Potential use of a serpin from arabidopsis for pest control

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Abstract

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC50 = 637 μg ml-1). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control. © 2011 Alvarez-Alfageme et al.

Figures

  • Table 1. Ecological function and main digestive proteases of the invertebrate species tested in vitro against AtSerpin1.
  • Table 2. In vitro inhibitory activity of the protease inhibitor AtSerpin1 against trypsin- and chymotrypsin-like serine, and cathepsin B- and cathepsin L-like cysteine activities in extracts of several pest and non-target invertebrate species.
  • Figure 1. Weight gain of Spodoptera littoralis larvae fed on a diet containing 65 or 650 mg g21 AtSerpin1 or control diet without inhibitor. Feeding assays were performed for 6 days with third-instar larvae. Bars represent mean 6 SE. Bars with different letters on the same day are significantly different (P,0.05; one-way ANOVA followed by Student-Newman-Keuls) (N = 48). doi:10.1371/journal.pone.0020278.g001
  • Figure 2. Serine-like proteolytic activities of Spodoptera littoralis third-instar larvae fed for 6 days on a diet containin 65 mg g21 AtSerpin1 or control diet without inhibitor. Bars represent mean 6 SE. Bars with different letters are significantly different (P,0.05; MannWhitney U test) (N = 24). doi:10.1371/journal.pone.0020278.g002
  • Figure 3. Western blot immunoassay showing the expression of AtSerpin1 in leaves of the transgenic Arabidopsis lines AtSerpin1OE1, AtSerpin1OE2, and AtSerpin1OE3, and the non-transformed line Col-0. Lanes: (1) Page ruler plus protein standard; (2) 100 ng AtSerpin1; (3) 50 ng AtSerpin1; (4) 25 ng AtSerpin1; (5) 12.5 ng AtSerpin1; (6) 5 ng AtSerpin1; (7) 0 ng AtSerpin1; (8) overproducing line AtSerpinOE3 (6 ng); (9) overproducing line AtSerpinOE2 (6 ng); (10) overproducing line AtSerpinOE1 (6 ng); (11) non-transformed line Col-0. In lanes 7–9, the upper band is the full-length and active form of AtSerpin1, while the lower band is the cleaved form after interaction with a protease. doi:10.1371/journal.pone.0020278.g003
  • Figure 4. Weight gain of Spodoptera littoralis larvae fed on transgenic Arabidopsis plants overproducing AtSerpin1 (lines AtSerpinOE1, AtSerpinOE2, and AtSerpinOE3) or on non-transformed plants (line Col-0). Feeding assays were performed for 4 days with second-instar larvae. Bars represent mean 6 SE. Bars with different letters on the same day are significantly different (P,0.05; one-way ANOVA followed by Student-Newman-Keuls) (N = 24). doi:10.1371/journal.pone.0020278.g004
  • Figure 5. Concentration-response curve for mortality of newborn Acyrthosiphon pisum nymphs fed for 3 days with artificial diet containing increasing concentrations of the protease inhibitor AtSerpin1. Points represent mean 6 SE. Three to six replicates with 15 nymphs each were used per concentration. doi:10.1371/journal.pone.0020278.g005
  • Table 3. Proteolytic activities of Acyrthosiphon pisum adults after 1 day of feeding on a control diet (AtSerpin12) or a diet containing 1000 mg ml21 AtSerpin1 (AtSerpin1+).

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Alvarez-Alfageme, F., Maharramov, J., Carrillo, L., Vandenabeele, S., Vercammen, D., van Breusegem, F., & Smagghe, G. (2011). Potential use of a serpin from arabidopsis for pest control. PLoS ONE, 6(5). https://doi.org/10.1371/journal.pone.0020278

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