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Chapter 6
Pesticides on the Inside: Exploiting the Natural Chemical Defenses of Maize against Insect and Microbial Pests Shawn A. Christensen,* Charles T. Hunter, and Anna Block Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608, United States *E-mail:
[email protected] Maize (Zea mays) is one of the most significant and abundantly grown crop plants in the United States with yields exceeding $50 billion annually. Despite major improvements in productivity, it is estimated that billions of bushels are lost per year due to the combined effects of herbivory and disease. The use of insecticides/fungicides has been a long-standing strategy to manage pests; however, broad public recognition of the environmental and human-health concerns associated with synthetic pesticides has heightened interest in the use of genetic tools to provide immunity. Breeding programs continue to work towards optimizing maize lines for effective resistance, but insufficient knowledge of the molecular mechanisms and specific defense chemicals that regulate pest resistance limits rapid progress in this area. To address this issue, efforts have been made to elucidate and characterize important maize defense chemicals including benzoxazinoids, terpenoid phytoalexins, free fatty acids, hormones, signaling peptides, inducible volatiles, and phenylpropanoids. In this chapter, we review the existing knowledge of these maize defense chemicals and discuss current and future strategies to exploit them for improved resilience to biological threats.
© 2018 American Chemical Society Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Introduction Maize (Zea mays) is a key agricultural commodity both in the United States and throughout the world, accounting for the majority of animal feedstocks, while also being important for biofuel production, human consumption, and dozens of industrial applications. While advances in maize breeding and biotechnology have resulted in unprecedented yields, biotic challenge from pathogens and insect pests continue to cause major losses. Recent surveys show that disease pressure alone accounts for approximately 10% of reduced production in the United States, averaging 1.5 billion bushels (valued at over $5 billion) in losses annually (1). Common fungal pathogens responsible for these losses include mycotoxigenic ear-rotting (e.g. Fusarium verticillioides and Aspergillus flavus), stalk-rotting (e.g. Fusarium graminearum and Colletotrichum graminicola), root-rotting (e.g. F. verticillioides and Pythium aristosporum) and leaf blight-causing (e.g. Cochliobolus heterostrophus and C. graminicola) fungi that devastate plant organs and contaminate seed with carcinogenic mycotoxins. Under recent climate conditions, post-harvest losses due to fungal derived mycotoxin contamination has been endemic, averaging an additional 1.1 billion bushels per year (1). In addition to fungal diseases, insect pests continue to cause significant damage to maize crops. Fall armyworm (Spodoptera frugiperda) and beet armyworm (Spodoptera exigua) defoliate plants, whereas the corn earworm (Helicoverpa zea) targets the developing cob, causing extensive damage to the seeds. Stem-boring insects such as the European corn borer (Ostrinia nubilalis) lead to structural damage (i.e. lodging) and microbial colonization through the creation of humid and contaminated tunnels conducive to stalk rot infections (2, 3). Also problematic are phloem-feeding insects such as the corn leaf aphid (Rhopalosiphum maidis), which causes wilting and curling of leaves. Moreover, below-ground herbivores like the western corn rootworm (Diabrotica virgifera virgifera) damage roots and lead to poor water and nutrient uptake. In an attempt to control losses from pathogen and insect attack, billions of dollars are spent annually on insecticides and fungicides. Despite these investments, insect and pathogen damage continues to be a widespread problem. Ineffective insect/disease management strategies coupled with environmental and human health concerns associated with the use of fungicides/insecticides has led to a heightened interest in the use of genetic tools to develop more pest resistant plants. The most successful and widely adopted technology for insect resistance in maize has been the engineering of Bt toxins to provide protection against Lepidoptera larvae (caterpillars) (4). These natural toxins, originally isolated from the bacteria Bacillus thuringiensis, are selectively operative to avoid harming non-Lepidopteran insects (5), and their use has led to dramatic reductions in the application of broad-spectrum insecticides (6). While historically an effective strategy for maize resistance to earworms, the emergence of Bt resistance has reduced its effectiveness against certain maize pests (7). Considering the potential for further Bt resistance and the widespread reluctance to grow and consume genetically modified plants in some parts of the world, there is a pressing need for new multipronged approaches to pest management. One strategy is to exploit the naturally occurring chemical defenses of maize to defend against attacking 48 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
organisms. In this perspective we will give a brief overview of the predominant chemical defenses of maize and discuss possible strategies for utilizing them.
Maize Defense Chemicals Benzoxazinoids One of the first chemical defenses discovered in maize was a class of compounds collectively referred to as Benzoxazinoids (BXs). They were identified in the 1950s as important components of host resistance against Fusarium rot in rye (8) and O. nubilalis herbivory in maize (9). They have since been characterized as anti-insect and antimicrobial hydroxamic acids found in a variety of grasses and other plant species (10). In maize, BXs are constitutively produced in immature tissues such as young seedlings (11, 12), but are also inducible by pathogen infection (13, 14), insect herbivory (15, 16), aphid feeding (14), and wounding in mature tissue. The BXs are composed of a class of related metabolites whose biosynthesis, beginning with indole, is modulated by a series of enzymes, denoted as BX1 through BX14 (17–19). The BX cascade produces an array of compounds with varying levels of toxicity (20, 21), the most widespread of which in maize is DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one) (Figure 1).
Figure 1. Structures of select chemical defense compounds from maize. Glycosylation by BX8 and BX9 to convert the toxic aglycones into less-toxic glucosides allows for storage of BXs in vacuoles (22). When plant cells are damaged and vacuoles disrupted, glucosidases convert the glucosides back to 49 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
the biocidal aglycone toxins (23), whereupon they delay growth and potentially kill attacking pests (20, 21, 24, 25). Interestingly, the BX biosynthetic genes are clustered in maize, with Bx1 through Bx8 all localized to the short arm of chromosome 4 (18), perhaps allowing for co-regulation of gene expression and co-inheritance of the functionally-related loci. The BXs and their breakdown products also play important roles in allelopathy by suppressing growth of competing plants (26), and have thus attracted attention for utilization in weed control (27, 28). Terpenoid Phytoalexins Nonvolatile acidic terpenoids constitute a more recently discovered class of maize anti-insect and antimicrobial compounds. Initially observed in maize stems infested with O. nubilalis, six acidic diterpenoids were identified as ent-kaurane-related structures, termed kauralexins. Common maize pathogens such as C. heterostrophus, F. graminearum, and Rhizopus microsporus elicit transcript accumulation of the kauralexin biosynthesis gene ent-copalyl diphosphate synthase known as Anther Ear 2 (An2), leading to significant increases in kauralexin concentrations (29, 30). In vitro studies provided initial evidence for the antimicrobial activity of select kauralexins against leaf-, ear-, and stalk-rot pathogens (29, 30). This activity was later confirmed in vivo using null mutations in An2, demonstrating that kauralexins play an important role in maize defense against pathogens (29). In addition to kauralexins, novel acidic sesquiterpenoids, termed zealexins, were also recently identified (31, 32). Zealexins are pathogen-elicited β-macrocarpene derivatives that appear to be ubiquitous in maize. Like kauralexins, zealexins have demonstrated strong anti-fungal growth activity against pathogens such as R. microsporus, A. flavus, and F. graminearum, supporting a broad role for nonvolatile terpenoid defenses in maize (29, 31, 32). The comparative accumulation of phytoalexins can vary widely and be organ- and stress-dependent (29–32). For example, the nonvolatile oxygenated sesquiterpene β-costic acid was recently characterized as one of the predominant root phytoalexins, promoting below-ground fungal and herbivore resistance (33). β-costic acid is produced by cyclization of farnesyl diphosphate to β-selinene by Terpene Synthase 21 (TPS21), and can accumulate to concentrations >100 µg·g-1 fresh weight in pathogen-challenged roots. Such concentrations were sufficient to inhibit the in vitro growth of several fungal pathogens and the corn root worm (Diabrotica balteata) (33). An even more recent discovery in maize root phytoalexins revealed non-acidic diterpenoids, termend dolabralexins (34). These newly identified derivatives of An2 and kaurene synthase-like 4 are elicited by pathogen attack and oxidative stress, demonstrating yet another class of maize phytoalexins with functional roles in defense. Free Fatty Acids Free fatty acids (FAs) and their oxygenated and cyclized derivatives represent a broad class of lipid constituents that play protective roles against biotic stress in dicot species (35–37), however, comparatively less is known about their function 50 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
in maize and other monocots. Plant FAs largely consist of long hydrophobic unbranched chains of hydrocarbons with hydrophilic carboxylic acid functional groups on one end of the molecule. Some FAs have been shown to have a role in pathogen resistance. For instance, transgenic eggplant overproducing palmitoleic acid (16:1) was more resistant to the fungus Verticillium dahliae (38). C18 FAs are involved in both basal resistance (39) and in resistance-gene mediated responses via stimulation of NADPH-oxidase activity and subsequent reactive oxygen species production (40). Very long chain FAs (VLCFAs; C ≥ 20) play roles in structural defenses such as cuticle formation, as well as sphingolipid generation, cell signaling, and pathogen resistance (36). While correlations between VLCFAs and plant defense have been reported, it is not clear whether they or their derivatives are directly involved in defense. As very little is known about the role of VLCFAs in maize defense, we refer to the following reviews that discuss their potential defensive functions in other plant species (35–37). Phenylpropanoids Phenylpropanoids are induced in maize in response to biotic attack. For instance, maize roots or leaves infected with C. graminicola accumulate high levels of flavonoids such as naringenin chalcone, apigenin and genkwanin as well as other phenylpropanoids including 3-caffeoyl-quinic acid, N-p-coumaryltryptamine and feruloyl-feruloyl-glycerol (41). The functional role (if any) of many of these compounds in pest resistance remains to be elucidated, however, bioactivity has been determined for select phenylpropanoids. Flavonoids, for instance, are small molecule derivatives of 2-phenyl-benzylγ-pyrone that form more than 9,000 isoforms in the plant kingdom (42). A class of flavonoids, termed flavones (distinguished by a 2-phenylchromen-4-one backbone), has several constituents that have demonstrated defensive roles against maize insects and pathogens. One example is the C-glycosyl flavone, maysin (2′′-O-α-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)-luteolin), which is constitutively produced in maize silks and provides resistance against H. zea (43). The biosynthetic pathway for the production of maysin in maize has recently been elucidated (44). Its production is highest in silk tissue where it can reach up to 2% of silk dry weight. Maysin is also produced to lower levels in leaves where its accumulation and that of it’s precursor rhamnosylisoorentin can be induced in response to UV-B exposure (45). Other C-glycosyl flavones isolated from maize silks (e.g. apimaysin and methoxymaysin) and phenylpropanoids, such as chlorogenic acid have also demonstrated important herbivore resistance properties (46–48). Mechanistically, flavones and other phenylpropanoids can be oxidized to quinones by polyphenol oxidase in tissues wounded by insect feeding. These quinones act as antinutritive compounds by binding proteins and thus reducing bioavailability to insects (49, 50). An unfortunate side effect of this process is the commercially-undesirable trait of silk browning (51, 52), which has led to the deselection of these compounds during sweetcorn breeding and increased insect susceptibility. Although less is known about the functional benefit of flavonoids in antipathogenic responses, both maysin and maysin-3′-methyl ether have demonstrated selective antimicrobial 51 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
activity against several bacterial pathogens (53). One could speculate that the antimicrobial activity of these C-glycosyl flavones may also be due to their protein binding activities. Another class of phenylpropanoids that have defensive roles are phenolic esters that function in the peroxidase-catalyzed cross-linking of cell wall polysaccharides, lignin, and proteins (54). These compounds are thought to strengthen the cell walls to restrict entry of stem boring caterpillars and movement of fungal pathogens. The levels of these compounds, such as dehydrodiferulate isomers, negatively correlates with O. nubilalis damage in maize (55, 56). The amount of diferulic acid in the pericarp and aleurone tissues of maize kernels negatively correlates with both F. graminearum disease serverity and fungal growth (57). Interestingly the production of phenylpropanoids can be benifical for pests. For instance p-coumaroyltyramine can be metabolized by Spodoptera littoralis and leads to increased larval growth, possibly by acting as a nitrogen source (58). Additionally, the maize bacterial pathogen Pantoea stewartii injects the effector protein WtsE into maize cells that leads to the accumulation of coumaroyl tyramine and heightened pathogen susceptibility (59). Similarly, the biotrophic fungus Ustilago maydis uses the effector protein Tin2 to induce anthocyanin production and reduce liginin production in maize to enhance U. maydis pathogenicity (60).
Herbivore-Induced Plant Volatiles Herbivore-induced plant volatiles (HIPVs) are volatile compounds produced in response to attack by plant-chewing insects. These metabolites are generally released as complex blends that can include green leaf volatiles (GLVs), monoterpenes, sesquiterpenes, homoterpenes, and indole. HIPVs have been shown to play important roles in indirect defense by helping parasitic wasps cue in on their lepidopteran hosts (61, 62) and in plant-plant signaling (63, 64). The genetic characterization of several HIPV regulatory and biosynthetic pathways has extended our knowledge regarding their defensive roles against herbivory. For instance, mutations in a maize 13-lipoxygenase (LOX) caused significant reductions in production of both GLVs and the plant hormone jasmonic acid (JA), leading to attenuated HIPV emissions and attractiveness to parasitoid wasps (65). Expression profiling, correlation of gene and metabolite presence, and recombinant protein analysis have revealed several other enzymes involved in HIPV production. For monoterpenes, recombinant TPS26 was reported to make limonene and myrcene in vitro (66). The biosynthesis pathway for the homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) were also recently discovered in maize, resulting from the sequential action of TPS2 and cytochrome p450s (67). Moreover, for sesquiterpenes, TPS23 is responsible for synthesis of (E)-β-caryophyllene (68), while TPS1, TPS3, TPS4, and TPS10 all function in β-farnesene biosynthesis (69–71). While many studies have demonstrated a role for HIPVs in above-ground interactions, their function in below-ground 52 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
defense is also important. For example, Köllner et. al., (2008) showed that Tps23 is induced in maize roots by D. v. virgifera feeding and that the resulting (E)-β-caryophyllene it produces attracts entomopathogenic nematodes, the natural enemies of D. v. virgifera (68).
Plant Hormones and Regulation of Defense Chemistry Most maize defense chemicals are either produced or activated in response to biotic attack. This requires tight regulation and is necessary both to conserve plant resources and to limit unintended self-harm due to the toxic nature of many of these compounds. A major stratagem that plants use to control the appropriate production or activation of these defense metabolites is regulation by hormones. These plant hormones form a complex network of synergistic or antagonistic signals, each with different rates and strengths of signal perception, promulgation, and downstream responses that govern resistance (Figure 2).
Figure 2. A generalized view of the complex network of plant hormone synergies (solid arrows) and antagonisms (dashed lines). Abbreviations: ABA-abscisic acid; Aux-Auxin; GA-Gibberellic acid; CK-cytokinin; BR-brassinosteroid; SA-salicylic acid; ET-ethylene; JA-jasmonic acid.
53 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
The core biosynthetic and primary response elements for some plant hormones and their signaling pathways have been determined in model plants like Arabidopsis thaliana, but many of the regulatory processes and downstream signaling components remain unclear. This is especially true for maize, where comparatively little is known about the signaling networks that regulate the perception and response of maize to biotic challenge. It is important to understand how these responses are regulated, as responses to infection or insect attack tend to vary widely between different plant species. Nearly all known plant hormones have been shown to participate in regulating plant stress responses to some degree and are, therefore, likely to impact the control of plant defense chemistry.
Jasmonates Jasmonates are peroxidation products of α-linolenic (18:3) acid produced by 13-LOXs. They include the well-studied products of the 13-LOX pathway 12-oxo-phytodienoic acid (12-OPDA), the hormone JA, and their derivatives. Unlike biotrophic pathogens that induce SA-mediated responses, necrotrophic pathogens elicit host responses that promote the accumulation of jasmonates. For example, C. heterostrophus-infected maize plants showed a rapid accumulation of 12-OPDA and JA in both infected tissues and in healthy tissues adjacent to infection sites (72). Evidence for JA regulation in direct antimicrobial chemical defenses has also been observed with the induction of the maize phytoalexins, kauralexins and zealexins (30, 32). Using Mutator transposon insertions in Lox10 and Lox12 and the JA biosynthesis genes, Lox8, Opr7, and Opr8, genetic evidence demonstrated that JA is required for pathogen-infected maize to survive under field and laboratory conditions, and further established its necessity for senescence, herbivore resistance, and reproductive development (65, 73–75). While research pertaining to the 13-LOX pathway has been extensive, particularly in the study of jasmonates, knowledge of the 9-LOX pathway has been more elusive. In the year 2000, analysis of potato (Solanum lycopersicum) homogenate revealed two inefficiently cyclized 9-allene oxide cyclase products, 10-oxo-11,15-phytodienoic acid (10-OPDA) and the 18:2-derived 10-oxo-11-phytoenoic acid (10-OPEA). Although structurally similar to the jasmonate 12-OPDA, the predictable downstream derivatives remained intangible for more than a decade until a recent study in maize helped elucidate their structures and functions. While profiling JA and other known FAs in fungal infected maize tissues, high levels of 10-OPEA and a novel series of related analytes were detected. Large scale purification and structure elucidation of these metabolites revealed 9-LOX derived 12-, 14- and 18-carbon cyclopente(a)none FAs that conceptually parallel jasmonates (Figure 3) (72). Intitial studies of these novel cyclopente(a)none FAs demonstrated that they exceed jasmonates in abundance within infected tissues, display signaling properties that mediate defense gene expression, activate programmed cell death, and promote direct phytoalexin activity against both insects and pathogens (72). Whether or not any of these newly identified molecules would specifically be classified as hormones remains to be determined. 54 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 3. Working model for the biosynthesis pathway of novel cyclopente(a)none fatty acids (FAs) in comparison to jasmonates. Superscript letters and symbols indicate the following: 1 = 18:3 derived; 0 = 18:2 derived; S = saturated cyclopente(a)none ring; and U = unsaturated cyclopente(a)none ring. Ethylene Ethylene functions synergistically with JA to promote defense responses in plants, particularly against insects and necrotrophic pathogens. The interconnected and mutually-stimulating signaling properties of ethylene and JA have made teasing apart the precise roles for each hormone difficult. In maize, ethylene has been shown to be important for promoting phytoalexin production (30, 32), inducing HIPV emissions (76), and in regulating resistance to R. maidis and S. frugiperda (77, 78). Salicylic Acid Salicylic acid (SA) also has well-documented roles in plant defense responses to biotrophic and hemibiotrophic pathogens, where it induces pathogenesis-related genes and activates systemic acquired resistance (79). SA engages in cross-talk with other hormones, including JA and ethylene. These interactions have been demonstrated to be either antagonistic or synergistic, depending on the specific plant-biotic interaction (80–82). The roles of SA have been extensively studied in 55 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
dicots and rice, but very little is known about them in maize. SA does appear to be involved in defense against fungal pathogens, as SA application induces cysteine proteases (83), PR-1 gene expression (83), and trypsin and lectin inhibitors (84). Additionally, SA is suppressed by the biotrophic pathogen U. maydis (85). On the other hand, application of methyl salicylate results in higher recruitment of Diabrotica speciosa to maize plants, suggesting a possible role for SA in conferring susceptibility to below-ground herbivory (86). Abscisic Acid Abscisic acid (ABA) is well-known for its roles in abiotic stress responses, particularly for drought and salinity stresses. More recently, ABA has been shown to be involved in regulation of chemical defense pathways as well (87). Studies in maize show that ABA appears to regulate the production of DIMBOA and certain phenolic acids including chlorogenic acid, caffeic acid, and ferulic acid (88). It is also induced by exposure to HIPVs, including indole (89). Application of exogenous ABA to maize roots elicited terpenoid phytoalexins (90) and resulted in increased resistance to foliar pathogens and insects, including C. graminicola (91), S. littoralis and Setosphaeria turcia (88), suggesting a possible role for ABA in systemic acquired resistance (91). Conversely, above ground exogenous ABA application led to increased susceptibility to C. graminicola (92). While ABA likely has a role in defense against biotic attack in maize, our current understanding is based solely on metabolite profiling or exogenous chemical applications. Studies using loss-of-function mutants in ABA biosynthesis or perception genes will help to better determine the role of ABA in maize defense. Other Plant Hormones and Enogenous Maize Signals Brassinosteroids, gibberellic acid, cytokinins, and auxins are all important for plant growth and development, but also appear to contribute to plant defense. In the maize relatives barley and brachypodium, brassinosteroids were found to be negative regulators of resistance to necrotrophic pathogens (93, 94). In rice, mutants of a gibberellic acid receptor accumulate gibberellic acid and have enhanced resistance to the blast fungus Magnaporthe grisea (95), though gibberellic acid appears to play a negative role in basal disease resistance (96). In maize, brassinosteroid activity has been shown to be important for antioxidant defense, but a role against biotic stress has not been determined. Evidence for a function of cytokinins in maize defense is also limited; however, recent evidence demonstrated a curious signaling role for DIMBOA in regulating cytokinin concentrations. The oxidative cleavage of DIMBOA via laccase and peroxidase generates transitional free radicals that mediate the activity of a dehydrogenase, leading to cytokinin degredation (97). Finally, the growth-promoting hormone auxin also appears to influence defense responses in plants, although results thus far have been contradictory (98, 99). Strongly integrated in plant hormone signaling are endogenous short-chain amino acid signals such as plant elicitor peptides (Peps) that effectively regulate herbivore and disease resistance. Similar to the potent signal systemin found in 56 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
the Solanaceae family (100), maize Peps activate defense responses including the production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivore and disease resistance (101, 102). In addition to Peps, the recently identified Z. mays immune signaling peptide (Zip1) functions as a biotroph-elicited signal that activates SA defense signaling (103), further demonstrating the interconnected role of petide signals in hormone-mediated maize resistance.
Functional Characterization of Defense Chemistry Maize makes a staggering array of compounds when faced with biotic challenge, many with unknown functions. Various approaches can be taken to determine whether a compound contributes to pest resistance. As an initial test, in vitro bioassays can be a helpful tool in determining biological activity. For example, recent analyses showed that a maize 9-oxylipin (i.e. 10-OPEA) had significant and dose-dependent insect and pathogen growth inhibitory activity (72), whereas diterpenoid phytoalexins demonstrated only minimal growth impairment against insects but had strong antifungal activity against ear, stem, and root pathogens of maize (29–32). While in vitro bioassays provide evidence for potential molecular function, in vivo analysis using genetic confirmation is needed to demonstrate a biological role. The identification of target genes involved in the biosynthesis of defense molecules can be accomplished by association mapping, co-expression analysis, and/or forward and reverse genetic approaches. A recent example in maize demonstrated the effectiveness of several of these approaches (33). Examination of transcript accumulation in response to F. graminearum found strong elicitation of the An2 gene, which was later genetically shown to be required for kauralexin production (90) and resistance to ear, stem, and leaf pathogens (29). In another study, metabolite-based genetic mapping using biparental populations, genome wide association studies (GWAS), and near-isogenic lines identified TPS21 to be essential for β-costic acid formation. Through both in vitro and genetic analyses, it was determined that β-costic acid plays a strong chemical defensive role against root pathogens and herbivores (33). The aforementioned studies provide proof of function, yet to engineer or incorporate the production of a particular compound into maize breeding lines, it is preferable to identify all of the genes involved in its production. As mentioned, one approach for identifying candidate genes of chemical defense pathways can be accomplished using co-expression network analysis. This approach works on the principal that genes with common/shared function will be co-regulated over a wide range of conditions as their products need to be present at the same time in the same tissue. This strategy was used in A. thaliana to identify P450s that co-expressed with geranyllinalool synthase that makes the substrate for TMTT synthase (104). Subsequent functional characterization of the candidates led to the identification of a bifunctional DMNT/TMTT homoterpene synthase (104). While not yet widely used in maize, this approach is promising for the elucidation of maize chemical defenses. Several online databases exist that perform co-expression analyses for maize genes. These include ATTED-ii (http://atted.jp/) (105), 57 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
CORNET (https://bioinformatics.psb.ugent.be/cornet/versions/cornet_maize1.0// main/precalc) (106) and PLANEX (http: //planex.plantbioinformatics.org/) (107). Collectively, these studies highlight current approaches to elucidate the function of maize chemical defenses and the pathways that produce them.
Engineering for Enhanced Chemical Defense Defense metabolites are differentially elicited in response to a variety of biological threats. While some plants are able to deploy effective tactics that result in immunity, pests have also evolved the ability to manipulate plant chemistry and suppress defense systems. Such chemical warfare can be observed in maize. For example, Figure 4 demonstrates that C. graminicola elicits far less production of many common maize defense metabolites than F. graminearum and C. heterostrophus, despite having demonstrated patterns of significantly greater fungal growth (29).
Figure 4. Heat map (n=4) displaying the relative abundance of maize defense metabolites in a B73-Mo17 hybrid under no treatment (control) or 24 h after damage or infection with C. graminicola (C. gram), F. graminearum (F. gram), and C. heterostrophus (C. het). Metabolites measured include salicylic acid (SA); jasmonic acid (JA); auxin (IAA); abscisic acid (ABA); 12-oxo-phytodienoic acid (12-OPDA); kauralexin A1 (KA1)-KA3 and KB1-KB3; zealexins A1 (ZA1), ZB1, and ZA4; and 6-methoxy-2-benzoxazoline (MBOA). Given the diversity of biological threats and the complexity of defense regulation, the challenge to engineer plants for broad resistance is substantial. Identification of defense-related quantitative trait loci (QTL) continues to inform breeding programs for enhanced resistance phenotypes; however, specific knowledge of chemical defenses has, until relatively recently, been unavailable. Recent studies in maize have demonstrated that targeted enhancement of chemical defenses can have significant impacts on plant-biotic interactions. For example, 1) overexpression of TPS10, responsible for a series of sesquiterpene HIPVs, resulted in increased recruitment of the parasitic wasp, Cotesia marginiventris (70); 2) upregulation of β-caryophyllene and α-humulene by introduction of an oregano TPS resulted in improved resistance against the root herbivore Diabrotica 58 Beck et al.; Roles of Natural Products for Biorational Pesticides in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
v. v. (108), but led to lower yield, higher above-ground herbivory (109), and increased susceptibility to C. graminicola (110); 3) expression of a sorghum MYB transcription factor involved in the production of 3-deoxyanthocyanidin phytoalexins resulted in improved resistance to C. graminicola (111); and 4) downregulation of ethylene-response factors by targeted disruption using CRISPR/Cas9 resulted in increased resistance to M. oryzae (112). These examples and others serve to demonstrate the potential for manipulating or enhancing maize chemistry to improve pest resistance. Still in its infancy, the engineering of enhanced resistance will require a greater understanding of the genetics, chemistry, and regulatory pathways of plant defense. As many defense chemicals are phytotoxic or require plant resources for their production, a means to control the production of defense chemicals to reduce potential negative impacts on growth, development, and yield will also be required.
Future Directions One of the perpetual challenges of developing crop resistance is the continuing evolution and adaptation of plant pests against engineered defense mechanisms. These evolutionary impacts are similar to those seen in the development of resistance to pesticides, where exposure to a particular compound limits the survival and reproductive capacity of a given organism only until natural seletion overcomes the treatment. Examples of acquired resistance to certain maize-produced defense chemicals already exist. For instance, F. verticillioides can effectively detoxify the benzoxazolinones, 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA) (113). Many other Fusarium species do not display such tolerance to these compounds (113). Insects have also developed resistance to certain BXs, as both S. littoralis and S. frugiperda are able to detoxify DIMBOA via glycosylation (21). Interestingly neither species can detoxify the related compound HDIMBOA (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one) (21). Such examples reveal the biochemical warfare currently underway between maize and its pests. Enhancing maize lines to produce controlled amounts of diverse defense chemicals that have antimicrobial and anti-herbivore activity, without compromising growth, development, and yield, could facilitate a more prolific agro-economic industry. Rapid progress and advancement of these intiatives will reduce the need for external pesticide application, reducing growing costs and increasing the safety of farm workers and consumers alike.
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