Marion O. Harris

Virulent Hessian Fly (Diptera: Cecidomyiidae) Larvae Induce a Nutritive Tissue During Compatible Interactions with Wheat

Abstract The compatible interaction between virulent Hessian fly, Mayetiola destructor (Say), larvae and susceptible wheat, Triticum aestivum L., plants was investigated at the light microscope and ultrastructural levels. During the first day of larval attack at the base of the sheath of the third leaf of a wheat seedling, small punctures of the appropriate size (0.1 μm in diameter) and spacing of the paired larval mandibles were found in the outer wall of epidermal cells. Inside epidermal cells, nuclei and cytoplasmic organelles seemed to be breaking down, and the number and size of cytoplasmic vacuoles had increased. Two to 3 d later, epidermal and mesophyll cells at the base of the third leaf showed signs of becoming nutritive. Nutritive cells were identified by an increase in cytoplasmic staining, increased numbers of cellular organelles (mitochondria, proplastids, Golgi, and rough endoplasmic reticulum), numerous small vacuoles, and an irregularly shaped nucleus. The Hessian fly nutritive tissue probably acts as a sink tissue within the wheat seedling, benefiting the growth of larvae by importing photoassimilates. Breakdown of nutritive cells began soon after they were first observed, indicated by a change in the shape and density of the cell nucleus. Contents of nutritive cells moved through compromised cell walls into adjacent cells that had a more complete breakdown and loss of cytoplasm. Structural changes were not restricted to the third leaf. The sixth leaf, a leaf more recently initiated by the shoot apical meristem that was not directly fed upon by larvae, was found to consist primarily of well-developed epidermal layers, with poorly developed mesophyll cells. The implications of these findings for understanding incompatible interactions between avirulent Hessian fly larvae and R gene-defended plants are briefly discussed.

Gene Expression of Different Wheat Genotypes During Attack by Virulent and Avirulent Hessian Fly (Mayetiola destructor) Larvae

Wheat and its relatives possess a number of resistance (R) genes specific for the Hessian fly (HF) [Mayetiola destructor (Say)]. HF populations overcome R gene resistance by evolving virulence. Virulent HF larvae manipulate the plant to produce a nutritionally enhanced feeding tissue and, probably, also suppress plant defense responses. Using two wheat R genes, H9 and H13, and three HF strains (biotypes) differing in virulence for H9 and H13, we conducted a genome-wide transcriptional analysis of gene expression during compatible interactions with virulent larvae and incompatible interactions with avirulent larvae. During both types of interactions, a large number of genes (>1,000) showed alterations in gene expression. Analysis of genes with known functions revealed that major targets for differential regulation were genes that encoded defense proteins or enzymes involved in the phenylpropanoid, cell wall, and lipid metabolism pathways. A combination of the enhancement of antibiosis defense, the evasion of nutrient metabolism induction, and the fortification and expansion of the cell wall are likely the collective mechanism for host-plant resistance observed during incompatible interactions. To overcome this resistance, virulent larvae appeared to suppress antibiosis defense while inducing nutrient metabolism, weakening cell wall, and inhibiting plant growth.

Gall midges (Hessian flies) as plant pathogens.

Gall midges constitute an important group of plant-parasitic insects. The Hessian fly (HF; Mayetiola destructor), the most investigated gall midge, was the first insect hypothesized to have a gene-for-gene interaction with its host plant, wheat (Triticum spp.). Recent investigations support that hypothesis. The minute larval mandibles appear to act in a manner that is analogous to nematode stylets and the haustoria of filamentous plant pathogens. Putative effector proteins are encoded by hundreds of genes and expressed in the HF larval salivary gland. Cultivar-specific resistance (R) genes mediate a highly localized plant reaction that prevents the survival of avirulent HF larvae. Fine-scale mapping of HF avirulence (Avr) genes provides further evidence of effector-triggered immunity (ETI) against HF in wheat. Taken together, these discoveries suggest that the HF, and other gall midges, may be considered biotrophic, or hemibiotrophic, plant pathogens, and they demonstrate the potential that the wheat-HF interaction has in the study of insect-induced plant gall formation.

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.

A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.

Does R Gene Resistance Allow Wheat to Prevent Plant Growth Effects Associated with Hessian Fly (Diptera: Cecidomyiidae) Attack?

Resistance genes (R genes) are an important part of the plants immune system. Among insects, the Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), larva is the target of the greatest number of characterized R genes (H1-H32). The biochemical/molecular mechanism of R gene resistance to Hessian fly is not well understood. In the absence of an effective R gene, larvae caused extensive growth deficits (> 30 cm) in wheat seedlings. In the presence of one of three effective R genes, H6, H9, or H13, larvae caused small growth deficits (approximately 3-4 cm) in two leaves (third and fourth) that were actively growing during the first days of larval attack. After larvae died on R gene plants, the fifth leaf and tiller leaves exhibited small increases in growth (2-4 cm). Growth responses of susceptible and resistant plants diverged at a time when Hessian fly larvae were establishing a nutritive gall tissue at feeding sites. The results of this study support the hypothesis that R gene resistance cannot prevent initial larval attack, but, by stopping the formation of the larval gall, it prevents the most serious consequences of larval attack.

Identification of 1-octacosanal and 6-methoxy-2-benzoxazolinone from wheat as ovipositional stimulants for hessian fly, Mayetiola destructor

Bioassay-guided fractionations of a dichloromethane extract of wheat epicuticular wax allowed the identification of 1-octacosanal and 6-methoxy-2-benzoxazolinone (MBOA) as the major components that stimulate oviposition by the Hessian fly, Mayetiola destructor. These compounds were identified by their mass spectral fragmentation patterns and by comparison of their gas chromatographic retention times with synthetic samples. Synthetic samples of 1-octacosanal or MBOA stimulated significant oviposition when compared with solvent controls. In combination, these compounds elicited a synergistic effect on the number of eggs laid by females compared to when they were presented alone. In a choice bioassay, a mixture of synthetic 1-octacosanal and MBOA in the approximate concentrations determined to be present in one plant equivalent of crude extract stimulated the same amount of oviposition as one plant equivalent of extract. This showed that together these two compounds appear to be responsible for the major proportion of the ovipositional stimulatory activity of the wheat epicuticular wax extract. Comparison of the activity of five straight-chain primary aldehydes with chain lengths from C22 to C30 revealed a relationship between chain length and the number of eggs laid by female Hessian flies, with 1-hexacosanal and 1-heptacosanal the most active of the aldehydes tested.

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.

Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae)

BackgroundThe chemical senses of insects mediate behaviors that are closely linked to survival and reproduction. The order Diptera contains two model organisms, the vinegar fly Drosophila melanogaster and the mosquito Anopheles gambiae, whose chemosensory genes have been extensively studied. Representing a third dipteran lineage with an interesting phylogenetic position, and being ecologically distinct by feeding on plants, the Hessian fly (Mayetiola destructor Say, Diptera: Cecidomyiidae) genome sequence has recently become available. Among plant-feeding insects, the Hessian fly is unusual in ‘reprogramming’ the plant to create a superior food and in being the target of plant resistance genes, a feature shared by plant pathogens. Chemoreception is essential for reproductive success, including detection of sex pheromone and plant-produced chemicals by males and females, respectively.ResultsWe identified genes encoding 122 odorant receptors (OR), 28 gustatory receptors (GR), 39 ionotropic receptors (IR), 32 odorant binding proteins, and 7 sensory neuron membrane proteins in the Hessian fly genome. We then mapped Illumina-sequenced transcriptome reads to the genome to explore gene expression in male and female antennae and terminal abdominal segments. Our results reveal that a large number of chemosensory genes have up-regulated expression in the antennae, and the expression is in many cases sex-specific. Sex-specific expression is particularly evident among the Or genes, consistent with the sex-divergent olfactory-mediated behaviors of the adults. In addition, the large number of Ors in the genome but the reduced set of Grs and divergent Irs suggest that the short-lived adults rely more on long-range olfaction than on short-range gustation. We also report up-regulated expression of some genes from all chemosensory gene families in the terminal segments of the abdomen, which play important roles in reproduction.ConclusionsWe show that a large number of the chemosensory genes in the Hessian fly genome have sex- and tissue-specific expression profiles. Our findings provide the first insights into the molecular basis of chemoreception in plant-feeding flies, representing an important advance toward a more complete understanding of olfaction in Diptera and its links to ecological specialization.

Pivoting from Arabidopsis to wheat to understand how agricultural plants integrate responses to biotic stress

In this review, we argue for a research initiative on wheats responses to biotic stress. One goal is to begin a conversation between the disparate communities of plant pathology and entomology. Another is to understand how responses to a variety of agents of biotic stress are integrated in an important crop. We propose gene-for-gene interactions as the focus of the research initiative. On the parasites side is an Avirulence (Avr) gene that encodes one of the many effector proteins the parasite applies to the plant to assist with colonization. On the plants side is a Resistance (R) gene that mediates a surveillance system that detects the Avr protein directly or indirectly and triggers effector-triggered plant immunity. Even though arthropods are responsible for a significant proportion of plant biotic stress, they have not been integrated into important models of plant immunity that come from plant pathology. A roadblock has been the absence of molecular evidence for arthropod Avr effectors. Thirty years after this evidence was discovered in a plant pathogen, there is now evidence for arthropods with the cloning of the Hessian flys vH13 Avr gene. After reviewing the two models of plant immunity, we discuss how arthropods could be incorporated. We end by showing features that make wheat an interesting system for plant immunity, including 479 resistance genes known from agriculture that target viruses, bacteria, fungi, nematodes, insects, and mites. It is not likely that humans will be subsisting on Arabidopsis in the year 2050. It is time to start understanding how agricultural plants integrate responses to biotic stress.