Modulation of nonessential amino acid biosynthetic pathways in virulent Hessian fly larvae (Mayetiola destructor), feeding on susceptible host wheat (Triticum aestivum).

Compatible interactions between wheat (Triticum aestivum), and its dipteran pest Hessian fly (Hf, Mayetiola destructor) result in successful establishment of larval feeding sites rendering the host plant susceptible. Virulent larvae employ an effector-based feeding strategy to reprogram the host physiology resulting in formation of a protein- and sugar-rich nutritive tissue beneficial to developing larvae. Previous studies documented increased levels of nonessential amino acids (NAA; that need not be received through insect diet) in the susceptible wheat in response to larval feeding, suggesting importance of plant-derived NAA in larval nutrition. Here, we investigated the modulation of genes from NAA biosynthetic pathways (NAABP) in virulent Hf larvae. Transcript profiling for 16 NAABP genes, annotated from the recently assembled Hf genome, was carried out in the feeding first-, and second-instars and compared with that of the first-instar neonate (newly hatched, migrating, assumed to be non-feeding) larvae. While Tyr, Gln, Glu, and Pro NAABP genes transcript abundance declined in the feeding instars as compared to the neonates, those for Ala, and Ser increased in the feeding larval instars, despite higher levels of these NAA in the susceptible host plant. Asp, Asn, Gly and Cys NAABP genes exhibited variable expression profiles in the feeding first- and second-instars. Our results indicate that while Hf larvae utilize the plant-derived NAA, de novo synthesis of several NAA may be necessary to: (i) provide larvae with the requisite amount for sustaining growth before nutritive tissue formation and, (ii) overcome any inadequate amounts in the host plant, post-nutritive tissue formation.

A Novel, Economical Way to Assess Virulence in Field Populations of Hessian Fly (Diptera: Cecidomyiidae) Utilizing Wheat Resistance Gene H13 as a Model.

Mayetiola destructor (Say) is a serious pest of wheat, Triticum aestivum L., in North America, North Africa, and Central Asia. Singly deployed resistance genes in wheat cultivars have provided effective management of Hessian fly populations for >50 yr. Thirty-five H genes have been documented. Defense mediated by the H gene constitutes strong selection on the Hessian fly population, killing 100% of larvae. A mutation in a matching Hessian fly avirulence gene confers virulence to the H gene, leading to survival on the resistant plant. As the frequency of virulence rises in the population, the H gene loses its effectiveness for pest management. Knowing the frequency of virulence in the population is not only important for monitoring but also for decisions about which H gene should be deployed in regional wheat breeding programs. Here, we present a novel assay for detecting virulence in the field. Hessian fly males were collected in Alabama, Georgia, North Carolina, and South Carolina using sticky traps baited with Hessian fly sex pheromone. Utilizing two PCR reactions, diagnostic molecular markers for the six alleles controlling avirulence and virulence to H13 can be scored based on band size. Throughout the southeast, all three avirulence and three virulence alleles can be identified. In South Carolina, the PCR assay was sensitive enough to detect the spread of virulence into two counties previously documented as 100% susceptible to H13. The new assay also indicates that the previous methods overestimated virulence in the field owing to scoring of the plant instead of the insect.

Effectiveness of Genes for Hessian Fly (Diptera: Cecidomyiidae) Resistance in the Southeastern United States.

The Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), is the most important insect pest of wheat (Triticum aestivum L. subsp. aestivum) in the southeastern United States, and the deployment of genetically resistant wheat is the most effective control. However, the use of resistant wheat results in the selection of pest genotypes that can overcome formerly resistant wheat. We have evaluated the effectiveness of 16 resistance genes for protection of wheat from Hessian fly infestation in the southeastern United States. Results documented that while 10 of the genes evaluated could provide protection of wheat, the most highly effective genes were H12, H18, H24, H25, H26, and H33. However, H12 and H18 have been reported to be only partially effective in field evaluations, and H24, H25, and H26 may be associated with undesirable effects on agronomic traits when introgressed into elite wheat lines. Thus, the most promising new gene for Hessian fly resistance appears to be H33. These results indicate that identified highly effective resistance in wheat to the Hessian fly is a limited resource and emphasize the need to identify novel sources of resistance. Also, we recommend that the deployment of resistance in gene pyramids and the development of novel strategies for engineered resistance be considered.

Use of microsatellite and SNP markers for biotype characterization in Hessian fly.

Exploration of the biotype structure of Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), would improve our knowledge regarding variation in virulence phenotypes and difference in genetic background. Microsatellites (simple sequence repeats) and single-nucleotide polymorphisms (SNPs) are highly variable genetic markers that are widely used in population genetic studies. This study developed and tested a panel of 18 microsatellite and 22 SNP markers to investigate the genetic structure of nine Hessian fly biotypes: B, C, D, E, GP, L, O, vH9, and vH13. The simple sequence repeats were more polymorphic than the SNP markers, and their neighbor-joining trees differed in consequence. Microsatellites suggested a simple geographic association of related biotypes that did not progressively gain virulence with increasing genetic distance from a founder type. Use of the k-means clustering algorithm in the STRUCTURE program shows that the nine biotypes comprise six to eight populations that are related to geography or history within laboratory cultures.

Hessian fly larval feeding triggers enhanced polyamine levels in susceptible but not resistant wheat.

BACKGROUND: Hessian fly (Mayetiola destructor), a member of the gall midge family, is one of the most destructive pests of wheat (Triticum aestivum) worldwide. Probing of wheat plants by the larvae results in either an incompatible (avirulent larvae, resistant plant) or a compatible (virulent larvae, susceptible plant) interaction. Virulent larvae induce the formation of a nutritive tissue, resembling the inside surface of a gall, in susceptible wheat. These nutritive cells are a rich source of proteins and sugars that sustain the developing virulent Hessian fly larvae. In addition, on susceptible wheat, larvae trigger a significant increase in levels of amino acids including proline and glutamic acid, which are precursors for the biosynthesis of ornithine and arginine that in turn enter the pathway for polyamine biosynthesis. RESULTS: Following Hessian fly larval attack, transcript abundance in susceptible wheat increased for several genes involved in polyamine biosynthesis, leading to higher levels of the free polyamines, putrescine, spermidine and spermine. A concurrent increase in polyamine levels occurred in the virulent larvae despite a decrease in abundance of Mdes-odc (ornithine decarboxylase) transcript encoding a key enzyme in insect putrescine biosynthesis. In contrast, resistant wheat and avirulent Hessian fly larvae did not exhibit significant changes in transcript abundance of genes involved in polyamine biosynthesis or in free polyamine levels. CONCLUSIONS: The major findings from this study are: (i) although polyamines contribute to defense in some plant-pathogen interactions, their production is induced in susceptible wheat during interactions with Hessian fly larvae without contributing to defense, and (ii) due to low abundance of transcripts encoding the rate-limiting ornithine decarboxylase enzyme in the larval polyamine pathway the source of polyamines found in virulent larvae is plausibly wheat-derived. The activation of the host polyamine biosynthesis pathway during compatible wheat-Hessian fly interactions is consistent with a model wherein the virulent larvae usurp the polyamine biosynthesis machinery of the susceptible plant to acquire nutrients required for their own growth and development.

A genome-wide survey of small interfering RNA and microRNA pathway genes in a galling insect.

Deployment of resistance (R) genes is the most effective control for Hessian fly, Mayetiola destructor (Say); however, deployment of R genes results in an increased frequency of pest genotypes that display virulence to them. RNA interference (RNAi) is a useful reverse genetics tool for studying such insect virulence pathways, but requires a systemic phenotype, which is not found in all species. In an effort to correlate our observed weak RNAi phenotype in M. destructor with a genetic basis, we have aggregated and compared RNAi related genes across M. destructor, three other insect species, and the nematode Caenorhabditis elegans. We report here the annotation of the core genes in the small interfering RNA (siRNA) and microRNA (miRNA) pathways in M. destructor. While most of the miRNA pathway genes were highly conserved across the species studied, the siRNA pathway genes showed increased relative variability in comparison to the miRNA pathway. In particular, the Piwi/Argonaute/Zwille (PAZ) domain of Dicer-2 (DCR-2) had the least amount of sequence similarity of any domain among species surveyed, with a trend of increased conservation in those species with amenable systemic RNAi. A homolog of the systemic interference defective-1 (Sid-1) gene of C. elegans was also not annotated in the M. destructor genome. Indeed, it is of interest that a Sid-1 homolog has not been detected in any dipteran species to date. We hypothesize the sequence architecture of the PAZ domain in the M. destructor DCR-2 protein is related to reduced efficacy of this enzyme and this taken together with the lack of a Sid-1 homolog may account for the weak RNAi response observed to date in this species as well as other dipteran species.

Effects of antinutrient proteins on Hessian fly (Diptera: Cecidomyiidae) larvae.

One strategy to enhance the durability of Hessian fly resistance (R) genes in wheat is to combine them with transgenes for resistance. To identify potential transgenes for resistance a protocol for rapidly screening the proteins they encode for efficacy toward resistance is required. However, the Hessian fly is an obligate parasite of wheat and related grasses. Consequently, no protocol for in vitro delivery of antinutrient or toxic proteins to feeding larvae is available. We report here the development of a Hessian fly in plantatranslocation (HIT) feeding assay and the evaluation of eight lectins and the Bowman-Birk serine proteinase inhibitor for potential in transgenic resistance. Of the antinutrient proteins evaluated, Galanthus nivalis L. agglutinin (GNA), commonly termed snowdrop lectin, was the most efficacious. Ingestion of GNA caused a significant reduction in growth of Hessian fly larvae, disruption of midgut microvilli, and changes in transcript level of genes involved in carbohydrate metabolism, digestion, detoxification, and stress response. These effects of GNA are discussed from the perspective of larval Hessian fly physiology.

Induced epidermal permeability modulates resistance and susceptibility of wheat seedlings to herbivory by Hessian fly larvae.

Salivary secretions of neonate Hessian fly larvae initiate a two-way exchange of molecules with their wheat host. Changes in properties of the leaf surface allow larval effectors to enter the plant where they trigger plant processes leading to resistance and delivery of defence molecules, or susceptibility and delivery of nutrients. To increase understanding of the host plant's response, the timing and characteristics of the induced epidermal permeability were investigated. Resistant plant permeability was transient and limited in area, persisting just long enough to deliver defence molecules before gene expression and permeability reverted to pre-infestation levels. The abundance of transcripts for GDSL-motif lipase/hydrolase, thought to contribute to cuticle reorganization and increased permeability, followed the same temporal profile as permeability in resistant plants. In contrast, susceptible plants continued to increase in permeability over time until the entire crown of the plant became a nutrient sink. Permeability increased with higher infestation levels in susceptible but not in resistant plants. The ramifications of induced plant permeability on Hessian fly populations are discussed.

Unusual conservation among genes encoding small secreted salivary gland proteins from a gall midge.

BACKGROUND: In most protein-coding genes, greater sequence variation is observed in noncoding regions (introns and untranslated regions) than in coding regions due to selective constraints. During characterization of genes and transcripts encoding small secreted salivary gland proteins (SSSGPs) from the Hessian fly, we found exactly the opposite pattern of conservation in several families of genes: the non-coding regions were highly conserved, but the coding regions were highly variable. RESULTS: Seven genes from the SSSGP-1 family are clustered as one inverted and six tandem repeats within a 15 kb region of the genome. Except for SSSGP-1A2, a gene that encodes a protein identical to that encoded by SSSGP-1A1, the other six genes consist of a highly diversified, mature protein-coding region as well as highly conserved regions including the promoter, 5'- and 3'-UTRs, a signal peptide coding region, and an intron. This unusual pattern of highly diversified coding regions coupled with highly conserved regions in the rest of the gene was also observed in several other groups of SSSGP-encoding genes or cDNAs. The unusual conservation pattern was also found in some of the SSSGP cDNAs from the Asian rice gall midge, but not from the orange wheat blossom midge. Strong positive selection was one of the forces driving for diversification whereas concerted homogenization was likely a mechanism for sequence conservation. CONCLUSION: Rapid diversification in mature SSSGPs suggests that the genes are under selection pressure for functional adaptation. The conservation in the noncoding regions of these genes including introns also suggested potential mechanisms for sequence homogenization that are not yet fully understood. This report should be useful for future studies on genetic mechanisms involved in evolution and functional adaptation of parasite genes.

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 3h of larval Hessian fly feeding on resistant wheat, midgut microvilli were disrupted, and after 6h, 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.

Virulence in Hessian fly (Diptera: Cecidomyiidae) field collections from the southeastern United States to 21 resistance genes in wheat.

Genetic resistance in wheat, Triticum aestivum L., is the most efficacious method for control of Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae). However, because of the appearance of new genotypes (biotypes) in response to deployment of resistance, field collections of Hessian fly need to be evaluated on a regular basis to provide breeders and producers information on the efficacy of resistance (R) genes with respect to the genotype composition of Hessian fly in regional areas. We report here on the efficacy of 21 R genes in wheat to field collections of Hessian fly from the southeastern United States. Results documented that of the 21 R genes evaluated only five would provide effective protection of wheat from Hessian fly in the southeastern United States. These genes were H12, H18, H24, H25, and H26. Although not all of the 33 identified R genes were evaluated in the current study, these results indicate that identified genetic resistance to protect wheat from Hessian attack in the southeastern United States is a limited resource. Historically, R genes for Hessian fly resistance in wheat have been deployed as single gene releases. Although this strategy has been successful in the past, we recommend that in the future deployment of combinations of highly effective previously undeployed genes, such as H24 and H26, be considered. Our study also highlights the need to identify new and effective sources of resistance in wheat to Hessian fly if genetic resistance is to continue as a viable option for protection of wheat in the southeastern United States.

Characterization and expression analysis of a gene encoding a secreted lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say).

In a salivary gland transcriptomics study we identified a cDNA with a full-length open reading frame for a gene (MdesL1) encoding a lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say). Fluorescent in situ hybridization on salivary polytenes positioned MdesL1 on the long arm of Autosome 1. BLASTp and conserved domain searches revealed the deduced amino acid sequence contained a lipase superfamily domain with similarity to lipases and phospholipases from other insects. A secretion signal peptide was identified at the amino terminus of the deduced amino acid sequence. Analysis of the transcript of MdesL1 in larval Hessian fly tissues by quantitative real-time PCR (qPCR) revealed the greatest abundance was in salivary glands. Analysis of transcript levels during development showed the greatest level was detected in feeding 1st-instar and early 2nd-instar larvae. Transcript levels increased dramatically over time in larvae feeding on susceptible wheat but were detected at low levels in larvae feeding on resistant wheat. These data suggest the protein encoded by MdesL1 is likely secreted into host-plant cells during larval feeding and could be involved in extra-oral digestion and changes in host-cell permeability or in generating a second messenger in a host-cell-signaling cascade.

Molecular characterization and responsive expression of a defender against apoptotic cell death homologue from the Hessian fly, Mayetiola destructor.

Apoptosis or programmed cell death is an active process occurring in multicellular organisms to maintain growth and development. The Hessian fly, Mayetiola destructor, is rapidly emerging as a model insect species to study insect-plant interactions and to decipher some exceptional physiological phenomena. In this study, we report the characterization and expression profiles of a putative Hessian fly defender against apoptotic cell death (DAD1) homologue designated MdesDAD1. The deduced amino acid sequence of MdesDAD1 revealed significant similarity (75% identity, 9e-42) to other insect and non-insect DAD1 sequences. Phylogenetic analysis grouped MdesDAD1 within a sub-clade consisting of other insect DAD1 homologues. Quantitative analysis indicated constitutive levels of MdesDAD1 mRNA in all the tissues examined but an altered expression pattern during development, wherein the highest mRNA levels observed were prior to pupation. Most interestingly, MdesDAD1 transcript was found to be up-regulated during incompatible (larvae reared on resistant wheat) Hessian fly/wheat interactions compared to compatible (larvae reared on susceptible wheat) interactions. These results suggest MdesDAD1 to have a putative role in the inhibition of unwanted apoptosis triggered during development and in incompatible Hessian fly/wheat interactions. The results obtained provide clues to plausible insect and host-plant factors that could be responsible for the induction of MdesDAD1.

Tissue and developmental expression of a gene from Hessian fly encoding an ABC-active-transporter protein: implications for Malpighian tubule function during interactions with wheat.

We report on the transcriptional patterns of a putative white (w) gene encoding an ABC-active-transporter protein during development in Hessian fly, Mayetiola destructor. The deduced amino acid sequence for the Hessian fly white showed 74-77% similarities to white/ATP-binding-cassette proteins and 52-57% similarities to scarlet/ATP-binding-cassette proteins from other dipterans. Conserved ATP-binding motifs and transmembrane alpha-helix segments were identified in the Hessian fly white protein further supporting its function as an ABC-active-transporter similar to the Drosophila white protein. Spatial analysis of transcript levels for white in larval Hessian fly tissues by quantitative real-time PCR revealed the greatest level of transcript in the Malpighian tubules, while analysis of temporal expression during development revealed the highest transcript levels in late 2nd- and early 3rd-instar larvae. Analysis of transcript levels for white in Hessian fly larvae feeding on susceptible and resistant wheat showed greater levels of the transcript in larvae feeding on resistant plants. We speculate the increased transcript level for white in larvae feeding on resistant wheat could be correlated with stress and increased Malpighian tubule activity associated with the metabolism and detoxification of toxic substrates generated either endogenously or encountered exogenously from the host plant.

Pyramiding of insecticidal compounds for control of the cowpea bruchid (Callosobruchus maculatus F.).

The cowpea bruchid (Callosobruchus maculatus F.) (Chrysomelidae: Bruchini) is a major pest of stored cowpea grain. With limited available technologies for controlling the bruchid, transgenic cowpeas with bruchid resistance genes engineered into them could become the next management tools. An investigation was made of two different sets of potential transgenic insecticidal compounds using an artificial seed system: (i) CIP-PH-BT-J and recombinant egg white avidin, and (ii) avidin and wheat alpha-amylase inhibitor. CIP-PH-BT-J (0.1%; 1000 mg kg(-1)) and recombinant egg white avidin (0.006%; 60 mg kg(-1)) incorporated separately into artificial seeds caused 98.2 and 99% larval mortality rates respectively. Combining CIP-PH-BT-J and avidin in the same artificial seed provided additional mortality compared with each factor incorporated singly; no insects survived in seeds with the combined toxins. Similarly, when avidin and wheat alpha-amylase inhibitor (alphaAI) (1%; 10 g kg(-1)) were incorporated separately into artificial seeds, this caused 99.8 and 98% mortality respectively. However, in combination, avidin and alphaAI did not increase mortality, but they did cause a significant increase in developmental time of the cowpea bruchids. These results emphasize that the joint action of potential insecticidal compounds cannot be predicted from results obtained separately for each compound, and they suggest potential transgenes for further consideration.

Antioxidant defense response in a galling insect.

Herbivorous insect species are constantly challenged with reactive oxygen species (ROS) generated from endogenous and exogenous sources. ROS produced within insects because of stress and prooxidant allelochemicals produced by host plants in response to herbivory require a complex mode of antioxidant defense during insect/plant interactions. Some insect herbivores have a midgut-based defense against the suite of ROS encountered. Because the Hessian fly (Mayetiola destructor) is the major insect pest of wheat worldwide, and an emerging model for all gall midges, we investigated its antioxidant responses during interaction with its host plant. Quantitative data for two phospholipid glutathione peroxidases (MdesPHGPX-1 and MdesPHGPX-2), two catalases (MdesCAT-1 and MdesCAT-2), and two superoxide dismutases (MdesSOD-1 and MdesSOD-2) revealed high levels of all of the mRNAs in the midgut of larvae on susceptible wheat (compatible interaction). During development of the Hessian fly on susceptible wheat, a differential expression pattern was observed for all six genes. Analysis of larvae on resistant wheat (incompatible interaction) compared with larvae on susceptible wheat showed increased levels of mRNAs in larvae on resistant wheat for all of the antioxidant genes except MdesSOD-1 and MdesSOD-2. We postulate that the increased mRNA levels of MdesPHGPX-1, MdesPHGPX-2, MdesCAT-1, and MdesCAT-2 reflect responses to ROS encountered by larvae while feeding on resistant wheat seedlings and/or ROS generated endogenously in larvae because of stress/starvation. These results provide an opportunity to understand the cooperative antioxidant defense responses in the Hessian fly/wheat interaction and may be applicable to other insect/plant interactions.

cDNA cloning and transcriptional expression of a peritrophin-like gene in the Hessian fly, Mayetiola destructor [Say].

One of the well-studied components of the insect gut is the peritrophic matrix (PM). This semipermeable structure primarily functions in digestion, and protection against invasive microorganisms and mechanical damage. We report the cDNA cloning and transcription profiles of a peritrophin-A like gene (designated MdesPERI-A1) in the Hessian fly Mayetiola destructor. The predicted amino acid sequence of MdesPERI-A1 revealed a putative secretion signal peptide at its amino terminus, similarity to peritrophins from other insects including dipterans, and the presence of two chitin binding domains each containing six cysteine residues. Quantitative expression analysis of MdesPERI-A1 mRNA in different larval tissues revealed the transcript to be predominantly present in the midgut (597.9-fold) compared to other tissues assayed including salivary glands and fat bodies. Spatial expression patterns during development showed a peak expression of MdesPERI-A1 in the feeding second-instars (146-fold) and a decline in expression in the pupal and adult stages. Transcription profiling of MdesPERI-A1 during compatible (larvae on susceptible plants) and incompatible (larvae on resistant plants) interactions with wheat revealed a greater level (1.7-fold) of MdesPERI-A1 transcript in larvae on resistant plants in the initial time point examined. However, MdesPERI-A1 expression declined in larvae on resistant plants at the later time points.

Tissue and life stage specificity of glutathione S-transferase expression in the Hessian fly, Mayetiola destructor: implications for resistance to host allelochemicals.

Two new Delta and Sigma glutathione S-transferases (GSTs) in the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), were characterized and transcription profiles described. The deduced amino acid sequences for the two M. destructor Delta GSTs (MdesGST-1 and MdesGST-3) showed high similarity with other insect Delta GSTs including the conserved catalytic serine residue. The deduced amino acid sequence for the M. destructor Sigma GST (MdesGST-2) showed high similarity with other insect Sigma GSTs including the conserved glutathione and substrate binding sites. Quantitative tissue expression analysis showed that mRNA levels for MdesGST-1 were predominant in fat body, whereas for MdesGST-2 and MdesGST-3 expression was predominant in the midgut. Temporal expression during development showed peak mRNA levels for MdesGST-1 during larval development, but in the pupal stage for MdesGST-2. MdesGST-3 showed a constitutive expression pattern throughout development. M. destructor feeds on wheat, and expression analysis after feeding indicated that mRNA levels for MdesGST-1 were significantly higher in incompatible interactions in which larvae fed on resistant wheat, while MdesGST-3 was significantly higher in compatible interactions when larvae fed on susceptible wheat. MdesGST-2 showed an equivalent expression pattern during both interactions. These results suggest that the M. destructor Delta GSTs are important in detoxifying wheat allelochemicals during feeding, while Sigma GST participates in metabolism of endogenous substrates.

Expression patterns of antibacterial genes in the Hessian fly.

We report on the transcriptional patterns of three antibacterial genes, a defensin (MdesDEF-1), a diptericin (MdesDIP-1) and a lysozyme (MdesLYS-1), during development in Hessian fly, Mayetiola destructor. Quantitative analysis by real-time PCR of mRNA levels in different tissues revealed a predominance of the transcripts for all three genes in the midgut, while analysis during development revealed greatest abundance in mRNA during the 3rd-instar. An evaluation of the midgut lumen revealed the presence of a diverse bacterial flora in larvae maintained on susceptible wheat. Further, the titer of bacteria in the midgut increased approximately 250-fold from the 1st-instar through the 2nd-instar. However, no detectable titer of bacteria was observed from the midgut lumen of larvae maintained on resistant plants. PCR amplicons produced using primers designed to conserved regions of the Pseudomonas 16S rRNA gene supported taxonomic identification for some of the bacteria comprising the midgut flora as belonging to the genus Pseudomonas. Analysis of mRNA for the Hessian fly antibacterial genes in larvae feeding on susceptible and resistant plants revealed an increase in the transcript level for MdesDEF-1 in 1st-instar larvae on susceptible plants, while the transcript levels for MdesDIP-1 and MdesLYS-1 were constant. Results suggest the transcriptional patterns of the Hessian fly antibacterial genes observed could be associated with the developing midgut bacterial flora present in larvae feeding on susceptible wheat as well as microbial challenge encountered at other stages in development.

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.