Responses of Wheat (Triticum aestivum) to Grain Aphid (Sitobion avenae) Infestation and Mechanical Wounding Using a cDNA Subtractitve Library Approach

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of commercial wheat (cv. Claire) to grain aphid (Sitobion avenae) infestation and mechanical wounding were investigated in this study, with the aim to eventually identify a source of molecular markers to breed wheat for enhanced insect resistance, and in particular for enhanced resistance to phloem-feeding insects. Mechanical wounding was included in this study as a comparison with aphid feeding to distinguish between insect-specific responses in wheat plants to those involved in a general wounding response. Wheat (Triticum spp.) is known to have partial resistance toward aphids [1]. The plant response and defence against insect feeding are complicated, but always follow the same principle: insect detection, signal transmission to initiate defence, changes in plant gene expression and subsequent production of defensive compounds, which may be targeted to the wound site to deter or kill insects. Defensive gene products/proteins reach the target area and deter or kill insects. Whether the last step is successful or not depends on the resistance and susceptibility of the plant towards that particular pest. In the light of this principle, it is important to detect changes in gene expression, first at the transcriptional level, which is useful for detection of early-stage responses, and then once sufficient time is allowed for the plant to produce defensive gene products, responses at the proteome level can be identified. Work presented in this study focuses on the changes at the transcriptional level; differential responses at the proteome level were investigated and presented in Ferry et al. 2011 [2] and Guan et al. 2015 [3]. Two cDNA subtractive hybridization libraries were constructed, one to identify transcripts involved in the responses to aphid infestation, and the second to identify transcripts involved in responses to mechanical wounding. Following subtractive hybridization, 520 and 800 clones were obtained from the subtractive hybridization between aphid-infested and un-infested wheat cDNAs and between mechanically wounded and un-wounded wheat cDNAs, respectively. Over 70% of the total clones were sequenced and 44% and 55% of sequenced clones were successfully identified by homology to known sequences held at NCBI with Blastx search engine in aphid-infested vs un-infested and mechanically wounded vs un-wounded cDNA subtractive libraries, respectively. These results reveal that the differences in the response of commercial wheat (cv. Claire) plants towards aphid infestation and mechanical wounding are subtle. Although the majority of differentially expressed putative genes after aphid infestation or mechanical wounding were involved in metabolic processes and photosynthesis, the majority of the genes expressed were different. Genes encoding glutathione transferase (GST), apoptosis and proteolysis were up-regulated after aphid feeding, suggesting their importance towards plant defence/tolerance against aphid attack. These results suggest that commercial wheat does have a certain degree of tolerance to aphids, but appears to lack a specific response to aphids; these findings are supported by those presented in Ferry et al. 2011 [2]

Differential protein expression in maize (Zea mays) in response to insect attack

Maize (Zea mays) is a major food stable in sub-Saharan Africa. However, yields are constrained by insect pests. Insect feeding induces a number of changes in genes encoding different proteins and the plant’s response can either be direct or indirect, or both. In this study, maize plants were infested with two insects with different feeding strategies (Spodoptera littoralis, chewing insect and Busseola fusca, stem borer) to investigate differential protein expression using the Proteomics technique. Infestation of S. littoralis (3rd instar larvae) resulted in 14 spots being up-regulated and 7 being down-regulated. Similarly, infestation of maize with B. fusca (3rd instar larvae) resulted in 12 spots being up-regulated and 9 spots being down-regulated. Interestingly, of those up-regulated only 9 were common to both insects, with only 4 common to both in terms of down regulation. Infestation of maize with S. littoralis resulted in a greater number of spots being up-regulated and less being down-regulated compared to maize infested with B. fusca. Unfortunately, we were unable to identify the proteins represented by these spots.Key words: Busseola fusca, down-regulation, expression, proteomics, Spodoptera littoralis, up-regulation

<em>Ex vivo</em> delivery of dsRNA targeting ryanodine receptors for control of <em>Tuta absoluta</em>

BACKGROUND: RNA interference (RNAi) is an endogenous eukaryote viral defence mechanism representing a unique form of post-transcriptional gene silencing. Due to its high specificity, this technology is being developed for use as dsRNA-based biopesticides for control of pest insects. Whilst many lepidopteran species are recalcitrant to RNAi, Tuta absoluta, a polyphagous insect responsible for extensive crop damage, is sensitive. Ryanodine receptors (RyRs) are intracellular calcium channels regulating Ca2+ release. The chemical pesticide class of Diamides functions agonistically against lepidopteran RyR, resulting in uncontrolled Ca2+ release, feeding cessation and death. Resistance to Diamides has emerged in T. absoluta, derived from RyR point mutations.RESULTS: RNAi was used to target RyR transcripts of T. absoluta. Data presented here demonstrate the systemic use of exogenous T. absoluta RyR specific (TaRy) dsRNA in tomato plants (Solanum lycopersicum) to significantly downregulate expression of the target gene, resulting in significant insect mortality and reduced leaf damage. Using a leaflet delivery system, daily dosing of 3 \ub5g of TaRy dsRNA for 72 h resulted in 50 % downregulation of the target gene and 50 % reduction in tomato leaf damage. Corrected larval mortality and adult emergence were reduced by 38% and 33 %, respectively. TaRy dsRNA demonstrated stability in tomato leaves up to 72h after dosing. CONCLUSIONS: This work identifies TaRy as a promising target for RNAi control of this widespread crop pest

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products

A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC50 values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins

A barley cysteine-protease inhibitor reduces teh performance of two aphid species in artificial diets and transgenic arabidopsis plants

Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 → G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzus persicae and Acyrthosiphon pisum. All the cystatins, except HvCPI-7, HvCPI-10 and HvCPI-12, inhibited in vitro the activity of cathepsin L- and/or B-like proteinases, with HvCPI-6 being the most effective inhibitor for both aphid species. When administered in artificial diets, HvCPI-6 was toxic to A. pisum nymphs (LC50 = 150 μg/ml), whereas no significant mortality was observed on M. persicae nymphs up to 1000 μg/ml. The effects of HvCPI-6 ingestion on A. pisum were correlated with a decrease of cathepsin B- and L-like proteinase activities. In the case of M. persicae, there was an increase of these proteolytic activities, but also of the aminopeptidase-like activity, suggesting that this species is regulating both target and insensitive enzymes to overcome the effects of the cystatin. To further analyze the potential of barley cystatins as insecticidal proteins against aphids, Arabidopsis plants expressing HvCPI-6 were tested against M. persicae. For A. pisum, which does not feed on Arabidopsis, a combined diet-Vicia faba plant bioassay was performed. A significant delay in the development time to reach the adult stage was observed in both species. The present study demonstrates the potential of barley cystatins to interfere with the performance of two aphid specie