CUKPGP:New pest resistance in rice, by breeding and genetic modification (GM) for constitutive and inducible levels of defence homoterpenes

Lead Research Organisation: Rothamsted Research
Department Name: Biological Chemistry & Crop Protection

Abstract

This project aims to develop and evaluate a new generation of rice crops in China with protection against major insect pest populations, that is delivered via a non-toxic mode of action, ie through modification of pest behaviour. The innovative science in this project will underpin the development of environmentally sustainable solutions for food production in China, extending to small-holder farms in rural areas.
The genes responsible for the biosynthesis of homoterpenes, known to be active against rice pests but not yet in any way exploited in sustainable pest control, will first be identified tentatively by next generation sequencing and bioinformatics, using information (including from UK-China Collaboration BB/J020281/1) from related genetics via wild and model plants, and identity confirmed by overexpression, including in rice, followed by chemical analysis. Elite rice cultivars and relatives will then be screened for high expression of these genes and new lines created overexpressing them under regulation by inducible promoter sequences, using chemical analysis and studies with pests, including the rice brown planthopper Nilaparvata lugens, and their parasites, including Anagrus nilaparvatae. The most promising lines from both routes will be developed by breeding, including production of synthetic crosses, and further genetic engineering for preliminary field evaluation in year 3. This will produce new sustainable pest resistance traits by breeding and genetic modification that will provide rice varieties that both resist pests but also exploit natural parasitism. In addition to the value in rice, these traits will be directly applicable to other crops, including in the long term perennial rain-fed rice. This new cooperation provides essential breeding opportunities and functional gene diversity (China) and secondary metabolite defence targeting (UK).

Technical Summary

New pest resistance in rice, but for eventual exploitation in other crops, will be developed by breeding and genetic modification (GM) for constitutive and inducible levels of defence active homoterpenes. The homoterpenes are naturally produced by plants on attack and reduce pest colonisation and promote natural pest control by parasitic wasps. These homoterpenes are produced at low levels by elite rice cultivars and these levels need to be raised for robust resistance, particularly by induction when specifically needed for pest control. The homoterpenes are biosynthesised from higher molecular weight precursors by oxidation and the enzymes, cytochromes P450, are known for some plants, e.g. Cyp82G1 from Arabidopsis thaliana and studied further in our UK-China collaboration BB/J020281/1. Next generation sequencing (NGS) and bioinformatics will be used tentatively to identify the rice genes with confirmation by chemical and behavioural analysis by over-expression in model systems, e.g. E. coli, and rice. Elite rice cultivars, their ancestors and wild relatives will be screened for high expression of these genes and for new lines created to overexpress them under regulation by inducible promoter sequences using chemical analysis, and behavioural and developmental studies with pests, including the rice brown planthopper, and their parasites, e.g. parasitic wasps. The most promising rice lines from both the screening and GM routes will then be developed by further breeding and more refined genetic engineering, particularly regarding the induced expression and response to elicitors for this induction. Preliminary field evaluation will be carried out in year 3 using the pest resistance assessments and also agronomic traits. This work will demonstrate the value of the new traits for exploitation in other food crops, but also in long term breeding for perennial growth, e.g. perennial upland/rain-fed rice for delivery of defence against pests solely via initial seed.

Planned Impact

The pressure currently to produce new resistance in rice to pests will guarantee impact if this project is successful in its overall aims. The impact on developing resistance by breeding and GM routes will result in numerous new projects following this and related to it by having secondary metabolite targets being funded if this project is successful. The exact route to impact will be via the State breeding agencies in China working under CAD and in the UK on wheat via the private breeding industry, with both of which this China-UK cooperation has extremely close links. The work will have another predictable impact, even if not fully practically successful in this project, in that there are many scientific groups, science funding agencies and those concerned with sustainable development of agriculture in both the developed and developing worlds who will be keen to see how a specific attempt to use stress plant secondary metabolites constitutively or inducibly expressed in crop pest management in a manner that can deliver novel defence traits via the seed.
We believe that replacement of pesticide use with the novel pest resistance traits described here would be both a plausible and highly desirable objective for Chinese agriculture, specifically with regard to rural area development.
The proposed activities aim to support agricultural and ecological development, strengthen agricultural infrastructure, ensure national food security, advance agro-processing production, and increase the income of farmers. The project uses a wide range of approaches including insect behaviour, natural product chemistry and state-of-the-art genomics, biochemistry and molecular biology. The scientist to be employed by the Chinese partner and based on extended placements at Rothamsted will benefit from being exposed to multi-disciplinary research and the diverse expertise of the applicants both in UK and China.
In Africa, where low-input systems are already required, a crop protection programme against pests and parasitic weeds exploits the defence homoterpenes effectively and sustainably by means of a companion cropping approach (push-pull). However, the resources and expertise of this China/UK cooperation are essential for access to the breeding opportunities and gene diversity for homoterpene biosynthesis as well as the success in targeting secondary metabolite/defence based evidence by this team and the ongoing UK-China Collaboration BB/J020281/1 on the genetics of homoterpene biosynthesis.

Publications

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Anderson JA (2016) Emerging Agricultural Biotechnologies for Sustainable Agriculture and Food Security. in Journal of agricultural and food chemistry

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Babikova Z (2013) How rapid is aphid-induced signal transfer between plants via common mycelial networks? in Communicative & integrative biology

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Birkett MA (2014) Prospects of genetic engineering for robust insect resistance. in Current opinion in plant biology

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Khan ZR (2014) Achieving food security for one million sub-Saharan African poor through push-pull innovation by 2020. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description We first identified, by NGS, potential target terpene synthase genes from lima bean following either infestation with spider mites or treatment with alamethicin and examined their expression profiles. Two such upregulated genes were transformed into an elite rice variety and secondary metabolite homoterpene production was quantified. It was demonstrated through behavioural bioassays that these homoterpenes were able to attract the natural enemy of rice pests, a parasitoid wasp.
Using coupled GC-electrophysiology (GC-EAG) recordings from planthoppers, the volatile profile of a rice variety susceptible to planthopper infestation has been shown to include 8 EAG-active compounds. Identification and quantification of these volatile compounds was performed and compared to 7 other rice varieties. The 8 rice varieties examined possessed a unique volatile profile that resulted in different planthopper behaviour. This provides further underpinning science for determining which volatile signals need to be deployed in modification of planthopper behaviour.
The transcriptomes of planthopper-infested and uninfested rice varieties have been compared. The homoterpenes, and several putative genes involved in the production of homoterpenes identified. Work is ongoing to prepare recombinant proteins for functional studies. If functionality is demonstrated, future work will aim to develop transgenic rice lines utilising these genes.
Exploitation Route This project specifically relates to new GM rice for pest resistance and should be developed, if successful, commercially.
Sectors Agriculture, Food and Drink