The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera

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Abstract

The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.

Highlights

► Antibodies were made against the major trypsins and chymotrypsins from Helicoverpa punctigera larvae. ► We used the antibodies to optimize procedures for extraction of proteases for immunoblot analysis and activity assays. ► Consumption of proteinase inhibitors did not lead to overproduction of trypsin or chymotrypsin. ► Ingestion of proteinase inhibitors resulted in excessive loss of proteases in the frass.

Introduction

Plant protease inhibitors (PIs) are part of a natural defense mechanism against herbivores and pathogens (Dunse and Anderson, 2011; Fan and Wu, 2005; Stevens et al., 2012), which has led researchers to examine their potential application in transgenic plants for protection against insect pests. There are many reports of lepidopteran species exhibiting reduced growth, development and survival after ingestion of protease inhibitors that have been incorporated into artificial diets (Bown et al., 2004; Srinivasan et al., 2005) or expressed in transgenic plants (Christeller et al., 2002; Dunse et al., 2010b). However, this response is not obtained with all PIs and no PIs have provided the level of sustainable plant protection required for commercial development (Abdeen et al., 2005).

Several mechanisms have been proposed to explain the detrimental impact of protease inhibitors on larval growth and survival. The most obvious mechanism is formation of a stable complex with digestive proteases which delays or blocks protein digestion, and limits the availability of essential amino acids required for insect growth, development and reproduction (Bown et al., 1997; Broadway, 1995; Gatehouse et al., 1997). Some insect species have been reported to respond to inhibition of gut proteases by hyperproduction of proteases to swamp the ingested PIs and allow digestion to occur (De Leo et al., 1998; Markwick et al., 1998). However, depending on PI levels this can lead to substantial loss of protein to the frass (Broadway, 1995; De Leo et al., 1998; Zhu-Salzman et al., 2003). This in turn limits the bioavailability of essential amino acids for protein synthesis and consequently leads to impairment of growth and development (Abdeen et al., 2005; Broadway, 1995; Markwick et al., 1998). In contrast, some insects are not severely affected by ingestion of PIs. These insects adapt to PIs in their diet by switching to different classes of proteases (Broadway, 1996; Jongsma and Bolter, 1997; Patankar et al., 2001; Winterer and Bergelson, 2001), or by producing inhibitor-insensitive proteases (Dunse et al., 2010b; Volpicella et al., 2003). Another adaptation to PIs is the production of proteases that degrade the PIs in the midgut (Giri et al., 1998; Gruden et al., 1998). Proteolysis of dietary PIs by insect proteases not only reduces the anti-nutritional effect of the PIs on digestive proteases, but also provides an additional source of amino acids (Telang et al., 2005).

It is often difficult to determine which of these adaptive mechanisms are operating in a particular insect pest due to limitations in techniques used to assay gut responses to PIs. Characterizing the effect of PI ingestion on insect digestion has been limited to measuring changes in protease activity or transcription of protease genes (Bown et al., 2004; Mazumdar-Leighton and Broadway, 2001). Protease activity is often difficult to measure accurately as activity can be affected by degradation of the proteases during extraction and storage or by the use of inappropriate substrates. Furthermore, protease activity measurements do not detect proteases that are complexed with inhibitors. In this paper we describe the use of enzyme assays and specific antibodies to quantify the levels of both active and inactive trypsins and chymotrypsins, after ingestion of the four trypsin and two chymotrypsin inhibitors produced from the multidomain NaPI precursor (proNaPI).

ProNaPI is a multidomain potato type II protease inhibitor from the ornamental tobacco Nicotiana alata. It is composed of six 6 kDa domains; two with chymotrypsin reactive sites (C1, C2) and four with trypsin reactive sites (T1–T4) (Atkinson et al., 1994; Lee et al., 1999). The precursor is processed during transit through the secretory pathway to release six 6 kDa PIs which are stored in the vacuole (Miller et al., 1999). When fed to Helicoverpa punctigera or Helicoverpa armigera larvae, the 6 kDa PIs were lethal to some larvae, but generally reduced growth and development without mortality (Charity et al., 1999; Dunse et al., 2010b; Heath et al., 1997). We have reported that H. armigera larvae that survive consumption of the six 6 kDa inhibitors (NaPIs) have elevated levels of a NaPI-resistant chymotrypsin (Dunse et al., 2010b). Here we describe the use of specific antibodies for the NaPIs as well as Helicoverpa chymotrypsins and trypsins to investigate the effect of NaPI consumption on protease production as well as stability of the NaPIs in the midgut of the major agricultural pest, H. armigera.

Section snippets

Production of NaPI affinity column

DNA encoding the chymotrypsin inhibitor (C1) and the trypsin inhibitor (T1) from NaPI was cloned into the pET11a expression vector (Novagen) and expressed in BL21 (DE3) cells as described by Schirra et al., 2001. The purified lyophilized C1-T1 dimer (7.2 mg) was immobilized onto cyanogen bromide-activated Sepharose 4 FastFlow resin (1140 mg, Sigma-Aldrich) according to the manufacturer's instructions.

Isolation of trypsins from H. punctigera gut

H. punctigera larvae (42) were reared to fourth instar on a haricot bean artificial diet (

Isolation of H. punctigera trypsins and encoding cDNAs

One major and one minor trypsin were purified from the excised midgut of 4th instar H. punctigera larvae using affinity chromatography on immobilized NaPIs followed by RP-HPLC (Supplementary Fig. S1). The N-terminal sequence of the major protein (IVGGSVTTIDQYPTIAALLYSWNLSTYW) was identical to the predicted N-terminal sequence of a trypsin from the closely related species H. armigera that is encoded by cDNA clone HaTC21 (GenBank ID: Y12269.1) (Bown et al., 1997). This 25 kDa protein was denoted

Discussion

Protease inhibitors are of great interest because of their negative effect on the growth and development of insect pests and their potential application for insect control in transgenic plants. A key component for the long term success of this technology is an understanding of the survival mechanisms insects use to overcome the negative effect of protease inhibitors in their diet. Earlier work has assessed the effect of NaPI on the growth and development of H. punctigera larvae (Dunse et al.,

Acknowledgements

This work was supported with funding from the Australian Research Council and Hexima Ltd. JAS was funded by a La Trobe University scholarship.

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