Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Abstract

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of P12-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by P12, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by P12. P12-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.The involvement of endogenous proteinaceous proteinase inhibitors (PIs) in plant defense against leaf-feeding insects has been well recognized (1, 2). Experiments with artificial diets and a wide range of insects confirmed the antinutritional effects of proteinaceous PIs (3-7), although some negative results were also reported (8). The expression of heterologous PIs in transgenic tobacco plants provided final confirmation in planta for their roles as resistance factors, although protection was only partial (9, 10).Recently, the effectiveness of proteinase inhibitors was suggested to depend on the affinity or specificity of an inhibitor for the main gut proteinases of an insect (4,11,12), but the mechanism of action and effect of proteinase inhibitors are only partially understood. Broadway and Duffey (3) showed that gut proteinase activities of Spodoptera exigua and Heliothis zea were similar or increased when larvae were chronically exposed to high levels of potato proteinase inhibitor II (PI2) or soybean trypsin inhibitor in artificial diets. The simple scenario that growth rates were reduced due to reduced rates of proteolysis (13) was, therefore, dismissed. Instead, these results were interpreted by Broadway and Duffey (3) to suggest that a feedback mechanism was leading to the hyperproduction of proteinases to compensate for the loss of activity, which in turn led to the depletion of essential amino acids and finally resulted in retarded growth rates.Our aim was to establish to what extent and how PI2 expressed in tobacco leaves would affect larval growth and digestive physiology of S. exigua. Our data show that S. exigua larvae adapt to PIs by induction of gut proteinase activity that is insensitive to inhibition.t MATERIALS AND METHODSPlant Transformation. A full-length PI2 cDNA clone, p303.5 1, was selected from a tuber-specific Solanum tuberosum cv. Bintje cDNA library by using p303 as a probe (14, 15). The region encoding the N-terminal part was amplified by PCR using primers 152FO (5'-GCGGGATCCACCATGGC...

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

“…However, when SBTI was incorporated into artificial diets and offered to C. capitata larvae at WD 50 of 3.01% was observed, with a slight effect on larval mortality, in disagreement with the results obtained in vitro. This probably happens because insect pests adapt to host plant proteinase inhibitors by synthesizing proteinases that are either insensitive to inhibitors (Jongsma et al, 1995;Broadway, 1997) or have the capacity to degrade them (Michaud, 1997;Giri et al, 1998). Zymogram using SBTI showed interactions between enzyme and inhibitor, with complex retardation during electrophoresis.…”

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

“…The necessity of such a strategy has become increasingly evident with the understanding that many insects possess very effective resistance mechanisms against individual PIs. Resistance has been attributed to complex proteolytic systems, allowing the insects to degrade PIs in Coleoptera (Girard et al, 1998a) and Lepidoptera (Giri et al, 1998), and to enhance the production of inhibitor-insensitive proteinases in response to PI ingestion in Coleoptera (Girard et al, 1998b;Bonade-Bottino et al, 1999;Cloutier et al, 1999) and Lepidoptera (Broadway 1995(Broadway , 1997Jongsma et al, 1995;Broadway, 1996b;Brown et al, 1997;Wu et al, 1997). It has been suggested (Orr et al, 1994;Broadway, 1996a) that such complex mechanisms are most likely to exist in polyphagous insects having generalized feeding habits compared to oligo-or monophagous insects.…”

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

“…Insect herbivores have developed over time effective strategies to elude the inhibitory effects of plant protease inhibitors (Broadway, 2000), including: (1) the use of complex digestive protease systems with proteases from different mechanistic classes acting in a complementary, coordinated manner (Terra and Ferreira, 1994;Brunelle et al, 1999Brunelle et al, , 2004; (2) the production of alternative, insensitive protease forms following ingestion of protease inhibitors (Jongsma et al, 1995;Bown et al, 1997;Cloutier et al, 1999Cloutier et al, , 2000Broadway, 2001a, 2001b;Zhu-Salzman et al, 2003;Brunelle et al, 2004); and (3) the degradation of defensive protease inhibitors using nontarget, insensitive digestive proteases (Michaud et al, 1995a;Michaud, 1997;Girard et al, 1998a;Giri et al, 1998;Gruden et al, 2003;ZhuSalzman et al, 2003). It is now generally recognized that protease/inhibitor interactions in plant-insect systems are the result of a long, coevolutive process triggering the continuous diversification of proteolytic and protease inhibitory functions in the competing organisms (Lopes et al, 2004;Valueva and Mosolov, 2004;Christeller, 2005;Kiggundu et al, 2006;Girard et al, 2007).…”