Convergent evolution of a metabolic switch between aphid and caterpillar resistance in cereals.
ABSTRACT: Tailoring defense responses to different attackers is important for plant performance. Plants can use secondary metabolites with dual functions in resistance and defense signaling to mount herbivore-specific responses. To date, the specificity and evolution of this mechanism are unclear. Here, we studied the functional architecture, specificity, and genetic basis of defense regulation by benzoxazinoids in cereals. We document that DIMBOA-Glc induces callose as an aphid resistance factor in wheat. O-methylation of DIMBOA-Glc to HDMBOA-Glc increases plant resistance to caterpillars but reduces callose inducibility and resistance to aphids. DIMBOA-Glc induces callose in wheat and maize, but not in Arabidopsis, while the glucosinolate 4MO-I3M does the opposite. We identify a wheat O-methyltransferase (TaBX10) that is induced by caterpillar feeding and converts DIMBOA-Glc to HDMBOA-Glc in vitro. While the core pathway of benzoxazinoid biosynthesis is conserved between wheat and maize, the wheat genome does not contain close homologs of the maize DIMBOA-Glc O-methyltransferase genes, and TaBx10 is only distantly related. Thus, the functional architecture of herbivore-specific defense regulation is similar in maize and wheat, but the regulating biosynthetic genes likely evolved separately. This study shows how two different cereal species independently achieved herbivore-specific defense activation by regulating secondary metabolite production.
Project description:Aphids are major pests in cereal crops that cause direct and indirect damage leading to yield reduction. Despite the fact that wheat provides 20% of the world's caloric and protein diet, its metabolic responses to aphid attack, in general, and specifically its production of benzoxazinoid defense compounds are poorly understood. The objective of this study was to compare the metabolic diversity of durum wheat seedlings (Triticum turgidum ssp. durum) under attack by three different cereal aphids: i) the English grain aphid (Sitobion avenae Fabricius), ii) the bird cherry-oat aphid (Rhopalosiphum padi L.), and iii) the greenbug aphid (Schizaphis graminum Rondani), which are some of the most destructive aphid species to wheat. Insect progeny bioassays and metabolic analyses using chromatography/Q-Exactive/mass spectrometry non-targeted metabolomics and a targeted benzoxazinoid profile were performed on infested leaves. The insect bioassays revealed that the plants were susceptible to S. graminum, resistant to S. avenae, and mildly resistant to R. padi. The metabolic analyses of benzoxazinoids suggested that the predominant metabolites DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin- 3-one) and its glycosylated form DIMBOA-glucoside (Glc) were significantly induced upon both S. avenae, and R. padi aphid feeding. However, the levels of the benzoxazinoid metabolite HDMBOA-Glc (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside) were enhanced due to the feeding of S. avenae and S. graminum aphids, to which Svevo was the most resistant and the most susceptible, respectively. The results showed a partial correlation between the induction of benzoxazinoids and aphid reproduction. Overall, our observations revealed diverse metabolic responses of wheat seedlings to cereal aphid feeding.
Project description:Highly adapted herbivores can phenocopy two-component systems by stabilizing, sequestering and reactivating plant toxins. However, whether these traits protect herbivores against their enemies is poorly understood. We demonstrate that the western corn rootworm Diabrotica virgifera virgifera, the most damaging maize pest on the planet, specifically accumulates the root-derived benzoxazinoid glucosides HDMBOA-Glc and MBOA-Glc. MBOA-Glc is produced by D. virgifera through stabilization of the benzoxazinoid breakdown product MBOA by N-glycosylation. The larvae can hydrolyze HDMBOA-Glc, but not MBOA-Glc, to produce toxic MBOA upon predator attack. Accumulation of benzoxazinoids renders D. virgifera highly resistant to nematodes which inject and feed on entomopathogenic symbiotic bacteria. While HDMBOA-Glc and MBOA reduce the growth and infectivity of both the nematodes and the bacteria, MBOA-Glc repels infective juvenile nematodes. Our results illustrate how herbivores combine stabilized and reactivated plant toxins to defend themselves against a deadly symbiosis between the third and the fourth trophic level enemies.
Project description: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-hydroxylinolenoyl)-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. Gene-sequence analysis indicates that Igl and Bx1 are evolutionarily related to the tryptophan synthase alpha subunit.
Project description:Plant disease can be effectively suppressed in intercropping systems. Our previous study demonstrated that neighboring maize plants can restrict the spread of soil-borne pathogens of pepper plants by secreting defense compounds into the soil. However, whether maize plant can receive benefits from its neighboring pepper plants in an intercropping system is little attention. We examined the effects of maize roots treated with elicitors from the pepper pathogen Phytophthora capsici and pepper root exudates on the synthesis of 1,4-benzoxazine-3-ones (BXs), the expression of defense-related genes in maize, and their ability to alleviate the severity of southern corn leaf blight (SCLB) caused by Bipolaris maydis. We found that SCLB was significantly reduced after the above treatments. The contents of 1,4-benzoxazine-3-ones (BXs: DIBOA, DIMBOA, and MBOA) and the expression levels of BX synthesis and defense genes in maize roots and shoots were up-regulated. DIMBOA and MBOA effectively inhibited the mycelium growth of Bipolaris maydis at physiological concentrations in maize shoots. Further studies suggested that the defense related pathways or genes in maize roots and shoots were activated by elicitors from the P. capsici or pepper root exudates. In conclusion, maize increased the levels of BXs and defense gene expression both in roots and shoots after being triggered by root exudates and pathogen from neighboring pepper plants, eventually enhancing its resistance.
Project description:Herbivore-induced plant responses have been widely described following attack on leaves; however, less attention has been paid to analogous local processes that occur in stems. Early studies of maize (Zea mays) responses to stem boring by European corn borer (ECB, Ostrinianubilalis) larvae revealed the presence of inducible acidic diterpenoid phytoalexins, termed kauralexins, and increases in the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-Glc) after 24 h of herbivory. Despite these rapidly activated defenses, larval growth was not altered in short-term feeding assays. Unexpectedly, ECB growth significantly improved in assays using stem tissue preconditioned by 48 h of larval tunneling. Correspondingly, measures of total soluble protein increased over 2.6-fold in these challenged tissues and were accompanied by elevated levels of sucrose and free linoleic acid. While microarray analyses revealed up-regulation of over 1100 transcripts, fewer individual protein increases were demonstrable. Consistent with induced endoreduplication, both wounding and ECB stem attack resulted in similar significant expansion of the nucleus, nucleolus and levels of extractable DNA from challenged tissues. While many of these responses are triggered by wounding alone, biochemical changes further enhanced in response to ECB may be due to larval secreted effectors. Unlike other Lepidoptera examined, ECB excrete exceedingly high levels of the auxin indole-3-acetic acid (IAA) in their frass which is likely to contact and contaminate the surrounding feeding tunnel. Stem exposure to a metabolically stable auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D), promoted significant protein accumulation above wounding alone. As a future testable hypothesis, we propose that ECB-associated IAA may function as a candidate herbivore effector promoting the increased nutritional content of maize stems.
Project description:Plants defend themselves against herbivores through the production of toxic and deterrent metabolites. Adapted herbivores can tolerate and sometimes sequester these metabolites, allowing them to feed on defended plants and become toxic to their own enemies. Can herbivore natural enemies overcome sequestered plant defense metabolites to prey on adapted herbivores? To address this question, we studied how entomopathogenic nematodes cope with benzoxazinoid defense metabolites that are produced by grasses and sequestered by a specialist maize herbivore, the western corn rootworm. We find that nematodes from US maize fields in regions in which the western corn rootworm was present over the last 50 y are behaviorally and metabolically resistant to sequestered benzoxazinoids and more infective toward the western corn rootworm than nematodes from other parts of the world. Exposure of a benzoxazinoid-susceptible nematode strain to the western corn rootworm for 5 generations results in higher behavioral and metabolic resistance and benzoxazinoid-dependent infectivity toward the western corn rootworm. Thus, herbivores that are exposed to a plant defense sequestering herbivore can evolve both behavioral and metabolic resistance to plant defense metabolites, and these traits are associated with higher infectivity toward a defense sequestering herbivore. We conclude that plant defense metabolites that are transferred through adapted herbivores may result in the evolution of resistance in herbivore natural enemies. Our study also identifies plant defense resistance as a potential target for the improvement of biological control agents.
Project description:Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.
Project description:Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription.
Project description:The biosynthesis of DIMBOA, a pesticidal secondary metabolite of maize, branches off the tryptophan pathway. We have previously demonstrated that indole is the last intermediate common to both the tryptophan and hydroxamic acid pathways. The earliest discovered mutant in the DIMBOA pathway, bxbx (benzoxazineless), is deficient in the production of DIMBOA and related compounds. This paper presents evidence that a gene identified by Kramer and Koziel [Kramer, V. C. & Koziel, M. G. (1995) Plant Mol. Biol. 27, 1183-1188] as maize tryptophan synthase alpha (TSA) is the site of the genetic lesion in the DIMBOA-deficient mutant maize line bxbx. We demonstrate that the TSA gene has sustained a 924-bp deletion in bxbx compared with its counterpart in wild-type maize. We report that the TSA gene maps to the same location as the bxbx mutation, on the short arm of chromosome 4. We present evidence that the very early and very high level of expression of TSA corresponds to the timing and level of DIMBOA biosynthesis but is strikingly different from the expression of the maize tryptophan synthase beta (TSB) genes. We show that feeding indole to bxbx seedlings restores their ability to synthesize DIMBOA. We conclude that the maize enzyme initially named tryptophan synthase alpha in fact is a DIMBOA biosynthetic enzyme, and we propose that it be renamed indole synthase. This work confirms and enlarges upon the findings of Frey et al. [Frey, M., Chomet, P., Glawischniq, E., Stettner, C., Grün, S., Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R. B., Briggs, S. P., Simcox, K. & Gierl, A. (1997) Science 277, 696-699], which appeared while the present paper was in review.
Project description:The wheat stem sawfly (WSS), Cephus cinctus Norton (Hymenoptera: Cephidae), is an important pest of wheat and other cereals, threatening the quality and quantity of grain production. WSS larvae feed and develop inside the stem where they are protected from the external environment; therefore, pest management strategies primarily rely on host plant resistance. A major locus on the long arm of wheat chromosome 3B underlies most of the variation in stem solidness; however, the impact of stem solidness on WSS feeding has not been completely characterized. Here, we used a multiomics approach to examine the response to WSS in both solid- and semi-solid-stemmed wheat varieties. The combined transcriptomic, proteomic, and metabolomic data revealed that two important molecular pathways, phenylpropanoid and phosphate pentose, are involved in plant defense against WSS. We also detected a general downregulation of several key defense transcripts, including those encoding secondary metabolites such as DIMBOA, tricetin, and lignin, which suggested that the WSS larva might interfere with plant defense. We comparatively analyzed the stem solidness genomic region known to be associated with WSS tolerance in wild emmer, durum, and bread wheats, and described syntenic regions in the close relatives barley, Brachypodium, and rice. Additionally, microRNAs identified from the same genomic region revealed potential regulatory pathways associated with the WSS response. We propose a model outlining the molecular responses of the WSS-wheat interactions. These findings provide insight into the link between stem solidness and WSS feeding at the molecular level.