Investigating the role of insect saliva in the plant-insect interaction as a basis for development of novel methodologies for control of homopteran pests of cereals

Phloem feeding insects (aphids, hoppers) are an important group of pests. They cause damage to their host plant not only by removing nutrients, but also more importantly by transmitting viral diseases. To date, there are no effective long-term control methods for managing the populations of these pests. To address this problem recent research has focused on studying endogenous defence mechanisms, with the aim of their future exploitation in crop protection. This project has studied both sides of plant-insect interactions (plant responses and insect means for invasion), using as a model system Oryza sativa (rice) as the host plant and Nilaparvata lugens (Brown planthopper) as the insect pest. The overall objective of the project was to investigate the molecular and biochemical nature of the 0. sativa - N. lugens interaction as a means of providing the basis for developing novel strategies for control of homopteran pests. For this purpose, insect feeding as the major determinant of host plant responses was studied with particular emphasis on the role of insect saliva, since recent studies on chewing insects have demonstrated that saliva is the main factor modulating host responses. Studying saliva from phloem feeding insects is extremely difficult and to date no salivary compounds from this insect order have been identified within host plants. Hydrogen peroxide (HzOz) is a part of the Reactive Oxygen Species (ROS) system, which includes superoxide (Oy), and hydroxyl radical (OH) as well. Several roles for HzOz have been assigned, such as signalling, cell wall modification and direct toxicity to pathogens and insects. Catalase is the main enzyme responsible for decomposition of hydrogen peroxide (HzOz). Using PCR with degenerate primers, a catalase cDNA (Kat-1) from Nilaparvata lugens salivary glands was identified. Kat-1 transcript abundance was higher in the gut than in salivary glands. Recombinant catalase (~57 kDa) was produced in E. coli, purified and used to produce antibodies in rats. lmmunodetection analysis indicated that there are three possible catalases with molecular masses of ~ 57 kDa, ~70 kDa and ~1 00 kDa in the salivary glands but only one ~57 kDa in the gut. A ~70 kDa immunoreactive protein was detected as well in infested host tissue from the resistant variety, suggesting that catalase is secreted from saliva into host plant tissue. Thus, the role of Nilaparvata lugens salivary catalase may be disruption of signalling and detoxification as well as conditioning of the host cells. Production of ROS occurs under both stressed and non-stressed conditions and ROS concentrations are modulated by enzymatic and non-enzymatic components within the antioxidant system. Key enzymes from the antioxidant system include superoxide dismutase (SOD; EC 1.15.1.1), catalases (CAT; EC 1.11.1.6), ascorbate peroxidases (AsPOX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2) and class III peroxidases (POX; EC 1.11.1.7). The effects of Nilaparvata lugens infestation and saliva on the antioxidant enzyme system in resistant and susceptible varieties were studied by assaying enzyme activity. Temporal and spatial analysis indicated different patterns of enzyme activation in susceptible and resistant host varieties, suggesting that the antioxidant enzyme system is controlled differently in the two rice varieties. At systemic level during infestation, increase of the HzOz-detoxifying system activity (SOD, CAT and AsPOX) was demonstrated only in the resistant variety. Further, saliva induced GR and AsPOX activity in the resistant variety only. These two enzymes are part of the AA-GSH (Ascorbate- Glutathione) cycle, the most important HzOz-detoxifying system in the chloroplasts, peroxisomes and mitochondria. At the mRNA level, there were local differential effects of infestation and saliva in the susceptible and resistant varieties on the expression of SOD and CatA genes. Variation in the expression patterns of genes coding for different forms of SOD, as well as CatA, was demonstrated only in the susceptible variety. Systemic expression of Nilaparvata lugens saliva-responsive genes was studied through subtractive hybridisation and Real Time RT-PCR confirmed the differential expression of several genes. Functional analyses indicated that Nilaparvata lugens saliva altered expression of genes involved In primary metabolism, signalling, transport, translation and regulation and with unknown functions. More than one third of up-regulated genes were implicated in senescence, including biosynthesis of exported amino acids and transport.EThOS - Electronic Theses Online ServiceGBUnited Kingdo