Mobilization of lipids and fortification of cell wall and cuticle are important in host defense against Hessian fly

BackgroundWheat – Hessian fly interaction follows a typical gene-for-gene model. Hessian fly larvae die in wheat plants carrying an effective resistance gene, or thrive in susceptible plants that carry no effective resistance gene.ResultsGene sets affected by Hessian fly attack in resistant plants were found to be very different from those in susceptible plants. Differential expression of gene sets was associated with differential accumulation of intermediates in defense pathways. Our results indicated that resources were rapidly mobilized in resistant plants for defense, including extensive membrane remodeling and release of lipids, sugar catabolism, and amino acid transport and degradation. These resources were likely rapidly converted into defense molecules such as oxylipins; toxic proteins including cysteine proteases, inhibitors of digestive enzymes, and lectins; phenolics; and cell wall components. However, toxicity alone does not cause immediate lethality to Hessian fly larvae. Toxic defenses might slow down Hessian fly development and therefore give plants more time for other types of defense to become effective.ConclusionOur gene expression and metabolic profiling results suggested that remodeling and fortification of cell wall and cuticle by increased deposition of phenolics and enhanced cross-linking were likely to be crucial for insect mortality by depriving Hessian fly larvae of nutrients from host cells. The identification of a large number of genes that were differentially expressed at different time points during compatible and incompatible interactions also provided a foundation for further research on the molecular pathways that lead to wheat resistance and susceptibility to Hessian fly infestation.

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In this issue of PNAS, Dodds et al . (4) provide evidence for direct, allele-specific interaction between alleles of a particular R protein and the corresponding pathogen-encoded Avr proteins. In a remarkable arc of history, they do so by using cloned alleles from flax and flax rust loci that were defined genetically five decades ago by Flor (2, 3, 5, 6). Combined with other recent breakthroughs, this work suggests that plant R proteins can function either by directly detecting … 

‡To whom correspondence should be addressed at: Department of Biology, University of North Carolina, CB# 3280, 108 Coker Hall, Chapel Hill, NC 27599.  

dangl{at}email.unc.edu

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title: 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.

title: Functional Characterization of HFR1, a High-Mannose N-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae1[C][W]
abstract: We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His6-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His6-HFR1 to Manα1-6(Manα1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His6-HFR1 in the diet. Thus, HFR1 is an antinutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants.

title: Differential Responses of Wheat Inhibitor-like Genes to Hessian Fly, Mayetiola destructor, Attacks During Compatible and Incompatible Interactions
abstract: Four groups of inhibitor-like genes that encode proteins with diverse structures were identified from wheat. The majority of these genes were upregulated by avirulent Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), larvae during incompatible interactions, and were downregulated by virulent larvae during compatible interactions. The upregulation during incompatible interactions and downregulation during compatible interactions resulted in four- to 30-fold differences between the expression levels in resistant plants and those in susceptible plants. The increased expression of inhibitor-like genes during incompatible interactions suggested that these genes are part of defense mechanisms in wheat against Hessian fly attacks, whereas the downregulation during compatible interactions suggested that virulent larvae can suppress plant defenses. Both the upregulation of the inhibitor-like genes during incompatible interactions by avirulent larvae and the downregulation during compatible interactions by virulent larvae were through mechanisms that were independent of the wound response pathway.

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

title: Reactive Oxygen Species Are Involved in Plant Defense against a Gall Midge[C][W][OA]
abstract: 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 (H2O2) was detected in wheat plants at the attack site during incompatible interactions. Increased accumulation of both H2O2 and superoxide was detected in rice plants during nonhost interactions with the larvae. No increase in accumulation of either H2O2 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.

title: Changes in properties of wheat leaf cuticle during interactions with Hessian fly.
abstract: Infestation of wheat by Hessian fly larvae causes a variety of physical and biochemical modifications of the host plant. Changes occur in cuticle permeability, lipid composition and gene transcript abundance, and these responses differ substantially between resistant and susceptible wheat lines. Staining assays revealed that susceptible plants exhibited a generalized increase in leaf sheath epidermal permeability during infestation; whereas, epidermal permeability was only minimally affected in resistant plants. Furthermore, temporal profiling using gas chromatographic methods revealed that changes in cuticle lipid (wax and cutin) composition correlated well with differing levels of epidermal permeability in susceptible and resistant plants. Temporal analysis of cuticle-associated gene mRNA levels, by quantitative real-time PCR, indicated a relationship between transcript abundance and changes in cuticle lipid profiles of resistant and susceptible plants. Results suggest that conserving cuticle integrity via induction of specific wax constituents and maintenance of cutin amounts, determined by the accumulation of cuticle-associated transcripts, could be important components of wheat resistance to Hessian fly larvae.

title: Rapid mobilization of membrane lipids in wheat leaf sheaths during incompatible interactions with Hessian fly.
abstract: Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time polymerase chain reaction analyses of 168 lipid-metabolism-related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.

title: Feeding of Hessian fly (Diptera: Cecidomyiidae) larvae on resistant and susceptible wheat.
abstract: The duration of feeding and relative amounts consumed by Hessian fly, Mayetiola destructor (Say), larvae on resistant and susceptible seedling wheat plants labeled with [35S]L-methionine was investigated. Plants were labeled over 48-h periods from initial infestation to 8 d after infestation, and the uptake of 35S by larvae determined. With the H3 and H5 genes for resistance, the uptake of 35S by larvae was significantly less than on susceptible controls, and suggested that the larvae were unable to initiate or maintain sustained feeding on the resistant plants. With the H6 gene for resistance, uptake of 35S by larvae during the first 48 h was not significantly different from that on susceptible controls, but then declined between 48 and 96 h. These data indicate that with the H6 gene, larvae fed actively for the first 48 h after infesting the plants, then ceased their feeding between 48 and 96 h. All larvae on resistant plants were alive 48 h after infesting plants and approximately 60% were alive 96 h after infesting resistant plants. All larvae on resistant plants were dead by 144 h after infestation. Results are discussed in the context of possible mechanisms of action for the resistance genes investigated.

title: The phenylpropanoid pathway and plant defence-a genomics perspective.
abstract: The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex flavonoids, isoflavonoids, and stilbenes. The enzymatic steps involved in the biosynthesis of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned. Less is understood about the regulatory genes that orchestrate rapid, coordinated induction of phenylpropanoid defences in response to microbial attack. Many of the biosynthetic pathway enzymes are encoded by gene families, but the specific functions of individual family members remain to be determined. The availability of the complete genome sequence of Arabidopsis thaliana, and the extensive expressed sequence tag (EST) resources in other species, such as rice, soybean, barrel medic, and tomato, allow, for the first time, a full appreciation of the comparative genetic complexity of the phenylpropanoid pathway across species. In addition, gene expression array analysis and metabolic profiling approaches make possible comparative parallel analyses of global changes at the genome and metabolome levels, facilitating an understanding of the relationships between changes in specific transcripts and subsequent alterations in metabolism in response to infection.

title: Inducible direct plant defense against insect herbivores: A review
abstract: Plants respond to insect herbivory with responses broadly known as direct defenses, indirect defenses, and tolerance. Direct defenses include all plant traits that affect susceptibility of host plants by themselves. Overall categories of direct plant defenses against insect herbivores include limiting food supply, reducing nutrient value, reducing preference, disrupting physical structures, and inhibiting chemical pathways of the attacking insect. Major known defense chemicals include plant secondary metabolites, protein inhibitors of insect digestive enzymes, proteases, lectins, amino acid deaminases and oxidases. Multiple factors with additive or even synergistic impact are usually involved in defense against a specific insect species, and factors of major importance to one insect species may only be of secondary importance or not effective at all against another insect species. Extensive qualitative and quantitative high throughput analyses of temporal and spatial variations in gene expression, protein level and activity, and metabolite concentration will accelerate not only the understanding of the overall mechanisms of direct defense, but also accelerate the identification of specific targets for enhancement of plant resistance for agriculture.

title: Plant Defense Against Herbivores : Chemical Aspects
abstract: Plants have evolved a plethora of different chemical defenses covering nearly all classes of (secondary) metabolites that represent a major barrier to herbivory: Some are constitutive; others are induced after attack. Many compounds act directly on the herbivore, whereas others act indirectly via the attraction of organisms from other trophic levels that, in turn, protect the plant. An enormous diversity of plant (bio)chemicals are toxic, repellent, or antinutritive for herbivores of all types. Examples include cyanogenic glycosides, glucosinolates, alkaloids, and terpenoids; others are macromolecules and comprise latex or proteinase inhibitors. Their modes of action include membrane disruption, inhibition of nutrient and ion transport, inhibition of signal transduction processes, inhibition of metabolism, or disruption of the hormonal control of physiological processes. Recognizing the herbivore challenge and precise timing of plant activities as well as the adaptive modulation of the plants’ metabolism is important so that metabolites and energy may be efficiently allocated to defensive activities. 431 A nn u. R ev . P la nt B io l. 20 12 .6 3: 43 145 0. D ow nl oa de d fr om w w w .a nn ua lr ev ie w s. or g by S ta te U ni ve rs ity o f N ew Y or k C ol le ge o f E nv ir on m en ta l S ci en ce a nd F or es tr y on 0 8/ 26 /1 4. F or p er so na l u se o nl y. PP63CH18-Boland ARI 31 March 2012 9:52 Toxicity: general term for the property of substances indicating the degree to which a compound can damage a cell or organism; it is dose dependent and measured by the effect on the particular target Trophic level: the position occupied by an organism in a food chain

title: Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex.
abstract: Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.

title: Mir1-CP, a novel defense cysteine protease accumulates in maize vascular tissues in response to herbivory
abstract: When lepidopteran larvae feed on the insect-resistant maize genotype Mp708 there is a rapid accumulation of a defensive cysteine protease, Maize insect resistance 1-cysteine protease (Mir1-CP), at the feeding site. Silver-enhanced immunolocalization visualized with both light and transmission electron microscopy was used to determine the location of Mir1-CP in the maize leaf. The results indicated that Mir1-CP is localized predominantly in the phloem of minor and intermediate veins. After 24 h of larval feeding, Mir1-CP increased in abundance in the vascular parenchyma cells and in the thick-walled sieve element (TSE); it was also found localized to the bundle sheath and mesophyll cells. In situ hybridization of mRNA encoding Mir1-CP indicated that the primary sites of Mir1-CP synthesis in the whorl are the vascular parenchyma and bundle sheath cells. In addition to the phloem, Mir1-CP was also found in the metaxylem of the leaf and root. After 24 h of foliar feeding, the amount of Mir1-CP in the root xylem increased and it appeared to move from xylem parenchyma into the root metaxylem elements. The accumulation of Mir1-CP in maize vascular elements suggests Mir1-CP may move through these tissues to defend against insect herbivores.

title: Overexpression of a Gene Encoding Hydrogen Peroxide-Generating Oxalate Oxidase Evokes Defense Responses in Sunflower1
abstract: Oxalate oxidase (OXO) converts oxalic acid (OA) and O2 to CO2 and hydrogen peroxide (H2O2), and acts as a source of H2O2 in certain plant-pathogen interactions. To determine if the H2O2 produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H2O2. Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H2O2, salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H2O2 increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.

title: Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene1
abstract: Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%–89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.

title: Cell wall loosening by expansins.
abstract: In his 1881 book, The Power of Movement in Plants , Darwin described a now classic experiment in which he directed a tiny shaft of sunlight onto the tip of a grass seedling. The region below the coleoptile tip subsequently curved toward the light, leading to the notion of a transmissible growth

title: Changes in Phytohormones and Fatty Acids in Wheat and Rice Seedlings in Response to Hessian Fly (Diptera: Cecidomyiidae) Infestation
abstract: Phytohormones and fatty acids (FAs) play important roles in plant resistance to insects and pathogens. In this study, we investigated the similarities and differences in the accumulations of phytohormones and FAs in the resistant wheat (Triticum aestivum L.) ‘Molly’ and the nonhost rice (Oryza sativa L.) ‘Niponbare’ in responses to Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), larval attacks. Using chemical ionization-gas-chromatography/mass spectrometry, we analyzed the concentrations of 13 phytohomones and FAs at the attack site of wheat and rice plants at 1, 6, 24, or 48 h after the initial attack. Hessian fly attack resulted in increases of salicylic acid (SA), 12-oxo-phytodienoic acid (OPDA), palmitic acid (FA16:0), but a decrease of abscisic acid in both wheat and rice plants. In addition, the accumulation of jasmonic acid (JA) increased, whereas the accumulation of cinnamic acid (CA) decreased in wheat plants, but no changes were observed in the accumulation of JA, and the accumulation of CA increased in rice plants after Hessian fly attack. However, the accumulations of benzoic acid, strearic acid (FA18:0), and oleic acid (FA18:1) increased in rice plants, but no changes were observed in wheat plants after Hessian fly attack. Hessian fly-induced changes were more rapid in wheat plants in comparison with those in rice plants. Our study suggests that SA and OPDA may be involved in resistance of wheat and rice plants to Hessian fly and that the R gene-mediated resistance responses are more rapid than nonhost resistance responses.

title: THE OXIDATIVE BURST IN PLANT DISEASE RESISTANCE.
abstract: Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. Emerging data indicate that the oxidative burst reflects activation of a membrane-bound NADPH oxidase closely resembling that operating in activated neutrophils. The oxidants are not only direct protective agents, but H2O2 also functions as a substrate for oxidative cross-linking in the cell wall, as a threshold trigger for hypersensitive cell death, and as a diffusible signal for induction of cellular protectant genes in surrounding cells. Activation of the oxidative burst is a central component of a highly amplified and integrated signal system, also involving salicylic acid and perturbations of cytosolic Ca2+, which underlies the expression of disease-resistance mechanisms.

title: The apoplastic oxidative burst in response to biotic stress in plants: a three-component system.
abstract: The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.

title: Reactive oxygen species: metabolism, oxidative stress, and signal transduction.
abstract: Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

title: Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species
abstract: Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.

title: Reactive oxygen gene network of plants.
abstract: Reactive oxygen species (ROS) control many different processes in plants. However, being toxic molecules, they are also capable of injuring cells. How this conflict is resolved in plants is largely unknown. Nonetheless, it is clear that the steady-state level of ROS in cells needs to be tightly regulated. In Arabidopsis, a network of at least 152 genes is involved in managing the level of ROS. This network is highly dynamic and redundant, and encodes ROS-scavenging and ROS-producing proteins. Although recent studies have unraveled some of the key players in the network, many questions related to its mode of regulation, its protective roles and its modulation of signaling networks that control growth, development and stress response remain unanswered.

title: Obviation of Wheat Resistance to the Hessian Fly Through Systemic Induced Susceptibility
abstract: 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.

title: Genetics and Biochemistry of Insect Resistance in Maize
abstract: Insects are a major concern for maize production worldwide. Host plant resistance to insects involves a number of chemical and biochemical factors that limit, but rarely eliminate insect damage. Most chemical and many biochemical factors involved in resistance to insects are synthesized independent of the pest as phytoanticipines. These factors are stored in sequestered forms that are modified to active structures upon insect infestation and tissue damage. Because the genetic basis of varietal responses to insects for maize is quantitative in nature, quantitative trait locus analysis has been a standard approach to describe insect resistance. These studies often examined correlated biochemical traits to link genetic loci with biological mechanisms. Recently there has been a realization of the importance of herbivore enemies that are attracted by maize in response to herbivore damage. Upon infestation, maize releases volatile chemicals to actively recruit parasitic wasps or nematodes to combat the insect pests. In this review we examine the current state of knowledge of the biochemical, genetic and plant-insect tritrophic mechanisms involved in maize resistance to insect pests.

title: Role of Dehydrodiferulates in Maize Resistance to Pests and Diseases
abstract: Phenolic esters have attracted considerable interest due to the potential they offer for peroxidase catalysed cross-linking of cell wall polysaccharides. Particularly, feruloyl residues undergo radical coupling reactions that result in cross-linking (intra-/intermolecular) between polysaccharides, between polysaccharides and lignin and, between polysaccharides and proteins. This review addresses for the first time different studies in which it is established that cross-linking by dehydrodiferulates contributes to maize’s defences to pests and diseases. Dehydrodiferulate cross-links are involved in maize defence mechanisms against insects such as the European, Mediterranean, and tropical corn borers and, storage pest as the maize weevil. In addition, cross-links are also discussed to be involved in genetic resistance of maize to fungus diseases as Gibberella ear and stalk rot. Resistance against insects and fungus attending dehydrodiferulates could go hand in hand. Quantitative trait loci mapping for these cell wall components could be a useful tool for enhancing resistance to pest and diseases in future breeding programs.

title: Virulence Analysis of Hessian Fly Populations From Texas, Oklahoma, and Kansas
abstract: In recent years, the number of wheat, Triticum aestivum L., fields heavily infested by Hessian fly, Mayetiola destructor (Say), has increased in the Great Plains of the United States. Historically, resistance genes in wheat have been the most efficient means of controlling this insect pest. To determine which resistance genes are still effective in this area, virulence of six Hessian fly populations from Texas, Oklahoma, and Kansas was determined, using the resistance genes H3, H4, H5, H6, H7H8, H9, H10, H11, H12, H13, H16, H17, H18, H21, H22, H23, H24, H25, H26, H31, and Hdic. Five of the tested genes, H13, H21, H25, H26, and Hdic, conferred high levels of resistance (>80% of plants scored resistant) to all tested populations. Resistance levels for other genes varied depending on which Hessian fly population they were tested against. Biotype composition analysis of insects collected directly from wheat fields in Grayson County, TX, revealed that the proportion of individuals within this population virulent to the major resistance genes was highly variable (89% for H6, 58% for H9, 28% for H5, 22% for H26, 15% for H3, 9% for H18, 4% for H21, and 0% for H13). Results also revealed that the percentages of biotypes virulent to specific resistance genes in a given population are highly correlated (r2 = 0.97) with the percentages of susceptible plants in a virulence test. This suggests that virulence assays, which require less time and effort, can be used to approximate biotype composition.

title: Controlling the false discovery rate: a practical and powerful approach to multiple testing
abstract: The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.

title: Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments
abstract: The problem of identifying differentially expressed genes in designed microarray experiments is considered. Lonnstedt and Speed (2002) derived an expression for the posterior odds of differential expression in a replicated two-color experiment using a simple hierarchical parametric model. The purpose of this paper is to develop the hierarchical model of Lonnstedt and Speed (2002) into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples. The model is reset in the context of general linear models with arbitrary coefficients and contrasts of interest. The approach applies equally well to both single channel and two color microarray experiments. Consistent, closed form estimators are derived for the hyperparameters in the model. The estimators proposed have robust behavior even for small numbers of arrays and allow for incomplete data arising from spot filtering or spot quality weights. The posterior odds statistic is reformulated in terms of a moderated t-statistic in which posterior residual standard deviations are used in place of ordinary standard deviations. The empirical Bayes approach is equivalent to shrinkage of the estimated sample variances towards a pooled estimate, resulting in far more stable inference when the number of arrays is small. The use of moderated t-statistics has the advantage over the posterior odds that the number of hyperparameters which need to estimated is reduced; in particular, knowledge of the non-null prior for the fold changes are not required. The moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom. The moderated t inferential approach extends to accommodate tests of composite null hypotheses through the use of moderated F-statistics. The performance of the methods is demonstrated in a simulation study. Results are presented for two publicly available data sets.

title: Global metabolic profiling of human osteoarthritic synovium.
abstract: Osteoarthritis (OA) is a debilitating disease associated with pain and loss of function in numerous diarthrodial joints of the body. Assessments of the severity and/or progression of OA are commonly based on radiographic stages and pain level, which aren't always correlated to severity of disease or joint dysfunction and may be confounded by other factors(1). There has been recent interest in identifying a biochemical signature of OA(1) that may be detected in serum, urine, and/or synovial fluid that would represent repeatable and predictable biomarkers of OA onset and/or progression. The objective of this study was to use global metabolic profiling to identify a distinct metabolic profile for cultured human synovial tissue from patients with end-stage OA compared to patients with little or no evidence of disease. While metabolic profiles from cultured tissues are not expected to reproduce in vivo profiles, it is expected that perturbations in metabolism caused by end-stage disease would result in differences in metabolic profiles in vitro compared to tissue with little or no evidence of disease. Because metabolomic perturbations often occur prior to alterations in the genome or proteome, metabolomic analysis possibly provides an earlier window to an altered biochemical profile for OA onset and/or progression, and may provide a unique set of potential drug targets. The synovium was targeted because it has been implicated in OA as a mediator of disease progression; osteoarthritic synovium has been demonstrated to express pro-inflammatory cytokines, such as Tumor Necrosis Factor - α (TNF-α), Interleukin-1 β (IL-1β), and IL-6(2), suggesting that a diseased synovial lining could produce an ideal set of biomarkers for diagnosing OA and/or monitoring disease progression. Media from the culture of synovial explants dissected from diseased human joints (early or end-stage OA) was subjected to global metabolic profiling with a liquid chromatography (LC)/and gas chromatography (GC)/mass spectrophotometry (MS)-based technology platform. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries developed at Metabolon, Inc (Durham, NC). Global metabolic profiling resulted in the identification of 105 distinct compounds across all sample groups, with 11 compounds showing significantly different relative concentrations between end-stage and no/early disease groups. Metabolites specific to collagen metabolism, branched-chain amino acid metabolism, energy metabolism and tryptophan metabolism were amongst the most significant compounds, suggesting an altered metabolic state with disease progression.