Potential Use of a Serpin from Arabidopsis for Pest Control

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

Barley cystain variants against phytopathogenic fungi pests and their impact on natural enemies

The goal of this study has been to know the effects of the barley cystatin Hv-CPI and seven derived variants generated by direct-mutagenesis, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata and to assess the potential impact of these proteins on the spiner soldier bug (SSB), Podisus maculiventris, a generalist hemipteran predator. Among the different cystatins tested, the variant C68 G, in which the only cysteine residue was changed to glycine, showed the highest inhibitory activity when tested in vitro against commercial cysteine proteases and CPB digestive enzymes. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing this variant, resulted in significant lower weight gains compared to those fed on non-transformed plants. No effects on survival, development, and weight, were observed when SSB nymphs fed on prey CPB reared with transgenic potato plants. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. The effects of the barley inhibitor Hv-CPI and its variants on the growth of the the necrotrophic fungus Fusarium oxysporum have been also analysed. The cystatin Hv-CPI inhibited fungal spore germination by 25%, the five point mutations inhibited spore germination by 18 to 40%, while the two truncated forms had no antifungal effect

Barley cystain variants against phytopathogenic fungi pests and their impact on natural enemies

The goal of this study has been to know the effects of the barley cystatin Hv-CPI and seven derived variants generated by direct-mutagenesis, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata and to assess the potential impact of these proteins on the spiner soldier bug (SSB), Podisus maculiventris, a generalist hemipteran predator. Among the different cystatins tested, the variant C68 G, in which the only cysteine residue was changed to glycine, showed the highest inhibitory activity when tested in vitro against commercial cysteine proteases and CPB digestive enzymes. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing this variant, resulted in significant lower weight gains compared to those fed on non-transformed plants. No effects on survival, development, and weight, were observed when SSB nymphs fed on prey CPB reared with transgenic potato plants. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. The effects of the barley inhibitor Hv-CPI and its variants on the growth of the the necrotrophic fungus Fusarium oxysporum have been also analysed. The cystatin Hv-CPI inhibited fungal spore germination by 25%, the five point mutations inhibited spore germination by 18 to 40%, while the two truncated forms had no antifungal effect

Ecological function and main digestive proteases of the invertebrate species tested <i>in vitro</i> against AtSerpin1.

<p>Abbreviations: SEP = serine endoproteases; CEP = cysteine endoproteases.</p><p>*Only SEP were tested against AtSerpin1.</p

<i>In vitro</i> 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.

<p>The percentage of inhibition was calculated as [(1 – activity with AtSerpin1/activity without AtSerpin1)×100]. Values represent mean+SE for duplicated independent determinations from a unique pool of extracts.</p><p>*pest species;</p>†<p>non-target species.</p><p>“ni” denotes no inhibition.</p

Weight gain of <i>Spodoptera littoralis</i> larvae fed on transgenic <i>Arabidopsis</i> plants overproducing AtSerpin1 (lines AtSerpin^OE1, AtSerpin^OE2, and AtSerpin^OE3) or on non-transformed plants (line Col-0).

<p>Feeding assays were performed for 4 days with second-instar larvae. Bars represent mean ± SE. Bars with different letters on the same day are significantly different (<i>P</i><0.05; one-way ANOVA followed by Student-Newman-Keuls) (<i>N</i> = 24).</p

Serine-like proteolytic activities of <i>Spodoptera littoralis</i> third-instar larvae fed for 6 days on a diet containin 65 µg g⁻¹ AtSerpin1 or control diet without inhibitor.

<p>Bars represent mean ± SE. Bars with different letters are significantly different (<i>P</i><0.05; Mann-Whitney <i>U</i> test) (<i>N</i> = 24).</p

Weight gain of <i>Spodoptera littoralis</i> larvae fed on a diet containing 65 or 650 µg g⁻¹ AtSerpin1 or control diet without inhibitor.

<p>Feeding assays were performed for 6 days with third-instar larvae. Bars represent mean ± SE. Bars with different letters on the same day are significantly different (<i>P</i><0.05; one-way ANOVA followed by Student-Newman-Keuls) (<i>N</i> = 48).</p

Concentration-response curve for mortality of newborn <i>Acyrthosiphon pisum</i> nymphs fed for 3 days with artificial diet containing increasing concentrations of the protease inhibitor AtSerpin1.

<p>Points represent mean ± SE. Three to six replicates with 15 nymphs each were used per concentration.</p