Barbara Hohn of Friedrich Miescher Institut (FMI), and Thomas Hohn, University of Basel, Switzerland, for their helpful and critical suggestions. We thank A. N. Lahiri Majumder for fruitful interaction and all departmental colleagues for cooperation. S. D. thanks Indo Swiss Collaboration in Biotechnology for providing an opportunity to interact with colleagues.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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