Functional Characterization of HFR1, a High-Mannose N-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae1[C][W] Subhashree Subramanyam, David F. Smith, James C. Clemens, Mary A. Webb, Nagesh Sardesai, and Christie E. Williams* Department of Agronomy (S.S.), Department of Biochemistry (J.C.C.), Department of Botany and Plant Pathology (M.A.W.), Department of Biological Sciences (N.S.), and Department of Entomology (C.E.W.), Purdue University, West Lafayette, Indiana 47907; Protein-Carbohydrate Interaction Core H, Emory University School of Medicine, Atlanta, Georgia 30322 (D.F.S.); and U.S. Department of Agriculture-Agricultural Research Service Crop Production and Pest Control Research Unit, West Lafayette, Indiana 47907 (C.E.W.)
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 His6-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 His6-HFR1 to Mana1-6(Mana1-3)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 His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His6-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.
In response to attack by herbivorous insects, plants produce a variety of defense proteins as an attempt to minimize additional damage. Some of these defense proteins are lectins (Chrispeels and Raikhel, 1991; Peumans and Van Damme, 1995; Czapala, 1997), which are widespread throughout the plant kingdom (Goldstein and Poretz, 1986). Plant lectins, also referred to as ‘‘agglutinins,’’ are a heterogenous group of carbohydratebinding proteins that are able to reversibly bind simple sugars and/or complex carbohydrates (for review, see Van Damme et al., 1998). However, over the past few years, novel sugar-binding domains have been identified (Kaku et al., 2006; Peumans et al., 2007; Van Damme et al., 2007a) that necessitated redefining the families of plant lectins. 1 This work was supported by funding from the U.S. Department of Agriculture (grant no. CRIS 3602–22000–014–00D). * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christie E. Williams (
[email protected]). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116145
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Man-binding lectins are widely found in higher plants and play a significant role in defense due to their ability to recognize high-Man-type glycans of foreign microorganisms or plant predators. However, recent investigations of two-domain Galanthus nivalis agglutinin (GNA)-related lectins revealed that these lectins have dual specificity and interact with both high-Man and complex N-glycans (Van Damme et al., 2007b). Thus, the conventional classification of this group of lectins as Man-binding lectins was modified to accommodate this broader specificity to complex N-glycans. However, in the absence of a redefined nomenclature for this group, we will continue to refer to this class of lectins as Man-binding lectins. The insecticidal activity of Man-binding lectins has been demonstrated against a spectrum of insects, both in artificial diets supplemented with the lectins (Powell et al., 1993; Fitches et al., 2001) and also when insects feed on natural and transgenic plants expressing lectins (Gatehouse et al., 1996; Nagadhara et al., 2004; Sadeghi et al., 2008). The snowdrop lectin, GNA, is toxic to brown planthopper (Nilaparvata lugens; Powell et al., 1998; Tang et al., 2001) and tomato moth (Lacanobia oleracea) larvae (Fitches et al., 1997; Gatehouse et al., 1997, 1999) as well as to several aphid species (Hilder et al., 1995; Rahbe´ et al., 1995; Gatehouse et al., 1996; Sauvion et al., 1996). Concanavalin A (lectin from
Plant Physiology, July 2008, Vol. 147, pp. 1412–1426, www.plantphysiol.org Ó 2008 American Society of Plant Biologists
Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
Canavalia ensiformis) shows significant antimetabolic activity toward third-instar nymphs of taro planthopper (Tarophagous proserpina; Powell, 2001). Lectin from garlic (Allium sativum) bulb is active against insects such as cowpea weevil (Callosobruchus maculates), Egyptian cotton leafworm (Spodoptera littoralis), and tomato moth, causing reduced larval weight gain and delayed development (Fitches et al., 1997, 2001). Some lectins, such as the garlic bulb lectin, play a dual role as storage protein and as defense proteins that are mobilized against attacking insects (Van Damme et al., 1995; Smeets et al., 1997; Dinant et al., 2003). Transgenic rice (Oryza sativa) plants expressing garlic leaf agglutinin have antifeedant properties that are insecticidal against sap-sucking insect pests such as the brown planthopper, the green leafhopper (Nephotettix virescens; Saha et al., 2006), and the Egyptian cotton leafworm (Sadeghi et al., 2008). Although the mechanisms by which these insecticidal lectins act on insects are still not well understood, ultrastructural studies reveal that binding to insect gut structures and resistance to proteolytic degradation by insect digestive enzymes are the two main prerequisites for the lectins to have deleterious effects on insects (Sauvion et al., 2004). The insecticidal activity is attributed to lectin recognition of Man glycans in the digestive tracts of insects (Fitches et al., 2001; Majumder et al., 2004). Immunohistochemical studies show that these lectins bind to carbohydrate moieties of glycoproteins on epithelial cells lining the luminal surface of insect midguts, such as the binding of GNA in brown planthopper (Powell et al., 1993, 1998) and concanavalin A in pea aphids (Acyrthosiphon pisum; Sauvion et al., 2004). An additional target is the peritrophic matrix lining the midgut region. Concanavalin A and GNA bind to many peritrophic matrix proteins in vitro (Fitches and Gatehouse, 1998). The binding of lectins to these tissues results in disruption of the digestive processes and nutrient assimilation (Eisemann et al., 1994) by damaging the structure of the insect brushborder epithelium (Harper et al., 1995) and altering the organization of the microvilli (Powell et al., 1998). Consequently, the insect may be repelled, retarded in its growth, or even killed. The effectiveness of lectins as antinutrients in insects is determined primarily by the strength of their binding to the gut-associated glycoproteins (for review, see Vasconcelos and Oliveira, 2004). Thus, the production of lectins showing affinity for high-Man and complex N-glycans is gaining prominence as an important mechanism involved in the resistance of economically important crop plants against phytophagous insects. The Hessian fly (Mayetiola destructor), belonging to the order Diptera (family Cecidomyiidae), is a major destructive pest of wheat (Triticum aestivum) worldwide. Hessian fly larvae hatch from the eggs and crawl down to the base of the plant (crown), where they feed on the abaxial surface of developing leaf sheaths (McColloch and Yuasa, 1917). The first-instar Hessian fly larvae have specialized mandibles that are capable Plant Physiol. Vol. 147, 2008
of injecting salivary fluids into cells at the surface of wheat leaf sheaths and meristems (Hatchett et al., 1990). Resistance to Hessian fly attack is achieved through R gene-mediated resistance (Hatchett and Gallun, 1970). So far, 32 distinct Hessian fly resistance genes have been identified (Williams et al., 2003; Sardesai et al., 2005a). Interactions with Hessian fly elicit two types of responses in wheat: susceptibility to the larvae, a compatible interaction; and resistance to the larvae, an incompatible interaction. During the compatible interaction, the larvae develop normally and complete their life cycle while the growth of the infested wheat plant is stunted. In the incompatible interaction, the larvae die within a few days after hatching and plant growth is normal (Gallun, 1977). Although considered a gall midge, the Hessian fly does not cause the formation of a typical gall in susceptible wheat. Instead, the larvae induce the formation of nutritive tissue in the region of their feeding sites. This modified tissue resembles the internal surface of a gall and provides a diet rich in soluble proteins and sugars for larval consumption (Harris et al., 2006). Over the last few years, several studies have been conducted to unravel the molecular events taking place as wheat responds to Hessian fly attack. Superficially, the gene-for-gene recognition event in wheat-Hessian fly interactions appears similar to those of plant interactions with microbial pathogens. However, it is now known that the mechanisms of plant defense in these interactions are very different. Classical pathogenesisrelated and other defense-response genes associated with plant-pathogen interactions are minimally responsive in resistant wheat during Hessian fly larval attack (Sardesai et al., 2005b). No evidence of a classical oxidative burst has been detected in wheat following attack by virulent or avirulent Hessian fly larvae (Giovanini et al., 2006). In fact, several unique genes have been identified in wheat that are specifically elicited by the Hessian fly (Williams et al., 2002; Puthoff et al., 2005; Giovanini et al., 2007). Previously, we cloned wheat cDNA corresponding to Hessian fly-responsive gene 1 (Hfr-1; GenBank accession no. AF483596). Characterization of the gene revealed an accumulation of transcripts in resistant plants, 1 to 3 d after egg hatch in the crown tissue (interaction site for the first-instar Hessian fly larvae), that was absent in susceptible and uninfested control plants (Williams et al., 2002; Subramanyam et al., 2006). Database searches suggested that HFR1 is a chimeric protein consisting of two domains, an N-terminal disease response or ‘‘dirigent’’ domain and a C-terminal jacalin-related lectin (JRL) domain (Supplemental Fig. S1). JRLs derive their name from jacalin, the first member of this lectin family, which was identified from jack fruit (Artocarpus integrifolia) seeds (Bunn-Moreno and Campos-Neto, 1981). Based on glycan-binding specificity, JRLs are divided into two groups: Galspecific and Man-specific agglutinins (Peumans et al., 2001). Jacalin has been shown to exhibit insecticidal activity against an important pest of maize (Zea mays), 1413
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by matrix-assisted laser desorption ionization mass spectrometry/tandem mass spectrometry (MALDI MS/MS-MS) yielded two large peptide sequences and three smaller fragments: ELLLHLYAYQNVQKTPDANQAVIVESK (representing amino acids 38–65) and GPWGKMSGELLDIPSTPQRLERITIRHGVVIDSLAFSFIDKAGEPYNVGPWGGR (representing amino acids 198–252), plus FTGSSFK, MELHVR, and YVEALGVYVR (representing amino acids 121–127, 171–176, and 331–340, respectively). This method confirmed the absence of contaminants and also verified the theoretical predicted HFR1 molecular mass (37.5196 kD), having an acidic pI of 5.81. His6-HFR1 Is a Lectin
Figure 1. Purification of His6-HFR1 protein. Using a Ni21-NTA affinity column, His6-HFR1 was purified from the total soluble protein fraction of cell lysate of E. coli transformed with the expression plasmid pETHFR1. The recombinant HFR1 was purified to apparent homogeneity and had the expected mass of 40.5 kD. Samples were resolved by SDSPAGE 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 mg); lane 2, affinity-purified His6-HFR1 (0.5 mg).
the Southern corn rootworm (Diabrotica undecimpunctata; Czapala and Lang, 1990), thus suggesting the role of plant defense for JRLs. In continuation of our previous work on the molecular characterization of wheat Hfr-1 (Subramanyam et al., 2006), we investigated the function of HFR1 protein. In this study, we expressed His6-HFR1 in Escherichia coli and purified the active recombinant His6-HFR1 protein. With the purified protein, we demonstrate lectin activity and glycan specificity of His6-HFR1 and provide evidence of its role in defense against Hessian fly larvae. RESULTS Purification and Identification of Recombinant HFR1 (His6-HFR1)
Following optimization of induction procedures, the soluble recombinant His6-HFR1 protein was purified for activity assays, based on the N-terminal 63 His tag affinity to a Ni21-nitrilotriacetic acid (NTA) column. The purified His6-HFR1 resolved as a homogenous band with molecular mass of 40.5 kD under reducing conditions (Fig. 1), which is consistent with the calculated sum of molecular masses of the 63 His tag (3 kD) and HFR1 (37.5 kD) from its predicted amino acid sequence. The purified fraction was assessed by mass spectrometry after enzymatic digestion with trypsin. Purification of peptide fragments and analysis 1414
Lectins are carbohydrate-binding proteins that may have the ability to agglutinate erythrocytes. The agglutination activity of His6-HFR1 was assayed using erythrocytes from several mammalian species. His6-HFR1 had the ability to agglutinate human group A (Supplemental Fig. S2, A and B) and rabbit erythrocytes (data not shown), but failed to agglutinate sheep, bovine, and guinea pig erythrocytes (data not shown). His6-HFR1 Preferentially Binds to High-Man N-Glycans
To screen the glycan specificity of His6-HFR1, we used version 3.0 of the glycan microarray, containing 320 glycan targets, printed by the Consortium for Functional Glycomics (www.functionalglycomics.org). The microarrays were composed of natural and synthetic glycans modified to contain amino linkers that were covalently coupled through amide linkages to N-hydroxysuccinimide-activated glass slides (Blixt et al., 2004). Each glycan was represented six times on each microarray. The glycan binding of His6-HFR1 was detected after incubation with anti-HFR1 antibodies and a fluorescently tagged secondary antibody. Screening of the glycan microarray with recombinant His6-HFR1 at a relatively high concentration (200 mg/ mL) confirmed that the lectin bound predominantly to high-Man N-glycans (Supplemental Table S1). At this concentration of protein, strong and weak binding affinities could not be distinguished for the different glycans due to binding saturation. To determine the relative affinities and specificity of His6-HFR1 to the diverse set of Man-containing glycans on the array, we carried out our analyses with 2-fold serial dilutions from 7.5 to 0.47 mg/mL His6-HFR1. As the lectin concentration was decreased, the glycan signals decreased into the linear range of the detector and relative affinities became apparent. In Figure 2, we show results from the binding of 1.88 mg/mL His6-HFR1 to Man-containing oligosaccharides. At this concentration, the relative affinities to the glycans were revealed (Fig. 2A). Similar patterns were observed at lower concentrations (data not shown). The results clearly indicated that His6-HFR1 was a high-Man N-glycan-binding lectin that is specific for the terminal Mana1-6(Mana1-3)Man Plant Physiol. Vol. 147, 2008
Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
Figure 2. Binding of His6-HFR1 lectin to glycan microarray. A, At a concentration of 1.88 mg mL21, His6-HFR1 bound with highest affinity (quantified as relative fluorescence units 6 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 a3 to a6 linkages (encircled). The names of each glycan structure represented in A and B are as follows: (1) Mana1-2Mana1-3Mana-Sp9; (2) Mana16(Mana1-2Mana1-3)Mana1-6(Mana2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp12; (3) Mana1-3(Mana1-6)Mana-Sp9; (4) Mana1-6(Mana1-3)Mana1-6(Mana2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp12; (5) Mana1-6(Mana1-3)Mana1-6(Mana13)Manb1-4GlcNAcb1-4GlcNAcb-Sp12; (6) Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12; (7) Mana1-6Manb-Sp10; (8) Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb-Sp10. [See online article for color version of this figure.]
trisaccharide (glycan 3; circled in Fig. 2B) that is commonly found on the a1-6 arm of the trimannosyl core in high-Man-type oligosaccharides of glycoproteins. The contribution of the Mana1-6(Mana1-3)Man portion to binding by His6-HFR1 is supported by the lower affinity of His6-HFR1 for glycan 7 (lacking the a1-3 arm) and glycan 1 (lacking the a1-6 arm). No significant difference was detected in the affinities of His6HFR1 to glycans 3, 5, 6, and 8, suggesting that the lectin does not recognize the chitobiosyl (dimer of b-1,4linked GlcNAc units) core structure. Thus, the diGlcNAc of the N-glycans was not required for strong binding of His6-HFR1. In fact, the lectin bound well where the trimannosyl structure was extended on the a1-6 arm of the glycan structure (glycan 5), but addition of another Man residue on the a1-4 arm (glycan 4) or on the a1-3 arm of the trimannosyl structure (glycan 2) both decreased binding. All other Man-containing glycans on the array (Supplemental Table S1) were not bound by the lectin. Half-maximal binding of the lectin to the highest affinity glycans (data not shown) was in the ,1 mg/mL range. Plant Physiol. Vol. 147, 2008
Immunodetection of Endogenous HFR1 in Wheat
Polyclonal antibodies were generated against a synthetic 15-residue peptide of HFR1 (Supplemental Fig. S1) and affinity purified. The anti-HFR1 antibodies were used to detect expression of HFR1 in wheat plant homogenates in response to Hessian fly larval feeding. Protein gel blots of leaf extracts treated with anti-HFR1 antibodies that had been preadsorbed with His6-HFR1 yielded no bands. As a control, blots that were incubated with anti-HFR1 antibodies that had been preadsorbed with storage buffer yielded a strong band of the expected size (37.5 kD), identical to the mass predicted from the amino acid sequence, and no other bands were detected by the antibodies. These studies confirmed the specificity of anti-HFR1 antibodies to His6-HFR1 (data not shown). To detect endogenous HFR1 protein in wheat, gel blots of protein extracts made from the crown tissue of different seedling genotypes were incubated with the anti-HFR1 antibodies. Plants were harvested over a time course (1, 2, and 3 d after egg hatch) during both 1415
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compatible and incompatible interactions. HFR1 protein was undetectable in both the uninfested controls and in samples from wheat plants involved in two different compatible interactions (H9-Iris infested with vH9 larvae [Fig. 3A] and Newton infested with biotype L larvae [Fig. 3B]). However, HFR1 protein was clearly present in samples from resistant H9-Iris plants involved in incompatible interactions with biotype L larvae (Fig. 3). Preimmune serum was used as a negative control to demonstrate that nonspecific binding of endogenous rabbit antibodies to His6-HFR1 did not occur (data not shown). The recombinant His6-HFR1 protein was used as a positive control, with a strong band indicating high specificity of the affinity-purified antibodies to HFR1. Quantitative gel-blot analysis revealed that the physiological levels of HFR1 during incompatible interactions in H9-Iris at 2 d after hatch of avirulent biotype L Hessian fly larvae was 17 mg/g leaf sheath tissue (data not shown). Our gel-blot analysis of serial dilutions of the recombinant His6-HFR1 protein showed that less than 30 ng per lane of His6-HFR1 was undetectable. The resistant plant sample was estimated to have 50 ng or more HFR1 on the gel blot, whereas we could not detect HFR1 in the susceptible plant sample. Thus, we estimate that the amount of HFR1 in resistant plants was at least two times more than that in the susceptible plant.
detected in samples from virulent and avirulent larvae corresponded to ingested wheat HFR1 and its degradation products. Recombinant His6-HFR1 Is a Developmental Retardant with Antifeedant and Insecticidal Activities
To determine whether HFR1 had detrimental effects on insect growth and development, we carried out an insect feeding bioassay using the recombinant His6HFR1 protein. Since Hessian fly larvae are obligate parasites of the plant and cannot be cultured in vitro, we used another dipteran insect, Drosophila melanogaster (fruit fly), in a diet incorporation bioassay. Comparison of larval length at 5 d after egg hatch showed no significant differences between the larvae fed the control diet and the lowest concentration of 0.3 mg of His6-HFR1 per gram of diet (P 5 0.09), but did show a significant inverse correlation (P , 0.0001) with the concentration of His6-HFR1 in all other treatments (Fig. 5A). The developmental time and mortality of D. melanogaster larvae increased with higher concentrations of His6-HFR1 in the diet. At the lowest concentrations of
Immunodetection of HFR1 in Hessian Fly Larval Tissue Homogenate
First-instar Hessian fly larvae were collected over a time course (1–3 d after hatch) and were thoroughly washed to remove plant products on their external surfaces. Gel blots of protein extracts from larval homogenates were incubated with anti-HFR1 antibodies to detect the presence of HFR1 in the internal tissues of the larvae. Even though the concentration of HFR1 was lower in susceptible plants that are host to virulent larvae, protein blot analyses detected multiple prominent bands in extracts of the virulent larvae. The largest band was very faint and had a molecular mass of 37.5 kD, corresponding to the size of the intact HFR1 protein. The smaller, more abundant bands appeared to be proteolytic products of HFR1 (Fig. 4). Only two very faint low molecular mass bands were seen in lanes containing extracts from avirulent larvae (Fig. 4). The high levels of HFR1 in resistant plants (Fig. 3) and the corresponding absence of intact HFR1 protein in the avirulent larvae that reside on but do not grow on these plants suggested that the avirulent larvae feed minimally. The anti-HFR1 antibodies did not detect HFR1, its degradation products, or any other bands on blots containing extract from neonate larvae (data not shown) that were allowed to emerge in water and had never fed on wheat plants. This result indicated that the bands detected in extracts from larvae that had fed on plants were not of larval origin. In addition, database searches never identified insect genes or proteins with similarity to the HFR1 sequence. Thus, bands 1416
Figure 3. Immunodetection of endogenous HFR1 lectin in resistant and susceptible wheat plants. Total plant proteins (8.0 mg) 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 affinitypurified 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). His6HFR1 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. Plant Physiol. Vol. 147, 2008
Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
His6-HFR1 (0.3 and 1.5 mg g21), the times to pupation and to eclosion as adults were not significantly different (P . 0.1) from those of the controls (Fig. 5B). Intermediate concentrations of His6-HFR1 (3, 6, and 9 mg g21) significantly lengthened the time to pupation (P , 0.001) and increased mortality (Fig. 5B; Table I). The highest concentrations (12 and 15 mg g21) led to death prior to pupation (Fig. 5B; Table I). Adult flies emerged approximately 4.3 d after pupation on the control diet as well as on the diet containing 0.3, 1.5, and 3 mg of His6-HFR1 per gram of diet. However, His6-HFR1 concentrations of 6 and 9 mg g21 resulted in arrested development in the puparium and inability to eclose as adults. At the highest concentrations tested (12 and 15 mg g21), on days 2 through 11, we observed larvae crawling up the walls of their tubes, presumably to avoid contact with the diet or to search for an alternative food source. These larvae remained in the first instar, did not pupate, and appeared to die slowly of starvation. In contrast, larvae remained in the diet until just prior to pupation when fed lower concentrations of His6-HFR1 or the control diet (data not shown). Table I shows the proportion of D. melanogaster larvae that survived to pupation and to adult eclosion when feeding on progressively higher concentrations of His6HFR1. The percentage of larvae that reached pupation and eclosed as adults differed significantly (P , 0.01) from the control at a concentration of 3.0 mg of His6HFR1 per gram of diet. The concentration at which 50% of the larvae died of His6-HFR1 for the larval stage was calculated to be 6.55 6 0.15 mg g21 diet. Behavioral Differences between Hessian Fly Larvae on Resistant versus Susceptible Plants
We compared the behaviors of developing biotype L Hessian fly larvae during the period when the defense response was activated and wheat HFR1 lectin was being produced in resistant but not in susceptible plants. The observed behaviors are defined in detail in ‘‘Materials and Methods,’’ whereas the frequency and duration of these behaviors, on which statistical analyses are based, are summarized in Table II. During the first 6 to 12 h after egg hatch, both virulent and avirulent larvae crawled to the base of the wheat seedling among the leaf sheaths and exhibited identical searching behavior and body contractions (Supplemental Video S1); no significant differences were detected in the frequency of either the searching events or body contraction patterns (P $ 0.05). Neither avirulent nor virulent larvae appeared to be oriented with respect to veins of the leaf sheath through 12 h after egg hatch. During this period, defense mechanisms in resistant plants, including the Hfr-1 gene, are just being activated by the presence of avirulent larvae and low levels of Hfr-1 mRNA are detectable (Subramanyam et al., 2006). One day after egg hatch (24-h time point), when HFR1 protein is clearly detectable in resistant plants (Fig. 3), the avirulent and virulent larvae did not differ in size or shape (Fig. 6, A and B) but behavioral Plant Physiol. Vol. 147, 2008
differences were evident. During this time, the virulent larvae showed signs of settling (Table II), indicated by fewer searching events per minute compared with the avirulent larvae (P , 0.00001) and the initiation of gut contractions (P , 0.00001; Supplemental Video S2; Table II). The virulent larvae aligned themselves in rows within the grooves corresponding to veins of the leaf sheath (Fig. 6B). This sessile behavior suggested that the larvae had begun to feed. In contrast, the avirulent larvae by 24 h after hatch exhibited more searching events per minute than the virulent larvae (P , 0.00001) and produced body contractions associated with locomotion. These avirulent larvae were often positioned perpendicular to the leaf veins (Fig. 6, A and E). By 24 h after egg hatch, a time when HFR1 protein was detectable and defenses were fully established in resistant plants, the avirulent larvae displayed writhing and head-rearing behaviors that were never exhibited by the virulent larvae during any developmental stage. Larvae that were rearing their heads did not display the searching behavior. This writhing (Fig. 6E; Supplemental Video S3) and head rearing (Supplemental Fig. S3) lasted until 72 and 96 h after hatch, respectively, after which these behaviors ceased. The avirulent larvae continued to appear disoriented with respect to their positions relative to leaf veins at 3 d after hatch (Fig. 6C). All virulent larvae were sessile, with heads attached to feeding sites from 48 h after egg hatch until pupation (Fig. 6, D and F), whereas the avirulent larvae became sessile only after 96 h. At no time did the avirulent larvae exhibit visible gut contractions indicative of food ingestion. By 96 h after hatch, the virulent larvae had successfully established feeding sites and fed voraciously (Supplemental Video S4). With careful observation near the end of this video, one sees evidence of saliva being expelled onto the leaf surface and then ingested. By 192 h after hatch, the avirulent larvae appeared flattened and desiccated, which suggested that they were dead (Supplemental Fig. S4), whereas the virulent larvae, now in their second instar, had greatly increased in size. Size Differences between Virulent and Avirulent Hessian Fly Larvae
During the first 24 h after egg hatch, first-instar virulent and avirulent Hessian fly larvae did not differ in length (P 5 0.37; Fig. 7). However, the first-instar virulent larvae showed a significant increase in length compared with the avirulent larvae (P 5 0.003) by 48 h after egg hatch and 72 h (Fig. 6, C and D). By 120 h after hatch, the virulent larvae had more than doubled in length compared with the avirulent larvae (P , 0.00001). The virulent larvae were in the second-instar stage by 192 h after hatch and had grown to over 2 mm in length. In contrast, at 192 h after hatch, the avirulent larvae were dead. They had not developed past the first-instar stage and had not increased in length (P 5 0.58, 0.41 6 0.02 mm) since hatching from the egg. 1417
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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 mg) 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.
plants, resulting in the formation of nutritive tissue (Harris et al., 2006). This tissue provides the virulent larvae with a diet rich in soluble proteins and sugars that sustain their growth and development until pupation. In contrast to the rapid initiation of feeding by the virulent larvae, the avirulent larvae exhibited prolonged activity (Table II), as a gene-for-gene recognition event triggers increased mRNA levels of defense genes (Sardesai et al., 2005b) along with genes encoding lectins, such as Hfr-1 (Subramanyam et al., 2006) and Hfr-3 (Giovanini et al., 2007). The specific elicitation of these wheat lectins, which previously have not been linked to defense, suggests the targeted involvement of this class of proteins during resistance to Hessian fly. In response to these changes in the resistant wheat plant, avirulent larvae exhibit a number of stress responses. Avirulent larvae have a nearly 8-fold increase in levels of mRNA encoding d-glutathione S-transferase, which persists for several days and is
DISCUSSION
Developmental outcomes diverge quickly in compatible and incompatible interactions for both the wheat plants and the Hessian fly larvae. Initially, virulent and avirulent larvae exhibit identical behaviors during the 12 h following egg hatch (Table II) as they migrate to the base of the plant searching for a permanent feeding site. During the migration, larvae exhibit searching behavior (Table II), puncture leaf sheath epidermal cells with their minute mandibles (0.5 mm long 3 0.1 mm diameter; Harris et al., 2006), and apply salivary secretions containing elicitors of plant responses. However, the plant responses diverge within about 6 h, with an induction of the first stages of susceptibility. During day 1 of compatible interactions, cell walls at the attack site of susceptible plants begin to thin and develop 4- to 10-mm ruptures (Harris et al., 2006) through which cellular contents flow. Virulent larvae rapidly became sessile and fed on the contents of these lysing cells (Table II; Harris et al., 2006) days before the nutritive tissue that will sustain larval development becomes well established. Two days after egg hatch, virulent larvae were noticeably larger than avirulent larvae (Figs. 6 and 7). Over the next few days, transcript levels from wheat genes involved in sugar transport (Liu et al., 2007; C.E. Williams, S. Subramanyam, K.D. Saltzmann, J.A. Nemacheck, and C. Zheng, unpublished data), amino acid synthesis (K.D. Saltzmann and C.E. Williams, unpublished data), HFR2 (a poreforming protein; Puthoff et al., 2005), and genes involved in polyamine biosynthesis (S. Subramanyam and C.E. Williams, unpublished data) increased in susceptible plants. These changes in wheat mRNA levels occur at the same time that physical changes in leaf sheath epidermal cells become evident in susceptible 1418
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 6 0.02 mm. Concentrations of His6-HFR1 at which larval lengths were significantly different from the control (0 mg g21) 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 mg g21 His6HFR1. Larvae died at 12 and 8 d after egg hatch without pupating at concentrations of 12 and 15 mg g21 His6-HFR1, respectively. Concentrations of His6-HFR1 at which pupation and eclosion were significantly different from the control (0 mg g21) 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. Plant Physiol. Vol. 147, 2008
Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
Table I. Proportion of D. melanogaster larvae surviving to pupation and eclosion as adults when fed His6-HFR1 Pupation and eclosion were scored until 15 d after egg hatch. Values represent percentage of larvae 6 SE for 30 larvae from three independent replicates. One-way ANOVA using SAS was carried out for calculation of significance. The concentrations of His6-HFR1 at which the percentage pupation and eclosion were significantly different from the control (0 mg g21) are indicated by asterisks. His6-HFR1 mg g
Larvae Reaching Pupation
Larvae Reaching Eclosion
21
0 0.3 1.5 3 6 9 12 15
96.7 83.3 76.7 70.0 63.3 6.6 0 0
6 6 6 6 6 6 6 6
3.3 3.3 8.8 0.0* 6.6* 3.3* 0.0 0.0
90.0 80.0 53.3 36.7 0 0 0 0
6 6 6 6 6 6 6 6
5.8 0.0 8.8 6.6* 0.0 0.0 0.0 0.0
presumed to function in an attempt to detoxify wheat allelochemicals (Mittapalli et al., 2007b). In addition, avirulent larvae experience oxidative stress, manifested as elevated levels of superoxide dismutase mRNA (Giovanini et al., 2006) along with phospholipid glutathione peroxidase mRNA (Mittapalli et al., 2007a). By 24 h after egg hatch, avirulent larvae exhibited two unusual behaviors that were never seen in virulent larvae: writhing (Supplemental Video S3) and head rearing (Table II; Supplemental Fig. S3). These behaviors may be another response to stresses from plant chemicals as well as to the stress of starvation. Avirulent larvae never settled or exhibited sustained feeding and did not increase in size after hatching from the egg. They appeared to die slowly of starvation (death occurs at approximately day 5; Gallun, 1977; Supplemental Fig. S4) rather than being killed quickly by the consumption of highly toxic plant substances. Although the avirulent larvae may be able to feed on individual cells that they puncture with their mandibles, plant defenses inhibit them from ingesting life-sustaining quantities of nutrients. Experiments examining the feeding habits of Hessian fly larvae on 32 P-labeled resistant and susceptible wheat seedlings showed that virulent Hessian fly larvae had ingested six times more radiation than avirulent larvae at 2 d after hatch (Gallun and Langston, 1963). Avirulent larvae stopped uptake of 32P by 3 d, whereas virulent larvae continued uptake for 14 d. Feeding or lack of feeding can result in physical changes within the insect midgut. The peritrophic matrix of the lepidopteran midgut is synthesized in response to feeding (Spence, 1991). A recent study shows that the mRNA level of virulent Hessian fly larval peritrophin A, a component of the peritrophic matrix, was twice that of avirulent larvae at 4 d after egg hatch and continued to rise through the second instar, corresponding with increasing consumption of nutrients (Mittapalli et al., 2007c). The authors suggest that cessation of feeding by avirulent larvae results in a lack of challenge to the Plant Physiol. Vol. 147, 2008
peritrophic matrix and a decreased need for peritrophin A. Thus, wheat defense against Hessian fly is a combination of activating chemical and physical feeding deterrents that lead to starvation and blocking the induction of plant processes that are beneficial to the insect. The behaviors of D. melanogaster larvae in the His6HFR1 feeding assay lent support to the assertion that HFR1 is one of the chemical feeding deterrents produced by resistant wheat plants. At low concentrations of His6-HFR1 (0.3 and 1.5 mg g21 diet medium; Fig. 5), the growth rate of D. melanogaster larvae was not affected, just as Hessian fly larvae develop normally on susceptible plants containing low constitutive levels of HFR1. But on an artificial diet containing higher concentrations of His6-HFR1, D. melanogaster larval development slowed. The highest concentrations of His6-HFR1 (12 and 15 mg g21 diet) were similar to the estimated concentration of HFR1 in resistant plants (17 mg g21); this is believed to be an average of the high levels of HFR1 released by the few wheat cells that surround each site of Hessian fly larval interaction and the constitutive low level that is maintained in the remaining cells at the base of the developing leaf (Subramanyam et al., 2006). At these highest concentrations of His6-HFR1, D. melanogaster larvae crawled out of the medium onto the wall of the vial, where they did not die immediately but resided without feeding for 8 to 12 d before death (Fig. 5). This behavior of D. melanogaster larvae appeared analogous to that of avirulent Hessian fly larvae, which continued to crawl in a disoriented searching manner for about 3 d (Table II) with minimal feeding, unable to move to a different plant or establish a feeding site, and eventually died. The antinutritional properties of several other plant lectins have been well documented against various homopteran, lepidopteran, dipteran, and coleopteran insects (Vasconcelos and Oliveira, 2004). Three modes of action of these dietary lectins have been identified: (1) binding of the lectins to the peritrophic matrix of the midgut and disrupting nutrient absorption (Harper et al., 1998); (2) binding to glycoproteins on epithelial cells of the midgut and disrupting tissue integrity (Powell et al., 1998; Sauvion et al., 2004); and (3) binding of lectins to carbohydrate moieties of the sensory receptors of the insect mouth parts, disrupting membrane integrity and interfering in the ability of the insects to detect food (Murdock and Shade, 2002). All three of these mechanisms result in decreased ability of the insects to ingest or absorb nutrients, leading to delayed development and premature death. But the third mechanism appears to be most consistent with our knowledge of Hessian fly feeding. The Hessian fly larval midgut appears to not be the target for these lectins and plant defenses, since during dual infestations, avirulent larvae that hatch first and induce an incompatible interaction are later able to feed and survive once nutritive tissue is established during the subsequent compatible interaction induced by virulent larvae nearby on the same leaf (Grover et al., 1989; C.E. 1419
Subramanyam et al.
Table II. Behavioral patterns of avirulent and virulent Hessian fly larvae on wheat plants Larval behaviors were scored for 60 s immediately following removal of the outer leaf during a time course of 6, 12, 24, 48, 72, 96, 120, and 192 h after egg hatch. Mean scores 6 SE represent average number of times each larva exhibited a behavior during the 60-s observation period. Hours after a N Hatch
Avirulent 6 12 24 48
Searchingb
Body Contractionsc
Gut Contractionsd
biotype L larvae on resistant H9-Iris seedlings 16 13.1 6 1.0 (16)h 11.6 6 1.3 (14) 0 18 12.2 6 0.5 (15) 16 6 0.4 (18) 0 30 14 6 0.8 (11) 13.6 6 0.7 (11) 0 31 21.2 6 0.5 (15) 9.0 6 0.0 (12) 0
6 6 6 6
0.0 0.0 0.0 0.0
(0) (0) (0) (0)
Writhinge
0 0 11.3 10.5
(0) (0) (9) (10)
0 3 4 0
0 0 15 16
11.7 6 0.2 (13) 0 6 0.0 (0) 0 6 0.0 (0)
0 32 21
15 12 0
6 6 6 6
0.0 0.0 0.4 0.5
No. of No. of Larvae Sessile Rearing Larvaef Headg
72 96 120
36 14.4 6 0.9 (20) 21.7 6 0.4 (21) 44 0 6 0.0 (0) 0 6 0.0 (0) 21 0 6 0.0 (0) 0 6 0.0 (0)
0 6 0.0 (0) 0 6 0.0 (0) 0 6 0.0 (0)
192
17
0 6 0.0 (0)
0 6 0.0 (0)
17
0
Virulent biotype L larvae on susceptible Newton seedlings 6 20 14.1 6 0.5 (20) 11.4 6 1.0 (20) 0 6 0.0 (0) 12 17 13.2 6 0.6 (14) 11.0 6 0.4 (17) 0 6 0.0 (0)
0 6 0.0 (0) 0 6 0.0 (0)
0 3
0 0
3.0 6 0 (18)
0 6 0.0 (0)
12
0
0 6 0.0 (0)
0 6 0.0 (0)
24
22
3.8 6 0.3 (18)
3.0 6 0.0 (20)
48
32
0 6 0.0 (0)
0 6 0.0 (0)
6.1 6 0.6 (29)
0 6 0.0 (0)
32
0
72 96 120
19 31 20
0 6 0.0 (0) 0 6 0.0 (0) 0 6 0.0 (0)
0 6 0.0 (0) 0 6 0.0 (0) 0 6 0.0 (0)
5.4 6 0.9 (17) 13.7 6 0.5 (31) 12.45 6 0.8 (20)
0 6 0.0 (0) 0 6 0.0 (0) 0 6 0.0 (0)
19 31 20
0 0 0
192
21
0 6 0.0 (0)
0 6 0.0 (0)
27.1 6 1.4 (21)
0 6 0.0 (0)
21
0
Comments
Indistinguishable from virulent Indistinguishable from virulent More active than virulent Many larvae exhibited both searching and writhing at the same time Increased rate of body contractions No body movement Alive but low activity, showing minute convulsions at the anterior end Larvae were dead, showed no movement, and appeared desiccated and flattened Indistinguishable from avirulent Indistinguishable from avirulent; some larvae showed body contractions but were sessile, as they did not display forward locomotion Some of the sessile larvae had no movement, others had fluttering body contractions but did not crawl All larvae were sessile at their permanent feeding sites – – Gut contraction with contents moving unidirectionally from the posterior to the anterior end of the larvae Gut contents showing bidirectional movement
a b N: Total number of larvae scored at the designated time point. Searching: active side-to-side movement, head low near the leaf surface c d probing the plant tissue. Body contractions: larva contracted its entire body, followed by a slow forward locomotion. Gut contractions: gut e contents moved either posterior to anterior, anterior to posterior, or both directions. Writhing: twisted or contorted motion, arching of the body f Sessile: larva attached at one location with entire ventral surface in contact with with anterior and posterior ends in contact with the leaf surface. g h the plant. Head rearing: larval head raised up away from the plant tissue with no detectable movement of the body. Numbers in parentheses indicate the number of larvae showing a specified behavior.
Williams and S.D. Baluch, unpublished data). Thus, the midgut is not severely damaged by the defenses of the wheat plant. Our immunodetection results also suggested that the midgut is not the target, since only minimal quantities of the HFR1 lectin and plant nutrients appeared to be ingested by avirulent Hessian fly larvae (Fig. 4; Table II). This observation implies that a component of deterrence occurs prior to nutrient ingestion, such as through blocking of sensory receptors in the larval mouth parts and interfering with the detection of food. Genetically, the virulent and avirulent Hessian fly larvae in our experiments were identical (biotype L), but their transcriptomes (Mittapalli et al., 2007a, 2007b, 2007c) and behaviors diverged rapidly due to changes 1420
induced in them by different cues from their resistant and susceptible host plants. One important difference was that although the mRNA and protein levels of HFR1 were higher in resistant plants (Subramanyam et al., 2006; Fig. 3), avirulent larvae were inhibited from feeding, so HFR1 and its proteolytic products were more abundant in virulent larvae that reside on susceptible plants. This ability of virulent larvae to accumulate HFR1 degradation products highlights the induced superior digestive abilities of virulent larvae that are receiving cues from their susceptible host plants. Levels of the digestive enzymes trypsin- and chymotrypsin-like Ser proteases are low in neonate Hessian fly larvae that have not fed on plants. But in the Plant Physiol. Vol. 147, 2008
Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
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-dayold 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.
gut of virulent larvae, the levels of these digestive enzymes increase over time (Shukle et al., 1985; Zhu et al., 2005), just as in the gut of another dipteran, the tsetse fly (Glossina morsitans), in which Ser proteases are induced by feeding (Yan et al., 2001). An induction of digestive enzymes may be responsible for the differing banding patterns of HFR1 proteolytic products observed in immunodetection experiments with virulent and avirulent larvae. Plant cues may also be responsible for stimulating larval feeding behavior. Extraintestinal digestion is a feeding behavior common among the Cecidomyiidae larvae, which includes Hessian fly (Mamaev, 1968; Roskam, 1992), when they are actively engaged in nutrient consumption. During this feeding behavior, salivary secretions and midgut enzymes are regurgitated onto the leaf sheath surface to mix with plant cell lysate (seen in Supplemental Video S4, which shows feeding of second-instar Hessian fly larva). Nutrients Plant Physiol. Vol. 147, 2008
become soluble and proteins degraded before the larva consumes the liquid diet. Extraintestinal digestion may protect the gustatory receptors and midgut of virulent larvae from the deleterious effects of plant products such as the HFR1 lectin, which may not be able to dimerize or bind to its target if degraded. HFR1 is a chimeric protein with an N-terminal dirigent domain and a C-terminal JRL domain (Williams et al., 2002), suggesting that the protein may be bifunctional. Dirigent domains are less well understood than are lectin domains, but they are thought to be involved in controlling stereoselective coupling of monolignols in the formation of lignans (Davin and Lewis, 2000) and are implicated in defense (Culley et al., 1995). Although true dirigent proteins possess a signal sequence associated with the secretory pathway (Gang et al., 1999), HFR1 does not have a signal sequence and it is possible that the dirigent domain in a chimeric protein is not functional. In our experiments, we used the entire HFR1 amino acid sequence to demonstrate feedingdeterrent properties of His6-HFR1 in D. melanogaster. It would be interesting to evaluate in the future whether the N terminus or the C terminus or both is responsible for the anti-insect activity. However, in a related chimeric protein in maize, b-glucosidase-aggregating factor, the JRL domain (C terminus) and not the dirigent domain (N terminus) is responsible for lectin and b-glucosidase-binding and aggregating activities (Kittur et al., 2007). The glycan array data (Fig. 2) demonstrated that His6-HFR1, like many of the so-called Man-specific lectins, is poorly reactive toward the Man monosaccharide and reacts exclusively with oligosaccharides or N-glycans. His6-HFR1 has highly specific lectin activity for binding the terminal Mana1-6(Mana1-3)Man trisaccharide of the a1-6 arm of the trimannosyl core in high-Man-type oligosaccharides of glycoproteins. This
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. Oneway ANOVA was carried out using SAS, and differences were considered significant at P , 0.05. 1421
Subramanyam et al.
structure is a predominant form of the N-linked glycosylated proteins found in dipteran relatives of the Hessian fly. For example, high-Man glycoproteins are integral to the midgut peritrophic matrix of mosquitoes (Anopheles gambiae and Aedes aegypti; Moskalyk et al., 1996). In addition, high-Man glycoproteins are components of salivary gland extracts from sandflies (Phlebotomus and Lutzomyia species; Volf et al., 2000). If high-Man glycoproteins are also present in Hessian fly larval salivary glands or gustatory receptors, then it is conceivable that high concentrations of His6-HFR1 may act as an antifeedant by binding to these structures; thus, they would not be ingested. His6-HFR1 does not require the core di-GlcNAc of the N-glycans for binding (Supplemental Table S1), as required by some other high-Man N-glycan-binding lectins such as tomato (Solanum lycopersicum) lectin (Lycopersicon esculentum agglutinin; Oguri, 2005) and tobacco (Nicotiana tabacum) lectin (Nicotiana tabacum agglutinin; Chen et al., 2002; Lannoo et al., 2006). The positive hemagglutination activity suggested that the presence of the His tag did not interfere with His6-HFR1 function (Supplemental Fig. S2). In this study, we have shown that the Hessian fly response protein HFR1 is a functional high-Man N-glycanbinding lectin. The rapid accumulation of this protein in the resistant plants indicated an early defense response to Hessian fly larval attack and correlated well with the behavior of the avirulent larvae on the wheat plants. HFR1 protein accumulation is one of several physiological and biochemical changes that contribute to the resistance response of wheat against Hessian fly larval attack. The predominant mode of action of HFR1 seems to be by contributing to conditions that starve the avirulent larvae, leading to antibiosis. This functionality of HFR1 opens up potential applications in engineering HFR1-expressing transgenic plant lines that will confer resistance against this and other devastating insect pests. MATERIALS AND METHODS Electrophoresis HFR1 protein was analyzed by SDS-PAGE as described by Laemmli (1970), using 10% or 4% to 20% Tris-HCl Polyacrylamide Ready Gels (Bio-Rad). Prior to electrophoresis, the protein samples were prepared by boiling for 5 min in 23 sample loading buffer (200 mM Tris-HCl, pH 6.8, 25% glycerol, 0.4% SDS, 0.02% bromphenol blue, and 200 mM dithiothreitol). Samples were resolved on SDS-PAGE gels in a Mini-Protean 3 apparatus (Bio-Rad) at 100 V for 1 h with 13 SDS running buffer (25 mM Tris-HCl, 192 mM Gly, and 0.1% SDS, pH 8.3). Total proteins were detected on gels by Coomassie Brilliant Blue (Bio-Rad), and the presence of His6-HFR1 protein was confirmed by staining the gels with Invision His-tag In-gel Stain (Invitrogen).
dilution used varied for different experiments from 1:1,000 to 1:5,000) custom synthesized in rabbit by Sigma in SuperBlock containing 0.05% (v/v) Tween 20 for 1 h with gentle agitation. Following three 10-min washes in TBST (20 mM Tris-HCl, 500 mM NaCl, pH 7.4, containing 0.05% Tween 20), the membranes were incubated for 1 h at room temperature in SuperBlock containing 1:30,000 dilution of goat anti-rabbit horseradish peroxidase (HRP)conjugated IgG (Pierce). After three washes with TBST, the membranes were incubated for 5 min with the SuperSignal West Pico Chemiluminescent substrate (Pierce), and luminescence was exposed to BioMax MR Film (Kodak).
Construction of a Plasmid (pET-HFR1) for Overexpression of Recombinant Protein To construct the expression vector, first the open reading frame of Hfr-1 (GenBank accession no. AF483596; Williams et al., 2002) was amplified by PCR using the following primers: Forward, 5#-CACCTCGCCTCAGTCTTTC-3#, and Reverse, 5#-CTAGGTTGAAACGGGTGAACG-3#. PCR was carried out in a 50-mL volume with 200 mM of each dNTP, 25 mM of each primer, and 2.5 units of Native Pfu DNA polymerase with 13 Pfu PCR buffer (Stratagene) using 20 ng of the Hfr-1 cDNA clone (UPW1Hfr1a; Williams et al., 2002) as template. The PCR conditions were as follows: 25 cycles of 45 s at 94°C, 45 s at 62°C, and 2 min at 72°C, followed by a final extension at 72°C for 10 min. The amplified product was directionally cloned into pET200/D-TOPO (Invitrogen) expression vector, in frame with a 63 N-terminal His tag. The resulting plasmid was designated pET-HFR1. The pET-HFR1 plasmid was sequenced (ABI PRISM DYEnamic ET Terminator Cycle Sequence Kit; Amersham) on an ABI PRISM 3700 sequencer (Applied Biosystems) to verify that the insert was in frame and in the proper orientation.
Purification of Recombinant His6-HFR1 Protein by Affinity Chromatography The expression plasmid pET-HFR1 was transformed into Escherichia coli BL21 (DE3) according to the manufacturer’s protocol (Invitrogen). Transformed cells were grown in Luria-Bertani medium (Sambrook et al., 1989) containing 50 mg/mL kanamycin. Protein expression was induced by the addition of 0.2 mM isopropyl-b-D-thiogalactopyranoside for 16 h at 20°C. Cells were harvested by centrifugation at 6,000g for 10 min. The pellet was suspended in precooled lysis buffer (50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) containing 10 mM imidazole (Sigma), 100 mM protease inhibitor cocktail for use in the purification of His-tagged proteins (Sigma), and 200 mM phenylmethylsulfonyl fluoride (Sigma) and subjected to lysis by a French pressure cell press (SIM AMINCO Spectronic Instruments) at 1,500 psi. Residual cells and debris were discarded after centrifugation (15,000g, 30 min, 4°C), and the recombinant protein from the supernatant lysate was allowed to bind via the His tag to 2 mL of Ni21-NTA resin (Invitrogen) at 4°C for 3 h. The bound resin-supernatant mix was packed in a purification column (Invitrogen). The column was then washed twice with 4 column volumes of lysis buffer containing 20 mM imidazole, twice with 4 column volumes of lysis buffer containing 60 mM imidazole, and twice with 4 column volumes containing 100 mM imidazole. The recombinant protein was finally eluted with lysis buffer containing 250 mM imidazole in 12 aliquots of 1 mL each and designated His6-HFR1 protein. The elutions containing the recombinant His6HFR1 protein were pooled together and dialyzed overnight at 4°C against storage buffer (50 mM Tris-HCl, pH 8.0, and 1 mM CaCl2) using a Slide-A-Lyzer (10,000 molecular weight cutoff) dialysis cassette (Pierce). His6-HFR1 protein was further concentrated using Centriprep (10,000 molecular weight cutoff) centrifugal filter tubes (Millipore) following the manufacturer’s protocol. All purification procedures were carried out at 4°C. Protein purification was verified by 10% SDS-PAGE (as described above) and confirmed by Invision His-tag In-gel Stain (Invitrogen) or by the protein gel-blot method described above using anti-His antibodies (Pierce) at 1:5,000 dilution in SuperBlock (Pierce).
Protein Gel Blot and Immunodetection Following electrophoresis, protein samples were electroblotted by standard procedures (Sambrook et al., 1989) onto a nitrocellulose membrane with a pore size of 0.45 mm (Bio-Rad) using a Protean II Mini Trans-Blot cell apparatus (Bio-Rad). Membranes were treated in blocking buffer (SuperBlock; Pierce) containing 0.05% Tween 20 at room temperature for 1 h. The membranes were then hybridized with the anti-HFR1 peptide antibodies (the
1422
Quantification of in Vitro-Expressed and Total Tissue-Extracted Protein The extracted protein was quantified using either Lowry’s method (Lowry et al., 1951) with the RCDC Protein Assay Kit (Bio-Rad) or by Bradford’s method (Bradford, 1976) using the Bradford Method Protein Assay Kit
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Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
(Amresco) with known concentrations of bovine serum albumin as standards according to the manufacturers’ protocols.
Peptide Sequencing and Analysis The affinity-purified recombinant His6-HFR1 was resolved by SDS-PAGE on a 10% gel (as described above). A discrete band corresponding to a molecular mass of 40.5 kD was excised from the Coomassie Blue-stained gel. Mass spectrometry of the excised band was carried out using MALDI MS/ MS-MS on a 4700 Proteomics Analyzer (Applied Biosystems). Analysis of the His6-HFR1 protein sequence from MALDI MS/MS-MS spectra was achieved using Global Proteome Server Explorer software (Applied Biosystems).
Preparation of HFR1-Specific Antibody Design, synthesis of peptide, and antibody production were done by Sigma using standardized procedures. Anti-HFR1 polyclonal antibodies were raised in New Zealand White rabbits against a 15-amino acid customsynthesized peptide from a region of the HFR1 lectin domain representing amino acids 206 to 220 (GELLDIPSTPQRLER) and affinity purified commercially by Sigma. To confirm the specificity of anti-HFR1 polyclonal antibodies to HFR1 protein, adsorption studies were carried out using recombinant His6HFR1 protein according to the protocol described by Caliskan and Cuming (2000) with slight modifications. Two nitrocellulose membrane strips of 1 cm 3 1 cm were taken in small plastic weighing boats. One of the nitrocellulose membranes was incubated with 10 mL of His6-HFR1 protein (3 mg) in storage buffer and the other with only 10 mL of storage buffer for 1 h. The membranes were then air dried. Once the membranes were completely dry, the membranes were blocked for 2 h with 5 mL of SuperBlock. Subsequently, the membranes were washed with TBST three times for 10 min each, and 50 mL of anti-HFR1 antibodies was added to both membranes at a dilution of 1:100 in SuperBlock and incubated for 2 h. The liquid antisera from both membranes (incubated with protein and incubated with buffer) were collected and labeled as adsorbed and control anti-HFR1 antibodies, respectively. Protein gel blots were made (as described above), and recombinant His6-HFR1 was detected with the adsorbed and control primary anti-HFR1 antibodies at a dilution of 1:500 in SuperBlock, followed by hybridization with a 1:30,000 dilution of goat anti-rabbit HRP-conjugated IgG (Pierce) in SuperBlock. His6-HFR1 protein was detected by chemiluminescence according to the protocol described above.
Agglutination Assays The ability of the His6-HFR1 recombinant protein to recognize cell surface glycoproteins was assessed using a hemagglutination assay. Erythrocytes from rabbit, bovine, sheep, and guinea pig (Hemostat Labs) plus human group A erythrocytes were collected, washed three times with buffer (50 mM Tris-HCl, pH 8.0, and 1 mM CaCl2), and suspended at a final concentration of 2% (v/v) in the same buffer. Agglutination of the erythrocytes as a measure of lectin activity was carried out on a glass slide in a final volume of 50 mL containing 25 mL of 2% erythrocyte suspension and 25 mL of purified recombinant His6-HFR1 protein and incubated at room temperature for 30 min. Hemagglutination was observed with a compound light microscope at a magnification of 203. Erythrocytes were incubated with the buffer only as the negative control.
Glycan Microarray Binding Affinity of His6-HFR1 The glycan microarray was printed by the Consortium of Functional Glycomics as described previously (Blixt et al., 2004). Version 3.0 of the array (http://www.functionalglycomics.org/static/consortium/resources/ resourcecoreh8.shtml), containing 320 glycans, each replicated six times on the array at different places, was used to determine the specificity and relative affinity of His6-HFR1 for oligosaccharides. His6-HFR1 recombinant protein was diluted in binding buffer (phosphate-buffered saline [PBS; Fisher Scientific] containing 1% bovine serum albumin and 0.05% Tween 20) to appropriate concentrations (200 mg/mL for glycan affinity screening, and dilutions of 7.5, 3.75, 1.88, 0.94, and 0.47 mg/mL for relative specificity assays). Aliquots (100 mL) of the His6-HFR1 dilutions were applied to separate array slides and incubated under coverslips for 60 min in a humidified chamber at 25°C. After incubation, the coverslips were gently removed in binding buffer and washed by dipping the slides four times in the same buffer. To detect His6-HFR1
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bound to the array, a monospecific rabbit anti-HFR1 antibody (IgG; described earlier) was diluted to 5 mg/mL in binding buffer, and aliquots (100 mL) were applied to the array slides and incubated as described above for the His6HFR1 incubation. After incubation with the antibodies, the slides were processed and washed as before, and the immune complexes were detected by applying 100 mL of a 5 mg/mL solution of Alexa488-labeled goat anti-rabbit IgG (Molecular Probes) in binding buffer to the microarray slide under a coverslip as described above. After incubation for 1 h, the coverslips were removed in a solution of PBS and gently washed by dipping the slides four times in successive washes of PBS buffer containing 0.05% Tween 20, PBS, and deionized water. After the deionized water wash, the slides were centrifuged for approximately 15 s to dry and immediately scanned in a Perkin-Elmer ProScanArray MicroArray Scanner using an excitation wavelength of 488 nm. ImaGene software (BioDiscovery) was used to quantify fluorescence. The data are reported as average relative fluorescence units from four of the six replicates (after removal of the highest and lowest values) for each glycan represented on the array.
Immunodetection of HFR1 Protein in Wheat Crown Tissue Homogenate Two experimental designs were used. For design 1 (fly genotype held constant), two nearly isogenic wheat (Triticum aestivum) lines, resistant H9-Iris (containing the H9 Hessian fly resistance gene) and susceptible Newton (no resistance gene), were infested with biotype L Hessian flies (avirulent to the H9-Iris wheat but virulent on Newton wheat), yielding an incompatible and a compatible interaction, respectively. For design 2 (wheat genotype held constant), one wheat genotype, H9-Iris, was infested with either vH9 Hessian flies (virulent to the H9 resistance gene) or biotype L Hessian flies, yielding compatible and incompatible interactions, respectively. Biotype L and vH9 flies were maintained as purified laboratory stocks in a 4°C cold room at the U.S. Department of Agriculture-Agricultural Research Service Crop Production and Pest Control Research Unit at Purdue University as described by Sosa and Gallun (1973). The wheat seedlings were grown and infested, and the crown tissue (the first 1 cm of tissue above the root junction) was harvested, as described by Subramanyam et al. (2006). Crown tissue harvested from these wheat seedlings was immersed in deionized water in a petri dish and carefully dissected to expose the larvae. Gentle agitation of seedlings in the water caused the larvae to be washed out to collect the plant tissue without the larvae. The plant tissue was collected, over a time course of 1, 2, and 3 d after larval hatch, in liquid nitrogen and kept frozen at 280°C. Total plant protein was extracted from 200 mg of frozen ground tissue using the Plant Total Protein Extraction Kit (Sigma) following the manufacturer’s instructions. For protein gel-blot analyses, equal amounts of plant protein samples (8.0 mg), along with 60 ng of His6-HFR1 as a positive control, were resolved by SDS-PAGE on a 4% to 20% gel and electroblotted onto a nitrocellulose membrane (as described above). Native HFR1 and recombinant His6-HFR1 were detected with anti-HFR1 antibodies at a dilution of 1:5,000 with SuperBlock, and HFR1-specific bands were detected as described previously. To detect the physiological levels of HFR1 in leaf sheath tissue, resistant wheat plants (line H9-Iris) were infested with biotype L Hessian flies as described by Subramanyam et al. (2006) to a density of 15 to 30 larvae per plant. On day 2 after egg hatch, plants were carefully dissected and the bottom 2 cm of leaf sheath 2 (on which the Hessian fly larvae were feeding) was collected in liquid nitrogen. Total plant protein was extracted from the leaf sheath tissue as described above, and gel-blot analysis was carried out by SDSPAGE on a 4% to 20% gel. Known concentrations of His6-HFR1 in a serial dilution (600, 300, 150, 60, and 30 ng) were detected with the anti-HFR1 antibodies and used to generate a standard curve. This standard curve was used for the quantification of HFR1 in 3 mg of leaf sheath from the resistant plant.
Immunodetection of HFR1 Protein in Larval Hessian Fly Homogenate One to 3 d after egg hatch, biotype L-infested wheat plants of H9-Iris (resistant) and Newton (susceptible) were immersed in deionized water in a petri dish and gently agitated to remove the larvae. Water containing the larvae was pipetted into 1.5-mL microcentrifuge tubes, and the larvae were allowed to settle to the bottom of the tubes for several minutes. The larvae were given several washes with 500 mL of deionized water inside the tubes.
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Water from the tubes was carefully removed and replaced with 200 mL of protein extraction buffer (0.125 M Tris-HCl, pH 6.8), and the larvae were crushed thoroughly in the extraction buffer with a sterile plastic pestle. The samples were then centrifuged at 10,000g for 15 min at 4°C to remove the debris. The supernatant was transferred into a fresh microcentrifuge tube. Protein concentration was estimated for each sample using the Bradford assay (described above). The samples were mixed with an equal volume of 23 Laemmli Sample Buffer (Bio-Rad) containing 5% (v/v) b-mercaptoethanol. Proteins were resolved by SDS-PAGE on a 4% to 20% gel and electroblotted onto a nitrocellulose membrane. HFR1 was detected with the anti-HFR1 antibodies at a dilution of 1:1,000 in SuperBlock, and bands were detected as described previously. To confirm that the anti-HFR1 antibodies did not bind to bands of insect origin on western blots, neonate larvae that had never fed on plants were collected as follows. Newton (susceptible) wheat plants were infested with biotype L as described earlier. Two days after infestation, the leaf blades containing the eggs were cut and placed in 1.5-mL microcentrifuge tubes containing deionized water. The larvae were allowed to hatch and crawl down into the water. Without disturbing the larvae, which had settled to the bottom of the tube, the water from the 1.5-mL microcentrifuge tube was removed. The larvae were given several washes with 500 mL of deionized water inside the tubes, and protein was extracted as described above. The proteins were resolved by SDS-PAGE, electroblotted onto a nitrocellulose membrane, and subjected to detection with the anti-HFR1 antibodies as described previously.
Drosophila melanogaster Feeding Assay Because Hessian fly larvae are obligate parasites and cannot be cultured on an artificial diet, the insecticidal activity of His6-HFR1 protein was assayed by monitoring its effect on the development of Drosophila melanogaster larvae. D. melanogaster and Hessian flies are both in the order Diptera. D. melanogaster w1118 female flies used in this study were allowed to lay eggs for a period of 3 h onto an embryo-collection grape-agar medium (Genesee Scientific) supplemented with an additional 20% (v/v) of commercially available grape juice (Welch’s). The embryos were incubated in a growth chamber (24 h of dark, 25°C, 60% relative humidity) to allow egg hatch for a period of 24 h. The feeding assay was carried out in 5-mL glass vials containing 0.115 g of Formula 4-24 Plain commercial diet (Carolina Biological Supply) with or without the recombinant His6-HFR1 protein in a final volume of 0.5 mL of protein storage buffer (50 mM Tris-HCl, pH 8.0, and 1 mM CaCl2). The experimental tubes contained the diet mixed with a dilution series of His6HFR1 protein (0.3, 1.5, 3, 6, 9, 12, and 15 mg g21). The control tube consisted of the diet containing protein storage buffer in a final volume of 0.5 mL. Ten neonate larvae were placed into each vial using a fine soft brush. Three replicates were performed for each treatment (with His6-HFR1) and control (with protein storage buffer). Mortality was estimated as the number of larvae that did not pupate out of the 10 larvae placed on the diet per tube. The developmental times from hatch of first-instar larvae to pupation and to emergence of adult flies (eclosion) were monitored. The percentage of mortality and eclosion was monitored as the larvae pupated and emerged as adult flies for each concentration of His6-HFR1. The percentage mortality was plotted against His6-HFR1 concentration to calculate the concentration at which 50% of the larvae died. The larvae were photographed at 5 d after egg hatch using a stereomicroscope (Olympus SZX12), and larval length was measured. One-way ANOVA using SAS (SAS Institute; version 8) was carried out to determine any significant differences within larval developmental time and length. Measurements were considered statistically significant at P , 0.05.
Scanning Electron Microscopy of Hessian Fly Larvae The crown tissue of H9-Iris and Newton wheat lines infested with biotype L was photographed at 1 and 3 d after egg hatch using a JOEL JSM-840 scanning electron microscope. Fresh crown tissue was excised, placed on an aluminum stub, and viewed with the scanning electron microscope under low vacuum at an accelerating voltage of 5 kV at room temperature.
Hessian Fly Larval Behavioral and Growth Studies Resistant H9-Iris and susceptible Newton near-isogenic wheat lines were grown and infested with biotype L Hessian flies (avirulent to H9-Iris but virulent on Newton), yielding an incompatible and a compatible interaction, respectively, as described by Subramanyam et al. (2006). The egg hatch was
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monitored with a stereomicroscope, and the time when the first larvae were observed at the ligule of the leaf on which the eggs were laid was considered the time of hatch. At selected time points (first instar: 6, 12, 24, 48, 72, 96, and 120 h; second instar: 192 h) after egg hatch, the first leaf sheath from each plant was carefully peeled off without disrupting or dislodging the larvae, and larvae on leaf sheath 2 were immediately viewed, photographed, and videographed for 60 s with a stereomicroscope (Olympus SZX12). The various behaviors of virulent and avirulent Hessian fly larvae that we observed were scored per unit of time (60 s). The behaviors seen were as follows. (1) Searching. The larva shows active side-to-side head movement. Its head is close to the surface of the leaf sheath and it is probing the plant tissue. Each lateral movement of the head to probe on the leaf surface was scored as an individual searching event. (2) Body contractions. The larva contracts its entire body, followed by a slow forward locomotion. These contractions occur simultaneously with the searching behavior. (3) Gut contractions. The larval gut contents move within the larva. The contents move either in one direction (posterior to anterior end) or in both directions. (4) Writhing. The larva exhibits a twisted or contorted motion with arching of the body. During arching, the anterior and posterior ends of the larva are in contact with the leaf surface but the middle part of the body is raised up and down. (5) Sessile. The entire ventral surface of the larva remains attached at one location on the plant, with mild contractions near the anterior end. (6) Head rearing. The larva raises its head up away from the plant tissue. Either the head moves up and down or remains in the raised position throughout the entire 60-s duration of the video. Between 15 and 32 larvae were scored for each behavior at each time point. Some larvae exhibited more than one behavior simultaneously. As an indicator of larval growth, the body length of larvae in the photomicrographs (16–22 larvae from six leaves at each time point) was also measured. One-way ANOVA using SAS was carried out to determine any significant differences in larval lengths. P , 0.05 was considered statistically significant.
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Domains of HFR1 deduced amino acid sequence. Supplemental Figure S2. Hemagglutination activity assay of His6-HFR1. Supplemental Figure S3. Avirulent Hessian fly larva displaying headrearing behavior. Supplemental Figure S4. Dead avirulent Hessian fly larva at 192 h after egg hatch. Supplemental Table S1. Glycan microarray (version 3.0) screening of His6-HFR1. Supplemental Video S1. Searching behavior in Hessian fly larva. Supplemental Video S2. Sessile behavior in virulent Hessian fly larva. Supplemental Video S3. Writhing behavior in avirulent Hessian fly larva. Supplemental Video S4. Feeding second-instar virulent Hessian fly larva.
ACKNOWLEDGMENTS We thank Jill Nemacheck and Sue Cambron (U.S. Department of AgricultureAgricultural Research Service) for help with growing and collecting the plant tissue and for maintaining Hessian fly stocks, respectively. We thank Dr. Herbert Ohm (Department of Agronomy, Purdue University) for supplying wheat seeds. We acknowledge the Consortium for Functional Glycomics (grant no. GM62116) for providing the resources for carrying out the glycan microarray analysis. Valuable discussions with Dr. Els Van Damme (Ghent University) are gratefully acknowledged. We thank Dr. Dorota Inerowicz (core proteomics facility of Bindley Bioscience Center, Purdue University) for assistance in MALDI-MS and Bill Kielhorn (Department of Entomology, Purdue University) in assisting with the videographic studies. We also thank Dr. Stanton Gelvin (Department of Biological Sciences, Purdue University) for allowing us the use of his microscopy facility. This publication is Purdue University Agricultural Experiment Station Journal Article Number 2007– 18252. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the U.S. Department of Agriculture. This is
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Properties of HFR1, a Hessian Fly-Induced Wheat Lectin
a joint contribution by the U.S. Department of Agriculture-Agricultural Research Service and Purdue University. Received January 10, 2008; accepted May 5, 2008; published May 8, 2008.
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