Vaughan A. Hilder

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests.

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.

Additive protective effects of different plant-derived insect resistance genes in transgenic tobacco plants

Abstract Plant genetic engineering has been proposed as a method of producing crop plants with enhanced resistance to insect pests. One strategy for attaining this goal is to exploit the inherent resistance shown by some plants against insect herbivores, which in some cases has been shown to involve potentially insecticidal proteins. Genes derived from higher plants encoding two such proteins, the cowpea trypsin inhibitor and the pea lectin, have been shown to enhance resistance to Heliothis virescens (tobacco budworm) when expressed in transgenic tobacco plants. Both genes were introduced together into individual plants by cross-breeding the two transgenic lines. Insect bioassays on the plants expressing both genes showed their effects on Heliothis to be additive. Thus, multigene, multimechanistic insect resistance can be built up in genetically engineered plants. Such multiple mechanisms are typical of natural insect resistance in plants and may offer an ecologically acceptable way of protecting crop plants from insect pests.

Protease activities in the larval midgut of Heliothis virescens: Evidence for trypsin and chymotrypsin-like enzymes

Protease activities in the midgut contents of larvae of the tobacco budworm, Heliothis virescens, were investigated. Two major activities were present: (i) a trypsin-like enzyme, hydrolysing the synthetic substrate N-benzoyl-arginine p-nitroanilide (BApNA), strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and not inhibited by chymostatin at concentrations where this inhibitor is chymotrypsin-specific; (ii) an enzyme which hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, but not N-benzoyl-tyrosine p-nitroanilide and was strongly inhibited by chymostatin. The latter activity was considered to represent a hitherto ill-characterised family of insect chymotrypsins, which differ from their mammalian counterparts in activity towards synthetic substrates. Both activities had strongly alkaline pH optima, in the pH range 10–11, but were shown to be due to distinct proteases. Low levels of elastase-like activity (hydrolysing succinyl (alanine)3 p-nitroanilide) were also detected. The two major enzyme activities were shown to account for almost all the protease activity of gut contents towards protein substrates. The proteases showed different sensitivities to inhibition by plant protein protease inhibitors, which were effective in protecting exogenous proteins from digestion by gut extracts.

Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens

The feeding activity of adult rice brown planthopper, Nilaparvata lugens (Stal) on selected proteins incorporated into artificial diets was examined, based on quantitative analysis of honeydew excretion, in order to determine the mechanism of action of antimetabolic proteins towards homopteran pests. The lectins Galanthus nivalis agglutinin (GNA), wheat germ agglutinin (WGA) and the enzyme soybean lipoxygenase (LPO), previously demonstrated to be toxic to this insect, reduced honeydew excretion levels of adult Nilaparvata lugens over a 24 h period when incorporated into artificial diet at 0.1% {w:v}, indicating that these proteins acted as antifeedants. Of the proteins tested GNA was the most effective antifeedant, reducing honeydew droplet production by 96%, although after 24 h there was some recovery in the honeydew excretion levels and thus the insects appeared to tolerate the presence of the antifeedant with time. The lectin Pisum sativum agglutinin (PSA), previously demonstrated to be non‐toxic, showed no antifeedant properties.

Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens

Insect feeding trials were carried out to determine the effects of a range of mannose‐specific lectins on third instar nymphs of the rice brown planthopper (BPH), Nilaparvata lugens. Stål. Dose response curves show that Galanthus nivalis agglutinin (GNA) has the strongest toxic effect of the lectins tested, and is effective at concentrations considerably lower than those previously reported. Narcissus pseudonarcissus agglutinin (NPA) and Allium sativum agglutinin (ASA) exhibit a significant antimetabolic effect towards the insect but were less effective (on a molar basis) than GNA. LC50 values for GNA, NPA and ASA are approximately 4 μM, 11 μM and >40 μM respectively. These mannose‐specific lectins are serologically identical, but differ in the number of subunits per protein molecule; ASA is a dimer, NPA is a trimer and GNA is a tetramer. The results obtained support the hypothesis, that the effectiveness of the mannose‐binding lectins as antimetabolites is determined by the number of subunits per molecule. Two N‐acetylglucosamine binding lectins, the dimeric Oryza sativa agglutinin (OSA) and the monomeric Urtica dioica agglutinin (UDA), were also tested but at a concentration of 0.1% w/v exhibited no significant antimetabolic effect towards BPH, although the related lectin wheatgerm agglutinin (WGA) has previously been demonstrated to be toxic towards the insect.

Genetic engineering of plants for insect resistance

Plants and insects have been co-evolving for at least 300 million years; during this time plant species have been under continuous selection pressure from herbivorous insect predators, and, in the absence of the ability to avoid predators by movement, have been forced to rely on physical and chemical defensive mechanisms. Physical mechanisms include the presence of hairs on the surfaces of plant tissues, spines, exuded gums and toughened surface layers, such as hardened seed coats. Chemical defensive mechanisms include all the cases where a plant metabolite present in the plant tissues is toxic to predators, or interferes with the normal growth and development of predators, or deters predators by its taste or smell (Norris and Kogan, 1980).

The inhibitory effects of the cysteine protease inhibitor, oryzacystatin, on digestive proteases and on larval survival and development of the southern corn rootworm (Diabrotica undecimpunctata howardi)

At least eight proteolytic activities have been identified in the midgut contents of larval Southern corn rootworm (Diabrotica undecimpunctata howardi). Around 70% of protease activity could be arrested by the cysteine protease inhibitors E‐64 and chicken egg‐white cystatin, while the aspartic acid protease inhibitor pepstatin caused 30% inhibition. The cysteine protease activity was found to be highly sensitive to inhibition by both chicken egg‐white cystatin and the rice cystatin, oryzacystatin I. Oryzacystatin I, expressed as a fully functional fusion protein in E. coli, was found to strongly inhibit larval gut protease activity. This recombinant oryzacystatin, incorporated into artificial diet at concentrations of 10 mM and above, caused significant decreases in larval survival and weight gain. E‐64 was also shown to cause a significant antimetabolic in vivo effect. These results demonstrate the great potential for cysteine protease inhibitors, such as oryzacystatin, as tools for exploitation in the control of the Southern corn rootworm.

Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea (Vigna unguiculata Walp.)

The protein and gene sequences of the cowpea Bowman-Birk type trypsin inhibitor which confers enhanced insect resistance to transgenic tobacco plants, and of cowpea trypsin/chymotrypsin inhibitors are presented. There are regions of high conservation and high divergence within the 5′ leader, mature protein and 3′ non-coding regions of the Bowman-Birk inhibitors and in the genes which encode them in different members of this family within the Leguminosae. The practical implications of this finding for studies on the evolution of plants and the utilization of these genes for enhancing insect resistance is discussed.