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In response to insect attack, many plants exhibit dynamic biochemical changes, resulting in the induced production of direct and indirect defenses. Elicitors present in herbivore oral secretions are believed to positively regulate many inducible plant defenses; however, little is known about the specificity of elicitor recognition in plants. To investigate the phylogenic distribution of elicitor activity, we tested representatives from three different elicitor classes on the time course of defense-related phytohormone production, including ethylene (E), jasmonic acid (JA), and salicylic acid, in a range of plant species spanning angiosperm diversity. All families examined responded to at least one elicitor class with significant increases in E and JA production within 1 to 2 h after treatment, yet elicitation activity among species was highly idiosyncratic. The fatty-acid amino acid conjugate volicitin exhibited the widest range of phytohormone and volatile inducing activity, which spanned maize (Zea mays), soybean (Glycine max), and eggplant (Solanum melongena). In contrast, the activity of inceptin-related peptides, originally described in cowpea (Vigna unguiculata), was limited even within the Fabaceae. Similarly, caeliferin A16:0, a disulfooxy fatty acid from grasshoppers, was the only elicitor with demonstrable activity in Arabidopsis thaliana. Although precise mechanisms remain unknown, the unpredictable nature of elicitor activity between plant species supports the existence of specific receptor-ligand interactions mediating recognition. Despite the lack of an ideal plant model for studying the action of numerous elicitors, E and JA exist as highly conserved and readily quantifiable markers for future discoveries in this field. ethylene | insect elicitor | jasmonic acid | plant defense | volatile organic compound

A previously unidentified class of compounds has been isolated from the regurgitant of the grasshopper species Schistocerca americana. These compounds (named here ''caeliferins') are composed of saturated and monounsaturated sulfated [alpha]-hydroxy fatty acids in which the [omega]-carbon is functionalized with either a sulfated hydroxyl or a carboxyl conjugated to glycine via an amide bond. The regurgitant contains a series of these compounds with fatty acid chains of 15-20 carbons and in varying proportions. Of these, the 16-carbon analogs are predominant and are also most active in inducing release of volatile organic compounds when applied to damaged leaves of corn seedlings. Caeliferins are nonlepidopteran elicitors identified in insect herbivores. This adds a category of insect herbivore-produced elicitors of plant responses, providing further evidence of the ability of plants to detect and respond to a broad range of insect herbivore-produced compounds. American grasshopper | insect herbivory | regurgitant | caeliferin

Plants can perceive a wide range of biotic attackers an respond with targeted induced defences. Specificity in plant non-self-recognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide ([sup.+][ICDINGVCVDA.sup.-], termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol [leaf.sup.-1] and triggers increases in the defense-related phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase [gamma]-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase [gamma]-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack. elicitor | guard hypothesis | indirect perception | insect herbivory | plant defense

Green leafy volatiles (GLV), six-carbon aldehydes, alcohols, and esters commonly emitted by plants in response to mechanical damage or herbivory, induced intact undamaged corn seedlings to rapidly produce jasmonic acid (JA) and emit sesquiterpenes. More importantly, corn seedlings previously exposed to GLV from neighboring plants produced significantly more JA and volatile sesquiterpenes when mechanically damaged and induced with caterpillar regurgitant than seedlings not exposed to GLV. The use of pure synthetic chemicals revealed that (Z)-3-hexenal, (Z)-3-hexen-1-ol, and (Z)-3-hexenyl acetate have nearly identical priming activity. Caterpillar-induced nocturnal volatiles, which are enriched in GLV, also exhibited a strong priming effect, inducing production of larger amounts of JA and release of greater quantities of volatile organic compounds after caterpillar regurgitant application. In contrast, GLV priming did not affect JA production induced by mechanical wounding alone. Thus, GLV specifically prime neighboring plants against impending herbivory by enhancing inducible chemical defense responses triggered during attack and may play a key role in plant--plant signaling and plant--insect interactions.

Phytohormones regulate the protective responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic actions referred to as signaling crosstalk. A bottleneck in crosstalk research is the quantification of numerous interacting phytohormones and regulators. The chemical analysis of salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid is typically achieved by using separate and complex methodologies. Moreover, pathogen-produced phytohormone mimics, such as the phytotoxin coronatine (COR), have not been directly quantified in plant tissues. We address these problems by using a simple preparation and a GC-MS-based metabolic profiling approach. Plant tissue is extracted in aqueous 1-propanol and mixed with dichloromethane. Carboxylic acids present in the organic layer are methylated by using trimethylsilyldiazomethane; analytes are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a sample vial. Analytes are separated by using gas chromatography and quantified by using chemical-ionization mass spectrometry that produces predominantly [[M+H].sup.+] parent ions. We use this technique to examine levels of COR, phytohormones, and volatile organic compounds in model systems, including Arabidopsis thaliana during infection with Pseudomonas syringae pv. tomato DC3000, corn (Zea mays) under herbivory by corn earworm (Helicoverpa zea), tobacco (Nicotiana tabacum) after mechanical damage, and tomato (Lycopersicon esculentum) during drought stress. Numerous complex changes induced by pathogen infection, including the accumulation of COR, salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid illustrate the potential and simplicity of this approach in quantifying signaling crosstalk interactions that occur at the level of synthesis and accumulation.

The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis. [ABSTRACT FROM AUTHOR]

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production. [ABSTRACT FROM AUTHOR]

Maize and a variety of other plant species release volatile compounds in response to herbivore attack that serve as chemical cues to signal natural enemies of the feeding herbivore. N-(17-hydroxy-linolenoyl)-L-glutamine is an elicitor component that has been isolated and chemically characterized from the regurgitant of the herbivore-pest beet armyworm. This fatty acid derivative, referred to as volicitin, triggers the synthesis and release of volatile components, including terpenoids and indole in maize. Here we report on a previously unidentified enzyme, indole-3-glycerol phosphate lyase (IGL), that catalyzes the formation of free indole and is selectively activated by volicitin. IGL's enzymatic properties are similar to BX1, a maize enzyme that serves as the entry point to the secondary defense metabolites DIBOA and DIMBOA. Genesequence analysis indicates that Igl and Bx1 are evolutionarily related to the tryptophan synthase alpha subunit.

Lysine acetylation is a key posttranslational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established, and it is known that pathogen effector proteins encoding acetyltransferases can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here, we investigate how the effector molecule HC-toxin (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes virulence in maize through altering protein acetylation. Using mass spectrometry, we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HCT as well as HCT-deficient or HCT-producing strains of C. carbonum. Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum. [ABSTRACT FROM AUTHOR]

Astigmatid mites depend on bioactive glandular secretions, pheromones, and defensive agents to mediate intra- and interspecies interactions. Aliphatic formates, such as (Z,Z)-8,11-heptadecadienyl formate (8,11-F17) and (Z)-8-heptadecenyl formate (8-F17), are rarely encountered natural products that are abundant in Sancassania sp. Sasagawa (Acari: Acaridae) mite secretions. Linoleic acid and oleic acid are predicted as key intermediates in the synthesis of the closely related aliphatic formates. To gain insight in this biosynthetic pathway, acarid mite feeding experiments were conducted using 13C-labeled precursors to precisely track incorporation. Analyses using 13C NMR spectroscopy demonstrated that the 13C-labeling pattern of the precursors was detectable on formates in exocrine secretions and likewise on fatty acids in total lipid pools. Curiously, the results demonstrated that the formates were biosynthesizedwithout the dehomologation of corresponding fatty acids. Careful examination of the mass spectra from labeling experiments revealed that the carbonyl carbon of the formates is originally derived from the C-1 position of the fatty acids. Consistent with a Baeyer-Villiger oxidation reaction, labeling studies support the insertion of an oxygen atom between the carbonyl group and carbon chain. Empirical data support the existence of a Baeyer- Villiger monooxygenase responsible for the catalyzation of the Baeyer-Villiger oxidation. The predicted existence of a Baeyer-Villiger monooxygenase capable of converting aliphatic aldehydes to formates represents an exciting opportunity to expand the enzymatic toolbox available for controlled biochemical synthesis. [ABSTRACT FROM AUTHOR]

Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. As the first described plant peptide signal, systemin regulates antiherbivore defenses in the Solanaceae, but in other plant families, peptides with analogous activity have remained elusive. In the current study, we demonstrate that a member of the maize (Zea mays) plant elicitor peptide (Pep) family, ZmPep3, regulates responses against herbivores. Consistent with being a signal, expression of the ZmPROPEP3 precursor gene is rapidly induced by Spodoptera exigua oral secretions. At concentrations starting at 5 pmol per leaf, ZmPep3 stimulates production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivory defense. These include proteinase inhibitors and biosynthetic enzymes for production of volatile terpenes and benzoxazinoids. In accordance with gene expression data, plants treated with ZmPep3 emit volatiles similar to those from plants subjected to herbivory. ZmPep3-treated plants also exhibit induced accumulation of the benzoxazinoid phytoalexin 2-hydroxy-4,7-dimethoxy-1,4-benzoxazjn-3-one glucoside. Direct and indirect defenses induced by ZmPep3 contribute to resistance against S. exigua through significant reduction of larval growth and attraction of Cotesia marginiventris parasitoids. ZmPep3 activity is specific to Poaceous species; however, peptides derived from PROPEP orthologs identified in Solanaceous and Fabaceous plants also induce herbivory-associated volatiles in their respective species. These studies demonstrate that Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing functional homologs of systemin outside of the Solanaceae. [ABSTRACT FROM AUTHOR]

Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane-related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 μg∙g-1 fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 μg∙mL-1, kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function. [ABSTRACT FROM AUTHOR]

A previously unidentified class of compounds has been isolated from the regurgitant of the grasshopper species Schistocerca americana. These compounds (named here ‘caeliferins’) are composed of saturated and monounsaturated sulfated α-hydroxy fatty acids in which the ω-carbon is functionalized with either a sulfated hydroxyl or a carboxyl conjugated to glycine via an amide bond. The regurgitant contains a series of these compounds with fatty acid chains of 15–20 carbons and in varying proportions. Of these, the 16-carbon analogs are predominant and are also most active in inducing release of volatile organic compounds when applied to damaged leaves of corn seedlings. Caeliferins are nonlepidopteran elicitors identified in insect herbivores. This adds a category of insect herbivore-produced elicitors of plant responses, providing further evidence of the ability of plants to detect and respond to a broad range of insect herbivore-produced compounds. [ABSTRACT FROM AUTHOR]

Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed "jasmonates." As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed "death acids." 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions.