Hessian fly (Diptera: Cecidomyiidae) virulence in Louisiana: assessment of field populations from 2023 to efficacy of 27 Hessian fly resistance genes in wheat.

The Hessian fly, Mayetiola destructor (Say), is one of the most important insect pest plaguing wheat (Triticum aestivum, L) producers across the United States and around the world. Genetic resistance is the stalwart for control of Hessian fly. However, new genotypes (biotypes) arise in deployment of wheat containing resistance genes, so field populations must be evaluated periodically to provide information on the efficacy of those deployed genes. Louisiana (LA), with its diverse agricultural landscape, is not exempt from the challenges posed by this destructive pest. We previously documented the resistance response of wheat lines harboring Hessian fly resistance (H) genes against field populations collected in 2008 from across the southeastern United States, including Iberville Parish, LA. In the spring of 2023, we reevaluated the resistance response of 27 H genes from the field populations collected from Iberville Parish, LA, and compared the results with those observed in 2008. Sixteen H genes showed comparable resistance to the field populations from both years. While 3 of the H genes, H11, H23, and H24, showed a significant decrease in resistance, 2 genes, H16 and H31, had marked increase in resistance. Furthermore, 6 additional H genes were evaluated in 2023, with 4 showing >70% resistance. Our results clearly identify a total of 20 H genes that are moderate to highly effective against the 2023 Hessian fly population from Iberville Parish, LA. The resistance response documented in this study offers valuable information to wheat breeders in the region for effective management of this insect pest.

Untapped Sources of Dual Resistance to Hessian Fly and Greenbug in Synthetic Hexaploid Wheats.

The Hessian fly (Hf) and greenbugs (Gb) are major pests of wheat, causing severe economic losses globally. Deploying resistant wheat is the most effective strategy for managing these destructive insects. However, the resistance is not effective against all Hf or Gb biotypes and can impose selection pressure on insects, resulting in the development of virulent biotypes. These challenges must be met through the discovery of new and novel sources of resistance to these pests. Synthetic Hexaploid Wheat (SHW)-developed cultivars are a rich source of resistance against a diverse array of pathogens and pests. In this study, 80 SHW lines were evaluated for their resistance to Hf and Gb under controlled environmental conditions. Of these, a total of 36 SHW lines showed resistance independently to Hf biotype L and Gb biotype E, while 27 lines showed combined resistance to both Hf and Gb. Further, a subset of 10 SHW lines showed resistance to additional Hf biotypes, Great Plains and vH13. The identification of SHW lines resistant to multiple insects and biotypes offers an invaluable resource to breeders who are looking to stack resistance traits to develop elite cultivars as a strategy to alleviate economic impacts upon global wheat production.

slag: A program for seeded local assembly of genes in complex genomes.

Although finished genomes have become more common, there is still a need for assemblies of individual genes or chromosomal regions when only unassembled reads are available. slag (Seeded Local Assembly of Genes) fulfils this need by performing iterative local assembly based on cycles of matching-read retrieval with blast and assembly with cap3, phrap, spades, canu or unicycler. The target sequence can be nucleotide or protein. Read fragmentation allows slag to use phrap or cap3 to assemble long reads at lower coverage (e.g., 5×) than is possible with canu or unicycler. In simple, nonrepetitive genomes, a slag assembly can cover a whole chromosome, but in complex genomes the growth of target-matching contigs is limited as additional reads are consumed by consensus contigs consisting of repetitive elements. Apart from genomic complexity, contig length and correctness depend on read length and accuracy. With pyrosequencing or Illumina reads, slag-assembled contigs are accurate enough to allow design of PCR primers, while contigs assembled from Oxford Nanopore or pre-HiFi Pacific Biosciences long reads are generally only accurate enough to design baiting sequences for further targeted sequencing. In an application with real reads, slag successfully extended sequences for four wheat genes, which were verified by cloning and Sanger sequencing of overlapping amplicons. slag is a robust alternative to atram2 for local assemblies, especially for read sets with less than 20× coverage. slag is freely available at https://github.com/cfcrane/SLAG.

Comparative Hessian Fly Larval Transcriptomics Provides Novel Insight into Host and Nonhost Resistance.

The Hessian fly is a destructive pest of wheat. Employing additional molecular strategies can complement wheat's native insect resistance. However, this requires functional characterization of Hessian-fly-responsive genes, which is challenging because of wheat genome complexity. The diploid Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly and displays phenotypic/molecular responses intermediate between resistant and susceptible host wheat, offering a surrogate genome for gene characterization. Here, we compared the transcriptomes of Biotype L larvae residing on resistant/susceptible wheat, and nonhost Bd plants. Larvae from susceptible wheat and nonhost Bd plants revealed similar molecular responses that were distinct from avirulent larval responses on resistant wheat. Secreted salivary gland proteins were strongly up-regulated in all larvae. Genes from various biological pathways and molecular processes were up-regulated in larvae from both susceptible wheat and nonhost Bd plants. However, Bd larval expression levels were intermediate between larvae from susceptible and resistant wheat. Most genes were down-regulated or unchanged in avirulent larvae, correlating with their inability to establish feeding sites and dying within 4-5 days after egg-hatch. Decreased gene expression in Bd larvae, compared to ones on susceptible wheat, potentially led to developmentally delayed 2nd-instars, followed by eventually succumbing to nonhost resistance defense mechanisms.

Insect derived extra oral GH32 plays a role in susceptibility of wheat to Hessian fly.

The Hessian fly is an obligate parasite of wheat causing significant economic damage, and triggers either a resistant or susceptible reaction. However, the molecular mechanisms of susceptibility leading to the establishment of the larvae are unknown. Larval survival on the plant requires the establishment of a steady source of readily available nutrition. Unlike other insect pests, the Hessian fly larvae have minute mandibles and cannot derive their nutrition by chewing tissue or sucking phloem sap. Here, we show that the virulent larvae produce the glycoside hydrolase MdesGH32 extra-orally, that localizes within the leaf tissue being fed upon. MdesGH32 has strong inulinase and invertase activity aiding in the breakdown of the plant cell wall inulin polymer into monomers and converting sucrose, the primary transport sugar in plants, to glucose and fructose, resulting in the formation of a nutrient-rich tissue. Our finding elucidates the molecular mechanism of nutrient sink formation and establishment of susceptibility.

Phenotypic and molecular characterization of Hessian fly resistance in diploid wheat, Aegilops tauschii.

BACKGROUND: The Hessian fly (Mayetiola destructor), belonging to the gall midge family (Cecidomyiidae), is a devastating pest of wheat (Triticum aestivum) causing significant yield losses. Despite identification and characterization of numerous Hessian fly-responsive genes and associated biological pathways involved in wheat defense against this dipteran pest, their functional validation has been challenging. This is largely attributed to the large genome, polyploidy, repetitive DNA, and limited genetic resources in hexaploid wheat. The diploid progenitor Aegilops tauschii, D-genome donor of modern-day hexaploid wheat, offers an ideal surrogate eliminating the need to target all three homeologous chromosomes (A, B and D) individually, and thereby making the functional validation of candidate Hessian fly-responsive genes plausible. Furthermore, the well-annotated sequence of Ae. tauschii genome and availability of genetic resources amenable to manipulations makes the functional assays less tedious and time-consuming. However, prior to utilization of this diploid genome for downstream studies, it is imperative to characterize its physical and molecular responses to Hessian fly. RESULTS: In this study we screened five Ae. tauschii accessions for their response to the Hessian fly biotypes L and vH13. Two lines were identified that exhibited a homozygous resistance response to feeding by both Hessian fly biotypes. Studies using physical measurements and neutral red staining showed that the resistant Ae. tauschii accessions resembled hexaploid wheat in their phenotypic responses to Hessian fly, that included similarities in larval developmental stages, leaf and plant growth, and cell wall permeability. Furthermore, molecular responses, characterized by gene expression profiling using quantitative real-time PCR, in select resistant Ae. tauschii lines also revealed similarities with resistant hexaploid wheat. CONCLUSIONS: Phenotypic and molecular characterization of Ae. tauschii to Hessian fly infestation revealed resistant accessions that shared similarities to hexaploid wheat. Resembling the resistant hexaploid wheat, the Ae. tauschii accessions mount an early defense strategy involving defense proteins including lectins, secondary metabolites and reactive oxygen species (ROS) radicals. Our results reveal the suitability of the diploid progenitor for use as an ideal tool for functional genomics research in deciphering the wheat-Hessian fly molecular interactions.

Multiple molecular defense strategies in Brachypodium distachyon surmount Hessian fly (Mayetiola destructor) larvae-induced susceptibility for plant survival.

The Hessian fly is a destructive pest of wheat causing severe economic damage. Numerous genes and associated biological pathways have been implicated in defense against Hessian fly. However, due to limited genetic resources, compounded with genome complexity, functional analysis of the candidate genes are challenging in wheat. Physically, Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly, and with a small genome size, short life cycle, vast genetic resources and amenability to transformation, it offers an alternate functional genomic model for deciphering plant-Hessian fly interactions. RNA-sequencing was used to reveal thousands of Hessian fly-responsive genes in Bd one, three, and five days after egg hatch. Genes encoding defense proteins, stress-regulating transcription factors, signaling kinases, and secondary metabolites were strongly up-regulated within the first 24 hours of larval feeding indicating an early defense, similar to resistant wheat. Defense was mediated by a hypersensitive response that included necrotic lesions, up-regulated ROS-generating and -scavenging enzymes, and H2O2 production. Suppression of cell wall-associated proteins and increased cell permeability in Bd resembled susceptible wheat. Thus, Bd molecular responses shared similarities to both resistant and susceptible wheat, validating its suitability as a model genome for undertaking functional studies of candidate Hessian fly-responsive genes.

Correction to: Agrobacterium: A Genome-Editing Tool-Delivery System.

By mistake the chapter was published with incorrect author name. The chapter has now been corrected.

Agrobacterium: A Genome-Editing Tool-Delivery System.

With the rapidly increasing global population, it will be extremely challenging to provide food to the world without increasing food production by at least 70% over the next 30 years. As we reach the limits of expanding arable land, the responsibility of meeting this production goal will rely on increasing yields. Traditional plant breeding practices will not be able to realistically meet these expectations, thrusting plant biotechnology into the limelight to fulfill these needs. Better varieties will need to be developed faster and with the least amount of regulatory hurdles. With the need to add, delete, and substitute genes into existing genomes, the field of genome editing and gene targeting is now rapidly developing with numerous new technologies coming to the forefront. Agrobacterium-mediated crop transformation has been the most utilized method to generate transgenic varieties that are better yielding, have new traits, and are disease and pathogen resistant. Genome-editing technologies rely on the creation of double-strand breaks (DSBs) in the genomic DNA of target species to facilitate gene disruption, addition, or replacement through either non-homologous end joining or homology-dependent repair mechanisms. DSBs can be introduced through the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspersed short palindromic repeats (CRISPR)/Cas nucleases, among others. Agrobacterium strains have been employed to deliver the reagents for genome editing to the specific target cells. Understanding the biology of transformation from the perspective not only of Agrobacterium, but also of the host, from processing of T-DNA to its integration in the host genome, has resulted in a wealth of information that has been used to engineer Agrobacterium strains having increased virulence. As more technologies are being developed, that will help overcome issues of Agrobacterium host range and random integration of DNA, combined with highly sequence-specific nucleases, a robust crop genome-editing toolkit finally seems attainable.

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.

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

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.

Avirulence effector discovery in a plant galling and plant parasitic arthropod, the Hessian fly (Mayetiola destructor).

Highly specialized obligate plant-parasites exist within several groups of arthropods (insects and mites). Many of these are important pests, but the molecular basis of their parasitism and its evolution are poorly understood. One hypothesis is that plant parasitic arthropods use effector proteins to defeat basal plant immunity and modulate plant growth. Because avirulence (Avr) gene discovery is a reliable method of effector identification, we tested this hypothesis using high-resolution molecular genetic mapping of an Avr gene (vH13) in the Hessian fly (HF, Mayetiola destructor), an important gall midge pest of wheat (Triticum spp.). Chromosome walking resolved the position of vH13, and revealed alleles that determine whether HF larvae are virulent (survive) or avirulent (die) on wheat seedlings carrying the wheat H13 resistance gene. Association mapping found three independent insertions in vH13 that appear to be responsible for H13-virulence in field populations. We observed vH13 transcription in H13-avirulent larvae and the salivary glands of H13-avirulent larvae, but not in H13-virulent larvae. RNA-interference-knockdown of vH13 transcripts allowed some H13-avirulent larvae to escape H13-directed resistance. vH13 is the first Avr gene identified in an arthropod. It encodes a small modular protein with no sequence similarities to other proteins in GenBank. These data clearly support the hypothesis that an effector-based strategy has evolved in multiple lineages of plant parasites, including arthropods.

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.

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.

Reactive oxygen species are involved in plant defense against a gall midge.

Reactive oxygen species (ROS) play a major role in plant defense against pathogens, but evidence for their role in defense against insects is still preliminary and inconsistent. In this study, we examined the potential role of ROS in defense of wheat (Triticum aestivum) and rice (Oryza sativa) against Hessian fly (Mayetiola destructor) larvae. Rapid and prolonged accumulation of hydrogen peroxide (H(2)O(2)) was detected in wheat plants at the attack site during incompatible interactions. Increased accumulation of both H(2)O(2) and superoxide was detected in rice plants during nonhost interactions with the larvae. No increase in accumulation of either H(2)O(2) or superoxide was observed in wheat plants during compatible interactions. A global analysis revealed changes in the abundances of 250 wheat transcripts and 320 rice transcripts encoding proteins potentially involved in ROS homeostasis. A large number of transcripts encoded class III peroxidases that increased in abundance during both incompatible and nonhost interactions, whereas the levels of these transcripts decreased in susceptible wheat during compatible interactions. The higher levels of class III peroxidase transcripts were associated with elevated enzymatic activity of peroxidases at the attack site in plants during incompatible and nonhost interactions. Overall, our data indicate that class III peroxidases may play a role in ROS generation in resistant wheat and nonhost rice plants during response to Hessian fly attacks.

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

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