Successful herbivore attack due to metabolic diversion of a plant chemical defense

Abstract

Plants protect themselves against herbivory with a diverse array of repellent or toxic secondary metabolites. However, many herbivorous insects have developed counteradaptations that enable them to feed on chemically defended plants without apparent negative effects. Here, we present evidence that larvae of the specialist insect, Pieris rapae (cabbage white butterfly, Lepidoptera: Pieridae), are biochemically adapted to the glucosinolate-myrosinase system, the major chemical defense of their host plants. The defensive function of the glucosinolate-myrosinase system results from the toxic isothiocyanates that are released when glucosinolates are hydrolyzed by myrosinases on tissue disruption. We show that the hydrolysis reaction is redirected toward the formation of nitriles instead of isothiocyanates if plant material is ingested by P. rapae larvae, and that the nitriles are excreted with the feces. The ability to form nitriles is due to a larval gut protein, designated nitrile-specifier protein, that by itself has no hydrolytic activity on glucosinolates and that is unrelated to any functionally characterized protein. Nitrile-specifier protein appears to be the key biochemical counteradaptation that allows P. rapae to feed with impunity on plants containing glucosinolates and myrosinases. This finding sheds light on the ecology and evolution of plant-insect interactions and suggests novel highly selective pest management strategies.

Fig. 1.

The glucosinolate–myrosinase system in plant defense and as manipulated by the P. rapae NSP. In damaged plant tissue, glucosinolates (1) are hydrolyzed by myrosinases yielding glucose and unstable aglucones (2). (A) In mechanically damaged plant tissue, the aglucones rearrange spontaneously into isothiocyanates [mustard oils (3)], which have frequently been shown to act as insecticides. Depending on the structure of the glucosinolate side chain R and the reaction conditions, nitriles (4) and other hydrolysis products can also be formed. (B) In plant material ingested by P. rapae larvae, the aglucones (2) do not undergo the rearrangement to the toxic isothiocyanates (3) but instead form nitriles (4) caused by the presence of NSP.

Fig. 2.

Spectroscopic identification of 4-hydroxyphenylacetonitrile sulfate isolated from P. rapae feces. Structure and numbering system of 4-hydroxyphenylacetonitrile sulfate are depicted. The signals in NMR, MS, and IR spectra were assigned as follows: ¹H NMR (D₂O, 400 MHz): δ 3.94 (s, H2); 7.32 (“d”,J = 9 Hz, H3′+ H5′); 7.43 (“d”,J = 9 Hz, H2′+ H6′). ¹³C NMR (D₂O, 100 MHz): δ 23.1 (C2); 120.8 (C1); 122.9 (C3′ + C5′); 129.3 (C1′); 130.3 (C2′ + C6′); 151.5 (C4′). Selected heteronuclear multiple bond correlation interactions are indicated by arrows. MS, m/z: 463 [M²⁺K]^–; 447 [M²⁺Na]^–; 212 [M]^–; 132 [M-SO₃]^–. MS/MS confirmed m/z 132 as a fragmentation product of m/z 212. IR, ν_max: 2,246 cm^–1 (w, CN). UV, λ_max: 208 nm (100%), 260 nm (4%).

Fig. 3.

Metabolism of glucosinolates in larvae of P. rapae. Analyses of glucosinolate hydrolysis products in feces of larvae fed A. thaliana (Col-0) rosette leaves (A) and in a comparable batch of leaves after mechanical disruption (B). Feces were extracted in a mixture of dichloromethane and water, whereas an equal amount of rosette leaves was macerated in water, and the aqueous phase was extracted with dichloromethane after 5 min. The organic phases were subjected to GC-MS. 1, 5-(methylsulfinyl)pentanenitrile; 2, 4-(methylsulfinyl)butanenitrile; 3, 4-(methylsulfinyl)butyl isothiocyanate (1-isothiocyanato-4-(methylsulfinyl)butane); 4, 3-(methylsulfinyl)propyl isothiocyanate (1-isothiocyanato-3-(methylsulfinyl)propane; IS, internal standard. Sections of total ion chromatograms are depicted.

Fig. 4.

Metabolism of benzylglucosinolate in vitro by myrosinase, P. rapae extracts, and heterologously expressed PrNSP. (A) Chemical structures of the hydrolysis products of benzylglucosinolate, phenylacetonitrile 1 and benzyl isothiocyanate 2. (B–F) Benzylglucosinolate was incubated with concentrated (B–D) or diluted (E) midgut extract (C, boiled midgut extract) in the presence (B, C, and E) or absence (D) of a commercial myrosinase preparation, or with myrosinase only (F). (G and H) Benzylglucosinolate was incubated with an extract of E. coli harboring a PrNSP expression construct (G) or the empty expression vector (H) in the presence of myrosinase. Assays were extracted with dichloromethane after 1-h (B–F) or 2-h (G and H) incubation at room temperature, and the organic phases were analyzed by GC-MS and GC-FID. Sections of GC-FID chromatograms are depicted. IS, internal standard.

Fig. 5.

Correlation of NSP activity with protein bands on SDS/PAGE after size exclusion chromatography of a purified NSP preparation. A plot showing total NSP activity per fraction (A) is superimposed with an image of a Coomassie-blue-stained SDS/PAGE gel showing the protein composition of each fraction (B). The eluate was collected in 2-ml fractions that were analyzed for NSP activity and by SDS/PAGE (an equivalent of 500 μl of fraction was loaded per lane). Arrows indicate bands analyzed by LC-MS/MS. The numbers on the left indicate the sizes of the molecular mass marker bands in kilodaltons.

Fig. 6.

Amino acid sequence of P. rapae NSP. A predicted extracellular targeting signal is shown in green. The protein contains two complete amino acid repeats (shown in red and blue) covering a stretch of ≈200 aa each and one incomplete repeat (shown in orange). Peptide sequences identified by LC-MS/MS analyses are underlined.

Fig. 7.

Expression analysis of PrNSP by RT-PCR. Agarose gel shows RT-PCR products obtained from different tissues of fourth instar P. rapae larvae. Lane 1, head; lane 2, body (without head, gut, fat body, and Malpighian tubules); lane 3, midgut; lane 4, fat body; lane 5, hindgut with Malpighian tubules; lane 6, adults. The PrNSP transcript is represented by a PCR band of 650 bp, the EF-1α transcript (control) by a band of 400 bp. M, DNA ladder.