Toxicity and mode of action of plant lectins with a ricin-B domain against pest insects

Abstract

In recent years a lot of attention was given to possible applications of genetic engineering within integrated pest management (IPM). Insect resistant transgenic crops have now been created for many of the world’s economically important crops including maize, cotton, rice and potatoes. Lectins are only one group of proteins that have received a lot of attention because of their insecticidal properties. Although the physiological role of lectins in plants remains enigmatic, it has been clearly shown that the specific carbohydrate-binding activity of lectins plays an important role in plant interactions with other organisms. Entomotoxic effects of different lectins against several orders of plant pest insects including Lepidoptera, Coleoptera, Diptera and Hemiptera have been demonstrated both in vitro and in planta. Since lectins are good candidates to confer insect resistance in transgenic crops, they can be considered of great economic potential for pest management. In this PhD project analyses were performed to investigate the entomotoxic effects of a specific plant lectin family, referred to as the ricin-B related lectins (previously called type 2 ribosome-inactivating proteins (RIPs)). In particular the insecticidal activity of Sambucus nigra agglutinin I (SNA-I) and I’ (SNA-I’) was investigated. SNA-I is a chimeric lectin of 240 kDa composed of an A-domain with N-glycosidase activity and a B-domain with carbohydrate-binding properties for NeuAc(α-2,6)Gal/GalNAc. Furthermore, experiments were carried out to study the mode of action of SNA-I and the hololectin SNA-II. SNA-II is a homodimeric Gal/GalNAc-binding lectin of 60 kDa that consists only of carbohydrate-binding B-domains but lacks enzymatic activity. Chapter 1 gives a detailed overview of the occurrence RIPs in plants and their biological properties. In addition, more information is given on the interaction of plant lectins and insects. In Chapter 2 the effects of the type 2 RIP SNA-I′ delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects, belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae), and Lepidoptera, the beet armyworm (Spodoptera exigua). It was shown that M. nicotianae fed on detached leaves of transgenic lines expressing SNA-I’ reduced adult survival and it was apparent that the type 2 RIP SNA-I’ influenced aphid development by a retardation in the onset of reproduction. In aphids fed on transgenic lines expressing SNA-I’, significant effects were observed on the life parameters, such as intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring. In addition, the experiment with caterpillars showed significant increases in mortality for larvae fed on the transgenic lines expressing SNA-I’ as compared to wild type plants. Further studies revealed a significant reduction in fresh larval mass of caterpillars as well as retardation in development. This study clearly showed the insecticidal activity of SNA-I’ on both insect species. Hence, this information provides further support for RIPs having a role in plant resistance to insect pests. In Chapter 3 the insecticidal activity of the type 2 RIP SNA-I was studied on two hemipteran insect species using both feeding assays with an artificial diet and transgenic lines overexpressing the protein. Feeding of the pea aphid (Acyrthosiphon pisum) on an artificial diet supplemented with different concentrations of the purified SNA-I resulted in reduced survival and fecundity. Similarly, analyses with tobacco aphids (M. nicotianae) on transgenic tobacco plants overexpressing the SNA-I gene revealed a delay in development and reduced adult survival. In addition, the fertility parameters of the surviving aphids were reduced. In the second part of this study a series of experiments was performed using transgenic lines in which a mutant form of SNA-I was expressed. The mutation of one carbohydrate binding site strongly lowered the insecticidal activity of SNA-I, whereas the mutation of both lectin sites in SNA-I abolished its entomotoxic effects on tobacco aphids. From these results it was evident that the carbohydrate-binding activity of SNA-I is essential for its insecticidal activity. At present, the mode of action of RIPs in general and SNA-I in particular towards insects is not known. In Chapter 4 we attempted to get insight into the mode of action of the type 2 RIPs SNA-I. Exposure of insect midgut CF-203 cells to SNA-I showed that this lectin is able to induce cell death with the typical characters of apoptosis such as cell shrinkage, plasma membrane blebbing, nuclear condensation and DNA fragmentation. Exposure of SNA-I to the cells induced caspase-3 like protease activity, suggesting that SNA-I can induce the apoptotic pathway. Interestingly, the hololectin SNA-II revealed a very similar activity towards CF-203 cells. Both SNA-I and SNA-II induced caspase-dependent apoptosis pathway in the low nM range, leading to typical symptoms of cell death. Since SNA-II only has carbohydrate-binding domains, it was concluded that the induction of apoptosis in CF-203 cells by treatment with SNA-I and SNA-II is independent of the activity of the A-chain of SNA-I. In further extend of the study on the mode of action of SNA-I and SNA-II the endocytosis and uptake mechanisms involved in the internalization of SNA-I and SNA-II in CF-203 cells were evaluated in the presence or absence of different inhibitors in Chapter 5. Therefore cytotoxicity of SNA-I and SNA-II on cells was evaluated and the effect of different inhibitors was scored. Furthermore, the internalization of FITC-labeled SNA-I and SNA-II was quantified. Microscopic studies revealed that the uptake of both SNA-I and SNA-II was dose dependent. In addition, it was shown that both SNA-I and SNA-II were taken up in the CF-203 cells by a receptor-mediated endocytosis mechanism that is clathrin- and caveolae-dependent. Since similar results were obtained for both SNA-I and SNA-II these results confirmed that the A-chain of the type 2 RIP is not involved in the internalization process. Taking into account the results of previous chapters which clearly showed the entomotoxic properties of SNA-I and its effect on insect cells experiments were set up in Chapter 6 to investigate the effect of SNA-I at insect level. Therefore, A. pisum and S. exigua were fed a diet containing SNA-I. Feeding bioassays with artificial diet supplemented with purified SNA-I or SNA-II on aphids showed insecticidal activities. Also caterpillars fed on diet mixed with SNA-I showed reduction in larval mass. In this study it was shown that SNA-I also induced the apoptotic characteristics at the level of the organism (insect). Caspase-3 like activity and DNA fragmentation were observed in the gut tissues of beet armyworms and pea aphids. In conclusion, the data of this PhD project demonstrated that the elderberry lectins SNA-I and SNA-II show insecticidal activity, which makes them potential tools for crop protection against pest insects