Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative

Abstract

Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

Fig. 1.

Zingiberene levels influence whitefly performance on F2 plants of an interspecific cross between S. lycopersicum cv Moneymaker and S. habrochaites PI127826 and both parents. (A) Leaf surface 7-epizingiberene (ng·mg⁻¹ FW leaf) (n = 5–8). (B) Whitefly mortality (% of total). (C) Whitefly fecundity displayed as the total number of eggs per leaf (n = 5–11). Bars represent means ± SE, different letters signify statistical differences (P < 0.05).

Fig. 2.

Enzymatic activity of recombinant ZIS enzymes. GC-MS chromatograms of ShZIS from S. habrochaites PI127826 (A) and ObZIS from O. basilicum (AY693646) (B) expressed in E. coli, assayed with sesquiterpene precursor E-E-FPP (FPP) or Z,Z-FPP (zFPP) and measured by SPME. Peaks: 1: 7-epizingiberene 2: β-sesquiphellandrene, 3: 7-episesquithujene, 4: sesquithujene, 5: (Z)-α-bergamotene, 6: (E)-α-bergamotene, 7: (E)-β-farnesene, 8: α-zingiberene 9: α-farnesene 10: β-bisabolene 11: (E)-nerolidol, 12: β-acoradiene, 13: (Z)-γ-bisabolene, 14: (E)-γ-bisabolene. The detector response for terpene-specific ion 93 is shown.

Fig. 3.

Identification of zingiberene stereo-isoforms. (A) GC-MS chromatograms of SPME sampling of S. habrochaites PI127826, ginger oil, recombinant ObZIS with E,E-FPP, and/or ShZIS with Z,Z-FPP. (B) Liquid injection of hexane leaf wash of F2 (plant 2) and parent S. habrochaites PI127826 compared with ginger oil. 1: 7-Epizingiberene and 2: α-zingiberene. The detector response for terpene-ion 93 is shown.

Fig. 4.

Effect of zFPS and ShZIS engineering in trichomes of cultivated tomato. (A) 7-epizingiberene levels in leaves (ng/mg) of transgenic lines expressing either zFPS alone (zFPS and line 12) or in combination with ShZIS as measured by SPME. Means ± SE (n = 3). (B) (Inset) GC-MS chromatogram of S. habrochaites PI127826, ginger oil and S. lycopersicum transgenic (line 2). 1: R-curcumene, 2: 7-epizingiberene, 3: S-curcumene, 4: α-zingiberene. The detector response for ion 119 is shown. (C) Distribution of whitefly eggs (%). (D) Spider mite (T. urticae) fecundity on Moneymaker, transgenic line 2 and PI127826 displayed as number of eggs per mite per 4 d. Means ± SE (n = 60). (E) Population growth rates of T. urticae on intact tomato plants. Shown are mean number of adult mites per plant (± SE; n = 4) on S. lycopersicum transgenic plants expressing either only zFps (gray symbols) or both zFps and ShZis (black symbols).