METABOLIC ENGINEERING OF PLANT VOLATILES TO INCREASE PLANT DEFENSE

An overview of reports on metabolic engineering to increase defense responses is described in Table 20.3.

Modification of Plant Volatiles via Constitutive Expression of Terpene Synthase Genes to Enhance Defense Responses

A very elegant demonstration of the importance of a volatile sesquiterpene in defense came from work using the natural biodiversity of maize (Zea mays). North American maize lines appeared not to have the ability to produce the sesquiterpene (£)-p-caryophyllene, while the European maize varieties and its common ancestor teosinte do and are resistant to corn rootworm (Diabortica vir- gifera virgifera) (Kollner et al. 2008). The production of this sesquiterpene was restored in North American lines by constitutive expression of an (£j-p-caryophyllene-synthase from Origanum vulgare. Lines with restored (£)-P-caryophyllene emission were now also effective in attracting below-ground entomopathogenic nematodes that parasitize on corn rootworm larvae and thus suffered less damage from the pest (Degenhardt et al. 2009), demonstrating the importance of (£)-p-caryophyllene in the plant’s indirect defense. Remarkably, transgenic maize seedlings producing (£j-p-caryophyllene exhibited an increase in susceptibility to a pathogenic fungus (Colletotrichum graminicola) (Fantaye et al. 2015), indicating that (E)-p-caryophyllene is involved in several defense processes.

A role for (E)-p-caryophyllene in defenses was also established in rice (Oryza sativa). Overexpression of sesquiterpene synthase OsTPS3 increased the amount of (£)-p-caryophyllene and these transgenic plants were preferred by Anagrus nilaparvatae, an egg parasitoid of the rice plant hopper Nilaparvata lugens (Cheng et al. 2007). Several other studies indicated that overexpression and downregulation of OsTPS3 in rice led to an increase or decrease of rice plant hopper incidence, respectively, indicating that both parasitoids and herbivores use the same volatile signal to find their host (Xiao et al. 2012; Wang et al. 2015). These studies indicate a role for (£)-p-caryophyllene in defense responses and emphasize that metabolic engineering of volatiles to increase plant defenses could also have unexpected or unwanted effects.

Detection of volatile P-ocimene by neighboring plants can induce hormonal signaling pathways (e.g., jasmonic acid; JA, salicylic acid; SA or ethylene; ET) and thus prime their defense responses. For example, exogenous application of allo-ocimene, a structural isomer of p-ocimene, to Arabidopsis thaliana leaves induced a subset of defense-related genes

Gene(s)

Origin

Engineered

Species

Strategy

Regulation

Changes in Volatile Spectrum-’

Effect on Plant Defenseb

Reference(s)

OS

Lima bean

Tobacco

Ox

Constitutive

methyl salicylate T. (E)-DMNT T. (Z)-DMNTT. TMTT t. when exposed to transgenic plants emitting (E)-P-ocimene

Spider mites (Mythimna separate) l Parasitic wasps (Cotesia kariyai) t

Muroi et al. (2011)

OS

Lima bean

Tomato

Ox

Constitutive

Methyl salicate t. (E)-3-hexenol t. when exposed to transgenic plants emitting (E)-(i-ocimene

Aphids (Macrosiphum euphorbiae) { Parasitic wasps (Apliidius en’i) t

Cascone et al. (2015)

TPS6

Oregano

Maize

Ox

Constitutive

(E)-(J-caryophyllene T, Ot-humulene f

Corn rootworm (Diabrotica virgifera) l

Pathogenic fungi (Colletotrichiun graminieola, Fusarium graminearum) l

Degenhardtet al. (2009), Fantaye et al. (2015), Robert et al. (2013)

HPL

Capsicum

anniium

A. t ha liana

Ox

Constitutive

(Z)-hexenal T. GLV biosynthesis T

Parasitic wasps (Cotesia glomerala) T Pathogenic fungi (Botrytis cinerea) l

Shiojiri et al. (2006)

ZIS. FPS

ZIS from Solatium habrvchaites; FPS from Tomato

Tomato

Ox

Trichome-

specific

New compound 7-epizingiberene

White fly (Bermisia tabaci) i Spider mites (Tetranychas evansi, Tetranychus urticae) i

Bleekeret al. (2012)

FPS

Tomato

Tomato

Ox

Trichome-

specific

No changes

No changes

Kortbeek et al.

  • (2016)
  • (Continued)

Gene(s)

Origin

Engineered

Species

Strategy

Regulation

Changes in Volatile Spectrum-'

Effect on Plant Defense1

Reference(s)

FPS

Chicken

Tomato

Ox

Trichome-

specific

(E)-nerolidol T. tt-famesene T. monoterpenes |

White fly (Bermisia tabaci) l

Kortbeek et al. (2016)

HPL

A. thaliana

A. thaliana

Ox

Constitutive

(Z)-3-hexenal T. other GLVs T

Pathogenic fungi (Botrytis cinereal) l Parasitic wasps (Cotesiaglomerata) T

Shiojiri et al. (2006)

HPL

A. thaliana

A. thaliana

Down-

regulation

Constitutive

(Z)-3-hexenal l, other GLVs l

Pathogenic fungi (Botrytis cinereal) T Parasitic wasps (Cotesia glomerata) i

Shiojiri et al. (2006)

HPU

Rice

Rice

Down-

regulation

Constitutive

(Z)-3-hexenal l, other GLVs l

Planthoppers (Nilaparvata lugens) t Moths (Chilo suppressalis) { Planthoppers (Sogatella furcifera) i Pathogenic fungi (Xanthonumas oryzae) {

Parasitic wasps (Anagrtts nilaparvatae) t

Tong et al. (2012)

HPL2

Rice

Rice

Ox

Constitutive

(E)-2-hexenal T. other GLVs t

Pathogenic fungi (Xanthomonas oryzae) 1

Gomi et al. (2010)

Abbreviations: CHS, chrysanthemol synthase; FPS, farnesyl diphosphate synthase; HPL, hydroperoxide lyase; NES1, linalool/nerolidol synthase; OS, (/:)-(}-ocimene synthase; Ox, overexpression; TPS, terpene synthase; ZIS, 7-epizingiberene synthases.

" Plant volatiles that have been increased IV or reduced (l) in amount.

b Incidence and performance of herbivores, pathogens or parasitic organisms that have been increased IV or reduced (1).

and increased resistance against the fungus Botrytis cinerea (Kishimoto et al. 2005). Also, application of synthetic p-ocimene on a cotton swab placed near Chinese cabbage (Brassica pekinensis) plants resulted in an increased accumulation of glucosinolates, increased attraction of parasitic wasps, reduced aphid performance and induction of JA and SA marker genes (Kang et al. 2018). A follow-up study used transgenic tobacco (Nicotiana tabacum) plants ectopically expressing (£)-p-ocimene synthase from lima bean (Phaseolus lunatus) as emitter. Upon perception of p-ocimene and infestation by herbivores, maize and lima bean receiver plants emitted higher amounts of monoterpenes, sequiterpenes, homoterpenes, methyl salicylate and hexyl acetate. Oviposition by the army worm Mythimna separata on maize and the two-spotted spider mite Tetranychus urticae on lima bean plants was reduced. Moreover, the increased volatiles resulted in higher presence of parasitoid wasps (Cotesia kariyai) on maize and predatory mites (Phytoseiuluspersimillis) on lima bean plants (Muroi et al. 2011). Interestingly, lima bean plants grown near lima bean plants infested with T. urticae emitted higher levels of the (£)-4,8-dimethyl-l,3,7-nonatriene (DMNT) and (£,£)-4,8,12-trimethyltrideca-l,3,7,11- tetraene (TMTT) and showed increased defense responses (Arimura et al. 2012). In a comparable study, tomato plants exposed to the mentioned above transgenic p-ocimene-emitter tobacco plants produced higher amounts of terpenes (p-ocimene and limonene), GLVs ((£)-3- hexenol and (Z)-3-hexenol) and benzenoids (methyl salicylate). Both indirect and direct defense responses against the potato aphid Macrosiphum euphorbiae were significantly enhanced, as more parasitoid wasps (Aphidius ervi) were attracted and thus impairing aphids reproduction (Cascone et al. 2015). These studies indicate that metabolic engineering to increase emission of airborne signals, such as terpenes, influenced plant-plant communication and improved direct and indirect plant defense responses.

Plant volatiles also play an important role in the so-called push-and-pull insect pest management system. The aim is to “push” herbivores from the field and “pull” them toward more attractive food sources outside the crop field by visual or chemical cues (Cook et al. 2007). It has been long known that aphids produce (£)-p-farnesene as an alarm signal to other conspecifics. By doing so, aphids betray themselves to predators and parasitoids who use (£)-p-farnesene for the localization of their food source. In transgenic A. thaliana, expression of a (£)-p-farnesene from peppermint (Mentha piperita) or maize (Zea mays), resulted in emission of (£)-p-farnesene and both repulsion of aphids and attraction of parasitoids was observed (Beale et al. 2006; Schnee et al. 2006). In a follow-up study, the peppermint (£)-p-farnesene-synthase was targeted to plastids of wheat (Triticum aestivum) to increase emission levels (Bruce et al. 2015). Under laboratory conditions, repulsion of different aphid species (Sitobion avenae, Rhopalosiphum padi, and Metopolophium dirhodum) was observed, and Aphidius ervi parasitoids spent more time on transgenic plants compared to wild type. To test whether this system could be a viable system for pest management in agriculture, a large-scale field trial was performed. During this trial neither enhanced direct nor indirect defense responses against aphids were observed (Bruce et al. 2015), suggesting that climate conditions, natural occurrence of both aphids and parasitoids and other ecological interactions might be important parameters affecting tritrophic interactions in the field.

Another field trial was performed with maize plants overexpressing an oregano (£)-p- caryophyllene synthase (TPS6). Maize plants emitted more (£)-p-caryophyllene and a-humulene, but suffered severe costs in terms of seed germination and biomass. Moreover, transgenic plants were more attractive to above-ground herbivores, while root damage by below-ground root nematodes was reduced (Robert et al. 2013). It appears that much needs to be learned about the ecological interactions in agricultural sites before such strategies can be applied in the field. Also, the need for tissue-specific terpene biosynthesis seems to be a desirable alternative to constitutive expression to reduce unwanted developmental and fitness costs.

 
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