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Adaptive evolution of threonine deaminase in plant defense against insect herbivores - PubMed

  • ️Sat Jan 01 2011

Adaptive evolution of threonine deaminase in plant defense against insect herbivores

Eliana Gonzales-Vigil et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

TD2-deficient tomato lines are compromised in resistance to S. exigua. (A) Three-day-old S. exigua larvae were transferred from an artificial diet to 4-wk-old WT plants or TD2-deficient lines (TDAs15 or TDAs7). One larva was caged per plant. At the indicated time after infestation, larvae were weighed and returned to their plant of origin. Values indicate the mean larval weight ± SE of 18–30 biological replicates. Means with a different italicized letter are significantly different at P ≤ 0.01. Similar results were obtained in two additional independent bioassays performed with both transgenic lines. (B) Western blot analysis of TD2 protein accumulation in undamaged control leaves (0) and damaged leaves from plants that were infested for 4 or 7 d.

Fig. 2.
Fig. 2.

TD2 is activated by a chymotrypsin-like protease in the lepidopteran midgut. (A) Total protein was extracted from tomato leaves that were damaged by L. decemlineata (Leaf), or from feces of M. sexta, T. ni, and L. decemlineata larvae reared on wild-type (WT) or jai1 plants (Frass). Proteins (20 μg) were separated by SDS/PAGE and stained with Coomassie Blue (Top). The same samples were used for immunoblot analysis with anti-TD2 (Middle) and anti-Rubisco large subunit (RbcL; Bottom) antibodies. Arrows denote polypeptides corresponding to RbcL, TD2, and pTD2. (B) Fourth-instar T. ni larvae were reared for 24 h on an artificial diet containing recombinant TD2, after which insect frass and the remaining diet were collected for protein extraction. Proteins were separated by SDS/PAGE and analyzed by immunoblotting for the presence of TD2. (C) M. sexta larvae (third instar) were allowed to feed on a TD2-containing diet as described above. Actively feeding larvae were frozen and then dissected. Protein extract prepared from the remaining diet, foregut (Fgut), midgut (Mgut), hindgut (Hgut), and frass were analyzed by immunoblotting for the presence of TD2. (D) Coomassie Blue-stained gel showing the TD2 cleavage products generated at various times (min) after incubation of recombinant TD2 with partially purified digestive proteases (TPP) isolated from frass of T. ni larvae grown on an artificial diet. (E) Dose-dependent effect of chymostatin on TD2 processing by T. ni digestive proteases. Chymostatin (at the indicated concentration in micromolar) was incubated with TPP for 15 min before addition of 0.4 μg TD2 substrate. Reactions were incubated at 37 °C for 1 h. Cleavage products were separated by SDS/PAGE, and the resulting gel was stained with Coomassie Blue. A reaction containing TD2 without the T. ni protease or chymostatin was included as a control (Mock).

Fig. 3.
Fig. 3.

Differential stability of TD isoforms. (A) Recombinant TD2 and TD1 were incubated at 37 °C for the indicated time (min) with partially purified T. ni TPP or an equivalent amount of assay buffer (Mock). Reaction products were analyzed by SDS/PAGE and staining with Coomassie Blue. (B) Differential temperature optimum of TD1 and TD2. Reaction mixtures containing recombinant TD1 (●) or TD2 (○) were incubated at the indicated temperature for 30 min for the activity assay. Activity levels are expressed relative to the activity observed at the TD1 and TD2 optimal temperature of 16 °C and 60 °C, respectively. (C) Differential heat inactivation of TD isoforms. Recombinant proteins were incubated at 55 °C for the indicated time before measuring TD activity at 30 °C. Activity is expressed relative to a control reaction that was not preincubated at 55 °C.

Fig. 4.
Fig. 4.

Crystal structure of pTD2. (A) Spatial arrangement of the four monomers (labeled A–D) that compose the pTD2 tetramer, with the helical linker regions that define the TD2 cleavage site shown in black. The box surrounding monomers A and B represents the asymmetric unit. (B) Cartoon diagram of pTD2 monomer with N1 domain (brown), N2 domain (green), helical linker (blue), and bound PLP cofactor molecule shown as stick models. Critical ion pairs present in pTD2 but not predicted by the TD1 homology model are shown as black cylinders. Critical ion pairs present in both proteins are shown as cylinders colored in both red (positively charged side chain) and blue (negatively charged side chain).

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