Insecticidal activity of wheat Hessian fly responsive proteins HFR-1 and HFR-3 towards a non-target wheat pest, cereal aphid (Sitobion avenae F.)

The interaction between Hessian fly (Mayetiola destructor) and wheat (Triticum aestivum) involves a gene-for-gene resistance mechanism. The incompatible interaction leading to resistance involves up-regulation of several Hfr (Hessian fly responsive) genes encoding proteins with potential insecticidal activity. The encoded proteins HFR-1, HFR-2 and HFR-3 all possess lectin-like domains. HFR-1 and HFR-3 were produced as recombinant proteins using Escherichia coli and Pichia pastoris, respectively as expression hosts. Purified recombinant proteins were assayed for insecticidal effects towards cereal aphid (Sitobion avenae), an insect to which wheat shows only tolerance. Both HFR-1 and HFR-3 were found to be insecticidal towards S. avenae when fed in artificial diet. Although HFR-3 has sequence similarity and similar chitin-binding activity to wheat germ agglutinin (WGA), the latter protein was almost non-toxic to S. avenae. HFR-3 binds strongly to aphid midguts after ingestion, whereas WGA binds but does not persist over a feed-chase period. Quantitative PCR showed that Hfr-3 mRNA does not increase in level after cereal aphid infestation. The results suggest that the lack of effective resistance to cereal aphid in wheat is not due to an absence of genes encoding suitable insecticidal proteins, but results from a failure to up-regulate gene expression in response to aphid attack.     

Gene-for-gene defense of wheat against the Hessian fly lacks a classical oxidative burst

Genetic similarities between plant interactions with microbial pathogens and wheat interactions with Hessian fly larvae prompted us to investigate defense and counterdefense mechanisms. Plant oxidative burst, a rapid increase in the levels of active oxygen species (AOS) within the initial 24 h of an interaction with pathogens, commonly is associated with defenses that are triggered by gene-for-gene recognition events similar to those involving wheat and Hessian fly larvae. RNAs encoded by Hessian fly superoxide dismutase (SOD) and catalase (CAT) genes, involved in detoxification of AOS, increased in first-instar larvae during both compatible and incompatible interactions. However, mRNA levels of a wheat NADPH oxidase (NOX) gene that generates superoxide (O2-) did not increase. In addition, inhibiting wheat NOX enzyme with diphenyleneiodonium did not result in increased survival of avirulent larvae. However, nitro blue tetrazolium staining indicated that basal levels of O2- are present in both uninfested and infested wheat tissue. mRNA encoded by wheat genes involved in detoxification of the cellular environment, SOD, CAT, and glutathione-S-transferase did not increase in abundance. Histochemical staining with 3,3-diaminobenzidine revealed no increases in wheat hydrogen peroxide (H2O2) during infestation that were correlated with the changes in larval SOD and CAT mRNA. However, treatment with 2',7'-dichlorofluorescin demonstrated the presence of basal levels of H2O2 in the elongation zone of both infested and uninfested plants. The accumulation of a wheat flavanone 3-hydroxylase mRNA did show some parallels with larval gene mRNA profiles. These results suggested that larvae encounter stresses imposed by mechanisms other than an oxidative burst in wheat seedlings.     

Modulation of defense-response gene expression in wheat during Hessian fly larval feeding

Abstract

Expression profiles of ten genes commonly up-regulated during plant defense against microbial pathogens were compared temporally during compatible and incompatible interactions with first-instar Hessian fly larvae, in two wheat lines carrying different resistance genes. Quantitative real-time PCR revealed that while a lipoxygenase gene (WCI-2) was strongly up-regulated during the incompatible interactions, genes encoding β-1,3 endoglucanase (GNS) and an integral membrane protein (WIR1) were moderately responsive. Genes for thionin-like protein (WCI-3), PR-17-like protein (WCI-5), MAP kinase (WCK-1), phenylalanine ammonia-lyase (PAL), pathogenesis-related protein-1 (PR-1), receptor-like kinase (LRK10) and heat shock protein 70 (HSP70) were minimally responsive. The application of signaling molecules, salicylic acid (SA), methyl jasmonate (MJ) and abscisic acid (ABA), to insect-free plants demonstrated association of these genes with specific defense-response pathways. SA-induced up-regulation of a gene related to lipoxygenases that are involved in jasmonic acid (JA)-biosynthesis is suggestive of positive cross-talk between SA- and JA-mediated signaling pathways. Data suggest that alternative mechanisms may be involved since few of these classical defense-response genes are significantly up-regulated during incompatible interactions between wheat and Hessian fly.     

Obviation of wheat resistance to the Hessian fly through systemic induced susceptibility

Unlike most documented plant-insect interactions, Hessian fly-resistance [Mayetiola destructor (Say)] in wheat (Triticum aestivum L.) is initiated by a gene-for-gene recognition event in which plants carrying a specific R gene recognize salivary effectors encoded by a corresponding larval avirulence gene. However, dual infestation resulting from oviposition by virulent insects from 5 d before to 3 d after oviposition by avirulent insects on the same host plant, lead to systemic induced susceptibility, obviation of resistance, and ultimately the survival of both virulent and genetically avirulent progeny to adulthood. Simultaneous oviposition allowed greater survival of avirulent progeny than ovipositions separated by larger intervals. Because of the induction of plant resistance, hatch of avirulent larvae before virulent was more detrimental to rate of development than hatch of virulent before avirulent larvae. Obviation of resistance was not localized to the leaf being attacked by the virulent larvae, but also functioned across spatial distance into younger leaves. This research suggests that virulent Hessian fly larvae directly suppress the defense response of wheat, thus providing a refuge for avirulent genotypes, preserving diversity in field populations and increasing durability of deployed resistance genes.     

Avoidance of occupied hosts by the Hessian fly: oviposition behaviour and consequences for larval survival

Abstract 1. Unoccupied wheat plants and wheat plants occupied by conspecific eggs or larvae were presented to ovipositing female Hessian flies in choice tests.
2. The presence of conspecific eggs on the leaf surfaces of wheat plants did not appear to have any effect on the responses of ovipositing Hessian fly females.
3. The presence of conspecific larvae at the base and nodes of wheat plants for 1, 6, or 11 days had significant effects on Hessian fly oviposition. Eggs oviposited on plants were inversely proportional to larval densities and days of larval occupation.
4. Feeding by Hessian fly larvae is associated with several changes in wheat plants. One of these changes, the growth arrestment of the plant, was measured by recording the heights of plants used in oviposition tests. Plant heights were inversely proportional to both larval densities and days of occupation. Plant heights were directly proportional to eggs oviposited on plants.
5. The consequences of adult female avoidance of plants occupied by conspecific larvae were investigated by allowing females to oviposit on unoccupied plants and 1-day, 6-day, and 11-day larval occupied plants, then scoring at the end of the first larval instar the survival of the offspring that resulted from this oviposition.
6. Survival during the first larval instar was 88% for the offspring of females that oviposited on unoccupied plants, decreasing to 82, 31, and 4% on the 1-day, 6-day, and 11-day occupied plant treatments. On these four plant treatments, a positive correlation was found between larval performance (i.e. survival) and the preferences of ovipositing females.
7. On the four plant treatments, relationships between first-instar larval density and first-instar larval survival varied significantly. On unoccupied plants, survival was inversely proportional to density. On plants oviposited on at 6 days of larval occupation, survival was directly proportional to density.     

Hessian fly (Mayetiola destructor) attack causes a dramatic shift in carbon and nitrogen metabolism in wheat

Carbon and nitrogen (C/N) metabolism and allocation within the plant have important implications for plant-parasite interactions. Many plant parasites manipulate the host by inducing C/N changes that benefit their own survival and growth. Plant resistance can prevent this parasite manipulation. We used the wheat-Hessian fly (Mayetiola destructor) system to analyze C/N changes in plants during compatible and incompatible interactions. The Hessian fly is an insect but shares many features with plant pathogens, being sessile during feeding stages and having avirulence (Avr) genes that match plant resistance genes in gene-for-gene relationships. Many wheat genes involved in C/N metabolism were differentially regulated in plants during compatible and incompatible interactions. In plants during compatible interactions, the content of free carbon-containing compounds decreased 36%, whereas the content of free nitrogen-containing compounds increased 46%. This C/N shift was likely achieved through a coordinated regulation of genes in a number of central metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and amino-acid synthesis. Our data on plants during compatible interactions support recent findings that Hessian fly larvae create nutritive cells at feeding (attack) sites and manipulate host plants to enhance their own survival and growth. In plants during incompatible interactions, most of the metabolic genes examined were not affected or down-regulated.     

Genotypic interaction between resistance genes in wheat and virulence genes in the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae)

The genotypic interaction between wheat resistance genes H3, H6, H7H8, H9 and virulence genes vH3, vH6, vH7vH8, vH9 of Hessian fly, Mayetiola destructor (Say), was studied in a growth chamber. Results showed that plants homozygous and heterozygous for the H3 gene expressed a high level of resistance against homozygous avirulent and heterozygous larvae carrying the vH3 virulence allele. The H7H8 genes were highly effective in the homozygous condition, but displayed a reduced level of resistance in the heterozygous condition. The H6 and H9 genes showed different levels of resistance against the reciprocal heterozygous larvae (vH6(a)vH6(A) versus vH6(A)vH6(a) and vH9(a)vH9(A) versus vH9(A)vH9(a)). Adults reared from vH6(a)vH6(A) and vH9(a)vH9(A) larvae were all males, consistent with the vH6 and vH9 X-linkage. Plants homozygous for H3, H6, H7H8, and H9 allowed for greater larval survival of heterozygous larvae, which suggests that avirulence to these resistance genes is incompletely dominant. Greater survival of homozygous avirulent larvae on heterozygous plants (H3h3, H6h6, H7h7H8h8, H9h9) suggests incomplete dominance of these wheat genes. Survival of heterozygous along with homozygous virulent larvae would reduce selection pressure for virulence in Hessian fly populations infesting fields of resistant wheat cultivars. This would be expected to slow the increase in frequency of virulence alleles that often results from deployment of resistant cultivars.     

Ultrastructural changes in the midguts of Hessian fly larvae feeding on resistant wheat

The focus of the present study was to compare ultrastructure in the midguts of larvae of the Hessian fly, Mayetiola destructor (Say), under different feeding regimens. Larvae were either fed on Hessian fly-resistant or -susceptible wheat, and each group was compared to starved larvae. Within 3 h of larval Hessian fly feeding on resistant wheat, midgut microvilli were disrupted, and after 6 h, microvilli were absent. The disruption in microvilli in larvae feeding on resistant wheat were similar to those reported for midgut microvilli of European corn borer, Ostrinia nubilasis (Hubner), larvae fed a diet containing wheat germ agglutinin. Results from the present ultrastructural study, coupled with previous studies documenting expression of genes encoding lectin and lectin-like proteins is rapidly up-regulated in resistant wheat to larval Hessian fly, are indications that the midgut is a target of plant resistance compounds. In addition, the midgut of the larval Hessian fly is apparently unique among other dipterans in that no peritrophic membrane was observed. Ultrastructural changes in the midgut are discussed from the prospective of their potential affects on the gut physiology of Hessian fly larvae and the mechanism of antibiosis in the resistance of wheat to Hessian fly attack.     

Biotype composition of Hessian fly (Diptera: Cecidomyiidae) populations from the southeastern, midwestern, and northwestern United States and virulence to resistance genes in wheat

Twenty-three Hessian fly, Mayetiola destructor (Say), populations collected in the southeastern (Alabama and Mississippi), midwestern (Indiana), and northwestern (Idaho and Washington) United States from 1995 to 1999 were evaluated for biotype composition based on response to Hessian fly resistance genes H3, H5, H6, and H7H8 in wheat, Triticum aestivum L. Biotypes L and O, combined, made up at least 60% of all Alabama populations. Biotype L was predominant in the northern third of Alabama and biotype O in the southern two-thirds of the state. Based on biotype data, wheat cultivars with H7H8 resistance should be highly effective in central and southern Alabama. Fifty-four percent of the Mississippi population consisted of biotype L, and the remaining virulent biotypes (B, D, E, G, J, and O) ranged in frequency from 1 to 17%. The Mississippi population also contained 4% of the avirulent biotype GP. Only biotypes D and L were found in Indiana populations, but biotype L was predominant. Hessian fly populations from Idaho and Washington contained one or more of the virulent biotypes D-H, J, and L-O; however, only biotypes E, F, and G occurred at frequencies > 12%. The avirulent biotype GP made up 25-57% of Idaho and Washington populations, a much higher percentage than found in populations from the eastern United States. Although the highest level of virulence in Idaho and Washington populations was found to resistance genes H3 and H6, the frequency of biotype GP would indicate that the currently deployed gene H3 would provide a moderate to high level of resistance, depending on location. Nine of the populations, plus populations collected from the mid-Atlantic state area in 1989 and 1996, also were tested against the wheat cultivar 'INW9811' that carries H13 resistance to Hessian fly biotype L and two Purdue wheat lines with unidentified genes for resistance. The H13 resistance in INW9811 was highly effective against all populations tested from the eastern and northwestern U.S. wheat production areas, except Maryland and Virginia. Population studies also indicated that wheat line CI 17960-1-1-2-4-2-10 likely carries the H13 resistance gene, based on the similarity of its response and that of INW9811 to eight fly populations. Continued monitoring of biotype frequency in Hessian fly populations is required for optimal deployment and management of resistance genes in all wheat production areas.     

Feeding by Hessian Fly (Mayetiola destructor [Say]) Larvae on Wheat Increases Levels of Fatty Acids and Indole-3-Acetic Acid but not Hormones Involved in Plant-Defense Signaling

Gall-inducing insects exert a unique level of control over the physiology of their host plants. This control can extend to host–plant defenses so that some, if not most, gall-inducing species appear to avoid or modify host plant defenses to effect production of their gall. Included among gall insects is Hessian fly (Mayetiola destructor [Say], Diptera: Cecidomyiidae), a damaging pest of wheat (Triticum aestivum L.) and an emerging model system for studying plant–insect interactions. We studied the dynamics of some defense-related phytohormones and associated fatty acids during feeding of first instar Hessian fly larvae on a susceptible variety of wheat. We found that Hessian fly larvae significantly elevated in their host plants’ levels of linolenic and linoleic acids, fatty acids that may be nutritionally beneficial. Hessian fly larvae also elevated levels of indole-3-acetic acid (IAA), a phytohormone hypothesized to be involved in gall formation, but not the defense-related hormones jasmonic (JA) and salicylic acids. Moreover, we detected in Hessian fly-infested plants a significant negative relationship between IAA and JA that was not present in control plants. Our results suggest that Hessian fly larvae may induce nutritionally beneficial changes while concomitantly altering phytohormone levels, possibly to facilitate plant-defense avoidance.     

Feeding by Hessian fly [Mayetiola destructor (Say)] larvae does not induce plant indirect defences

Abstract.  1. Recent research has addressed the function of herbivore-induced plant volatiles in attracting natural enemies of feeding herbivores. While many types of insect herbivory appear to elicit volatile responses, those triggered by gall insects have received little attention. Previous work indicates that at least one gall insect species induces changes in host-plant volatiles, but no other studies appear to have addressed whether gall insects trigger plant indirect defences.
2. The volatile responses of wheat to feeding by larvae of the Hessian fly Mayetiola destructor (Say) (Diptera: Cecidomyiidae) were studied to further explore indirect responses of plants to feeding by gall insects. This specialist gall midge species did not elicit a detectable volatile response from wheat plants, whereas a generalist caterpillar triggered volatile release. Moreover, Hessian fly feeding altered volatile responses to subsequent caterpillar herbivory.
3. These results suggest that Hessian fly larvae exert a degree of control over the defensive responses of their host plants and offer insight into plant-gall insect interactions. Also, the failure of Hessian fly larvae to elicit an indirect defensive response from their host plants may help explain why natural enemies, which often rely on induced volatile cues, fail to inflict significant mortality on M. destructor populations in the field.