Functional characterization of HFR1, a high-mannose N-glycan-specific wheat lectin induced by Hessian fly larvae

Abstract

We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His(6)-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His(6)-HFR1 to Manalpha1-6(Manalpha1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His(6)-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His(6)-HFR1 in the diet. Thus, HFR1 is an antinutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants.

Figure 1.

Purification of His₆-HFR1 protein. Using a Ni²⁺-NTA affinity column, His₆-HFR1 was purified from the total soluble protein fraction of cell lysate of E. coli transformed with the expression plasmid pET-HFR1. The recombinant HFR1 was purified to apparent homogeneity and had the expected mass of 40.5 kD. Samples were resolved by SDS-PAGE on a 10% gel and stained with Coomassie Brilliant Blue. Lane M, protein molecular mass markers; lane 1, total soluble protein fraction of E. coli transformed with pET-HFR1 (10 μg); lane 2, affinity-purified His₆-HFR1 (0.5 μg).

Figure 2.

Binding of His₆-HFR1 lectin to glycan microarray. A, At a concentration of 1.88 μg mL⁻¹, His₆-HFR1 bound with highest affinity (quantified as relative fluorescence units ± se) to eight of 320 glycans represented on the glycan microarray version 3.0. B, Representation of the glycan structures corresponding to the reference numbers in A. Man is represented by circles, and the GlcNAc core is represented by squares. His₆-HFR1 had highest affinity for structures containing α3 to α6 linkages (encircled). The names of each glycan structure represented in A and B are as follows: (1) Manα1-2Manα1-3Manα-Sp9; (2) Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; (3) Manα1-3(Manα1-6)Manα-Sp9; (4) Manα1-6(Manα1-3)Manα1-6(Manα2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; (5) Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; (6) Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12; (7) Manα1-6Manβ-Sp10; (8) Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ-Sp10. [See online article for color version of this figure.]

Figure 3.

Immunodetection of endogenous HFR1 lectin in resistant and susceptible wheat plants. Total plant proteins (8.0 μg) extracted from Hessian fly-infested and uninfested wheat crown tissue over a time course were resolved by SDS-PAGE on 4% to 20% gradient gels. Immunoblot analysis was carried out using an HFR1-specific affinity-purified polyclonal antibody and detected by chemiluminescence using a secondary anti-rabbit IgG-HRP-conjugated goat antibody. Arrowheads indicate the endogenous HFR1 protein (37.5 kD). His₆-HFR1 protein (60 ng) was used as a positive control and showed a single band of the expected 40.5-kD size. A, H9-Iris wheat infested with vH9 (virulent on H9 plants) or biotype L (avirulent on H9 plants) Hessian fly larvae. Uninfested and infested wheat crown tissues were collected at 1 and 2 d after egg hatch. B, Newton (susceptible and nearly isogenic to H9-Iris) and H9-Iris (resistant) wheat lines infested with biotype L Hessian fly larvae (avirulent on H9-Iris and virulent on Newton). Infested crown tissue was collected at 1, 2, and 3 d after egg hatch. Uninfested control plants were collected at 2 d after eggs hatched on the infested plants.

Figure 4.

Immunodetection of HFR1 lectin ingested by Hessian fly larvae. Total protein was extracted from virulent larvae (biotype L feeding on susceptible Newton plants) and avirulent larvae (biotype L present on resistant H9-Iris plants) at 1, 2, and 3 d after hatching from eggs. Larvae were washed to remove any HFR1 lectin that may have been present on the external surfaces of the larvae. The protein (1.0 μg) was resolved by SDS-PAGE on 4% to 20% gradient gels. The protein gel blot of these samples was incubated with the anti-HFR1 antibody. The presence of intact HFR1 (37.5 kD) and its proteolytic products (ranging from 10 to 30 kD) was detected in virulent larvae, while the avirulent larvae lacked a detectable band corresponding to the 37.5-kD HFR1 protein but contained proteolytic products of very low molecular mass only.

Figure 5.

Effect of His₆-HFR1 protein on the development of D. melanogaster larvae. A, Length of larvae at 5 d after hatch. Error bars represent se for the respective number of larvae (given above each bar) measured for each concentration from three independent replicates. The length of neonate larvae was 1.12 ± 0.02 mm. Concentrations of His₆-HFR1 at which larval lengths were significantly different from the control (0 μg g⁻¹) are marked with asterisks. B, Development time from hatch of neonate larvae to pupation is represented by black bars, and time from hatch until eclosion as adult flies is represented by white bars. No adult flies eclosed from vials containing 6 to 15 μg g⁻¹ His₆-HFR1. Larvae died at 12 and 8 d after egg hatch without pupating at concentrations of 12 and 15 μg g⁻¹ His₆-HFR1, respectively. Concentrations of His₆-HFR1 at which pupation and eclosion were significantly different from the control (0 μg g⁻¹) are indicated with asterisks. Error bars represent sd between three biological replicates, with each replicate having 10 larvae. One-way ANOVA was carried out using SAS, and all differences were considered significant at P < 0.05.

Figure 6.

Scanning electron micrographs of Hessian fly larvae on wheat plants. A, One-day-old avirulent biotype L larva oriented perpendicular to leaf sheath veins of H9-Iris wheat (resistant plant). B, One-day-old virulent biotype L larva oriented parallel to leaf sheath veins of Newton wheat (susceptible plant nearly isogenic to H9-Iris). C, Three-day-old avirulent biotype L larva showing a lack of growth and development on the leaf sheath of a H9-Iris wheat plant. D, Three-day-old virulent biotype L larva showing increased size on the leaf sheath of a Newton wheat plant. E, Frontal view of writhing 1-d-old avirulent biotype L larva oriented perpendicular to leaf sheath veins. F, Lateral view of the anterior end of a 3-d-old virulent biotype L larva showing mouth parts firmly attached to the plant tissue. Note the parallel orientation of the virulent larvae in the epidermal groove of the leaf sheath of the susceptible plants and the apparent disorientation of the avirulent larvae on the resistant plants.

Figure 7.

Length of biotype L Hessian fly larvae on resistant and susceptible wheat plants. The lengths of larvae (16–22 larvae from six leaves) were measured at 6, 12, 24, 48, 72, 96, 120, and 192 h after egg hatch. At early stages of infestation (6, 12, and 24 h after hatch), the differences in length of virulent larvae (biotype L on susceptible Newton wheat) and avirulent larvae (biotype L on resistant H9-Iris wheat) were not significant. Length differences became markedly significant (indicated by asterisks) by later stages of infestation, with avirulent larvae showing no increase in size over neonate larvae. One-way ANOVA was carried out using SAS, and differences were considered significant at P < 0.05.