Resistance: DNA methylation and the evolution of pesticide-resistance genes in aphids

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics

Abstract

Most insect species are specialist parasites that have adapted to colonize one or a few closely related plant species. Circa 10% of all insect herbivores are generalist ("polyphagous"), and these are often the most noxious pests, having evolved resistance to many pesticides. Generalist insects are prone to such "pesticide-breaking" because by being exposed to a wide range of hosts, they have already evolved resistance to many different plant chemicals. Worryingly, many of these phytochemicals have been used to derive pesticides, and this makes such generalist insects pre-adapted to pesticide-breaking.

The green peach aphid (GPA) Myzus persicae can colonize over 400 different plant species, and it has evolved resistance or tolerance to 71 chemicals in 50 years. GPA has become a model to understand how "pesticide-breaking" has evolved. In a former BBSRC funded project, we discovered the molecular mechanism underpinning the insect's remarkable adaptive potential; DNA methylation by two genes (DNMT3A and 3B) enables GPA to adjust to diverse plant species and overcome pesticide toxicity without genetic modification. Genetically identical clones can adjust to a new host plant and show pesticide resistance within hours or days after exposure, and furthermore, their (genetically identical) offspring becomes increasingly well-adjusted. We have shown that the expression of detoxification genes is controlled by DNA methylation (which is an epigenetic process). We also showed that when DNMT3A/B methylation is knocked-down, that the aphids can no longer adjust. We also know that different genes are being up- or down-regulated by DNMT3A/B methylation depending on the host plant or pesticide being encountered.

Building on this knowledge, our new proposal has two principal objectives:
1) Identify and describe the entire gene networks affected by de novo DNA methylation mediated by DNMT3A/B. This is important because these are the genes that enable the aphid to detect and respond to the pesticide, and hence, this will help the development of pesticides against novel insect targets. Our Project Partner Syngenta, and others, will thus be helped in their development of such pesticides.
2) Understand the evolutionary forces that generate and shape the DNA variation underpinning the genetic variation in these "resistance genes" in the DNMT3A/B pathway. This is important because the 50 years of pesticide usage will have left a decipherable signature in the genome of the aphids (and other pest insects). By studying this signature, we can optimise pest insect control strategies.

The hypotheses are:
1) GPA possesses genes that help the insect to detect the novel host plant / pesticides, which instigates DNMT3A/B upregulation.
2) The DNMT3A/B methylated genes enable the insect to detoxify these chemicals.
3) Generalist aphids (e.g., GPA) display a larger change in DNMT3A/B expression levels upon host switch and pesticide exposure than specialist aphids (such as the cabbage aphid Brevicoryne brassicae, and the English grain aphid Sitobion avenae).
4) Co-regulatory networks affected by DNMT3A/B are more extensive for generalist than specialist aphids.
5) Generalist insect pests (including herbivores, animal and human insect pests) show genome streamlining, lineage-specific gene families and gene duplication that is distinctive from specialist pest insects.
6) The historic use of pesticide treatment will have impacted the 5 evolutionary forces (mutation, recombination, gene flow, genetic drift and natural selection), which has shaped the genomic variation in GPA in populations across the world.

In collaboration with our Project Partner Syngenta, we have designed three exciting experiments that test these hypotheses. We believe the knowledge generated by this research is likely to uncover new targets for insect control, and will help to optimise species-specific insect control strategies, and hence secure sustainable agriculture.

Technical Summary

The green peach aphid (GPA) Myzus persicae has evolved resistance to 71 chemicals in 50 years, and it has become a model to study the evolution of "pesticide-breaking". In a former BBSRC project, we discovered that DNA methylation by two genes (DNMT3A and 3B) enables GPA to up- and down-regulate genes associated with resistance. In this proposal, we aim study the entire gene networks affected by de novo DNA methylation mediated by DNMT3A and 3B. We also analyse the evolutionary forces that generate and shape the DNA variation underpinning the existing genetic adaptations in an agricultural setting.

Aim 1 is to characterize the genes that depend on DNMT3A/B for differential expression (DE) upon GPA host change (Aim 1.1) and pesticide treatment (Aim 1.2). We will isolate and sequence RNA from aphids with and without host transfer / insecticide exposure to identified DE genes. We will then conduct gene-specific bisulphite sequence of DE genes to examine the link between gene body methylation and DE (Aim 1.3).

Aim 2 investigates how pesticide treatment and DNMT3A/B affect gene expression of two specialist aphid species. We will test how the knock down of DNMT3A/B affects GPA and two specialist aphid species to adjust to new plant hosts and pesticides. We will also identify the genes that depend on DNMT3A/B regulation in specialist and generalist aphids.

Aim 3 elucidates the evolution of genes in the DNMT3A/B pathway involved in response to plant host change and pesticides in GPA, and other (generalist and specialist) pest insects. Aim 3.1 examines the level of genome streamlining, the number of lineage-specific genes in multigene families, and number tandem-duplications of detoxifying genes, testing the hypothesis that these features are associated to rapid pesticide-breaking. Aim 3.2 will identify genes and genomic areas in GPA that are under positive selection across world populations with known history of insecticide usage (in collaboration with Syngenta).

Planned Impact

This proposal is for the BBSRC highlight call 'Understanding the challenge of resistance in agriculture' in which the green peach aphid (GPA, Myzus persicae) is specifically mentioned. This proposal responds to all aims in the call. Here we detail who will benefit from our proposed research and explain how, focusing on the specific aims from the BBSRC highlight call:

1. "Using new scientific approaches to address practical problems for agriculture or resistance to pesticides." and "Focusing on the molecular mechanisms of resistance, its evolutionary drivers and the ecological processes involved in the emergence and spread." - Our previous BBSRC grant (BB/L002108/1: "Functional Genomics of Aphid Adaptation to Plant Species") identified DNMT3A/B as an important molecular mechanism controlling the regulation of genes that enable GPA to parasitise new host plants and overcome pesticide toxicity. Building on this breakthrough, we now wish to dissect the entire gene pathway enabling GPA to detect and adapt to pesticides.
Beneficiaries: We believe this will help industry, such as our project partner Syngenta, to identifying new potential targets for pesticide development. In turn, this will aid agriculture and food security.

2. "Promoting collaboration between researchers with existing interests in resistance and others with wider relevant expertise in underpinning science." - The proposal involves researchers interested in molecular aspects of plant-insect interactions (Hogenhout. JIC), genomics / bioinformatics (Swarbreck. EI), and evolution (Van Oosterhout, UEA), and Syngenta, Jealott's Hill (Firth and colleagues). The integration of functional genetics and genomics with evolutionary theory enhances the proposed research project. We will be using population genetic theory to understand the processes that occur during the evolution of insecticide resistance in the field, resequencing the genomes of ~100 GPA individuals exposed to pesticides across the globe.
Beneficiaries: The research is of direct relevance to Syngenta, who have committed to support this proposal with a >10% contribution, and others. Besides this financial contribution, the project will benefit significantly from the knowledge of Syngenta about the previous usage of pesticides in locations that will be sampled for GPA to resequence. We believe that the knowledge generated will improve pest insect control strategies, promoting more sustainable usage of pesticides in agriculture, thereby helping long-term food security.

3. "Stimulating innovative research to understand resistance and inform interventions for enhancing effectiveness of existing products and optimizing the use of new ones" - The neonicotinoid TMX has been particularly effective at control of GPA for many years, but "pesticide breaking" appears to be an evolutionary inevitability, especially for generalist pests such as GPA. By using a comparative phylogenomic approach, this proposal investigates how GPA and a wide range of other pest insects have evolved resistance to pesticides in the agricultural setting.
Beneficiaries: The proposed research will aid Syngenta as well as other strategic research projects of the Hogenhout lab, such as the identification of plant resistance to GPA (funded by BBSRC-LINK and iCASE projects with the sugar beet seed breeding company SESVanderHave), development of control methods for the notorious insect pest, the tobacco whitefly Bemisia tabaci (iCASE project with Oxitec), and establishing global networks on vector-borne diseases (funded by the UK-US partnership award and GCRF network with East Africa).

4. "Raising the profile amongst the broader research community of the impact of resistance on agriculture and the scientific challenges it presents." - Here, we plan to engage to broader research community and the public by setting up the OpenINSECTVector website and engaging with the general public in a Citizen Science (see Pathways to Impact).

Publications

10 25 50
 
Description We used transcriptome data of M. persicae colonies on 9 plant species to reannotate the the M. persicae genome and identified novel aphid gene families that play a role in regulating aphid-plant interactions. The writing of a publication is in progress.

We optimized methods to generate and analyze bisulphite sequence data of aphids.

We developed the nanopore sequencing technology for aphids and generated Hi-C data that taken together enables us to generate a chromosome-level assembly for M. persicae clone O.

We collected M. persicae and other aphid species worldwide and resequenced the genomes of about 40 single M. persicae. These sequences will be aligned to the chromosome-level assembly.

We established SapFeedersHub that enables the downloading of aphid genome sequence data and annotations generated within this grant.

A manuscript describing some of the results generated within this award was submitted to BioRxiv and is being reviewed for publication in a journal.
Exploitation Route Data were shared our industrial collaborator. A manuscript describing some of the results generated within this award was submitted to BioRxiv. We established SapFeedersHub that enables the downloading of aphid genome sequence data and annotations generated within this grant.
Sectors Agriculture, Food and Drink,Chemicals,Education,Environment

 
Description We exchanged knowledge on research progress with industrial partner at several meetings. Progress made within this grant led to funding of a iCASE studenthip that starts in Oct 2020 and is co-funded by Syngenta and is in collaboration with Liverpool School of Tropical Medicine. Tools, resources and knowledge gained within this project led to the the approaches described in work package 3 of the successful BRIGIT consortium project that generates data and informs policy makers about mitigating the potential introduction of Xylella fastidiosa, which is transmitted by a sap-feeding insect species that is common in the UK.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Education,Environment,Retail,Transport
Impact Types Societal,Economic,Policy & public services

 
Description Project Leader of BRIGIT, a UK-wide consortium to mitigate the risks of Xylella fastidiosa outbreaks in the UK
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
Impact The BRIGIT consortium includes people from various layers of government, charities, research institutes and industry. The writing of the BRIGIT proposal and activities within BRIGIT so far increased the knowledge of the consortium members about the Xylella pathosystem and how Xylella fastidiosa may spread in the UK and harm the environment. This is likely to influence future regulations to maximize protection of the UK environment.
URL https://www.jic.ac.uk/brigit/
 
Description Recruitment of new head for the Entomology Facility at the institute
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
Impact Provided leadership to the JIC Entomology Facility steering committee upon retirement of Facility head and recruitment of a new Facility head. Developed a draft of a business plan, managed Entomology Facility staff in the transition period, developed a position description for the new Facility head, interviewed applicant and successfully recruited a new head.
 
Description BRIGIT - A consortium for enhancing UK surveillance and response to Xylella fastidiosa
Amount £5,000,000 (GBP)
Funding ID BB/S016325/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 03/2021
 
Description Cash contribution to IPA grant
Amount £131,700 (GBP)
Organisation Syngenta International AG 
Sector Private
Country Switzerland
Start 04/2018 
End 03/2021
 
Title BS-seq analyses on aphids 
Description We optimized bisulphite sequencing of aphids, including library construction, sequencing and data analyses. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Providing a useful method and strategy to the research community. 
 
Title Genome assembly 
Description Developed strategies to improve the assembly of aphid genomes that includes the identification and removal of contigs derived from microbial organisms that are abundantly present in aphids. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Assisted colleagues in the US with improving genome assembly of the soybean aphid. 
 
Title Nanopore for aphids 
Description We optimized DNA isolation methods for nanopore sequencing of aphid genomes. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact We are able to improve aphid genome assemblies. 
 
Title Developed and launched SapFeedersHub 
Description Database that provides access via an Ensemble platform to genome sequences and annotations of insect species of the order Hemiptera, including aphids, leafhoppers, planthoppers and froghoppers/spittle bugs. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact Colleagues can download sequence data and annotations of economically important insect pests, including for instance the Xylella insect vector Philaenus spumarius. Data can be used for population structure analyses and GWAS to study genomic regions involved in insect phenotypes or host preference. 
URL http://sapfeedershub.jic.ac.uk/index.html
 
Description Collaboration with East Malling Research 
Organisation East Malling Research
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are sequencing and annotating the genome of the woolly apple aphid, a serious pest of apple trees
Collaborator Contribution The collaborator provided frozen aphids for genome and RNA sequencing
Impact We will obtain the complete genomes and transcriptomes of the woolly apple aphid, which is in a distinct clade in the aphid phylogenetic tree and useful for comparative genome analyses among aphids.
Start Year 2018
 
Description Collaboration with Syngenta on an iCASE studentship 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution Wrote iCASE studentship. Advertised position for PhD student and hosted and interviewed applicant.
Collaborator Contribution Contributed to the writing of the iCASE studentship
Impact iCASE project that starts Oct '20.
Start Year 2019
 
Description Collaboration with University of Oxford 
Organisation University of Oxford
Department Department of Plant Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Visited colleagues at University of Oxford to discuss specific project and experimental approaches
Collaborator Contribution Contributed knowledge and resources for new experiments
Impact Progress with achieving research goals by graduate student and postdoctoral researcher in the lab. Making plans for a collaborative research proposal.
Start Year 2019
 
Description Engaged with colleagues at the International Centre of Insect Physiology and Ecology (ICIPE) 
Organisation International Centre of Insect Physiology and Ecology (ICIPE)
Country Kenya 
Sector Academic/University 
PI Contribution Involved colleague at ICIPE in the GCRF-funded project to study Napier grass stunt phytoplasma
Collaborator Contribution Provided plant and insect materials for sequencing
Impact Materials are being processed
Start Year 2018
 
Title Annotation of genes encoding long non-coding RNAs in aphid genomes 
Description We developed a pipeline to annotate genes for long non-coding RNAs in aphid genomes 
Type Of Technology Software 
Year Produced 2018 
Impact We have the sequences of aphid candidate long non-coding RNAs 
 
Title Improve aphid genome assembly pipeline 
Description We optimized methods to improve aphid genome assemblies using existing software 
Type Of Technology Software 
Year Produced 2018 
Impact We obtained high quality chomosome level assemblies of aphid genomes 
 
Title Improved aphid genome annotation pipeline 
Description We work with the Earlham Institute to optimize annotation of aphid genomes 
Type Of Technology Software 
Year Produced 2019 
Impact Improved annotation of aphid genomes. 
 
Description Attended Gatsby Plant Science Annual Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Attended the annual meeting on behalf of Gatsby-funded graduate student
Year(s) Of Engagement Activity 2019
 
Description Attended Introductory programme for UvA professors 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Participated in an introductory programme for UvA professors. The Executive Board of the University of Amsterdam offers this programme to all newly appointed UvA professors, to facilitate them in their new role in the academic community and as UvA representatives. The programme offered insights and tools that helped strengthen personal effectiveness in daily work situations including extending network within the UvA. It also allows to gain more insight into the role and position of a UvA professor, meet colleagues and exchange experiences plus learn more about Dutch academic leadership codes. Furthermore, organizational and financial aspects of the university will be highlighted as well as current developments within the UvA and the role of academic leadership.
Year(s) Of Engagement Activity 2019
 
Description Engagement with Syngenta 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Regular meetings with colleagues at Syngenta, Jealott's Hill, UK, and Switserland and USA to discuss project proposals and research progress on aphids.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Invited research seminar at CAS institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a research seminar at IGDB, Beijing, China on 16 Mar 2018. Research fellow Thomas Mathers in my lab also contributed a talk.
Year(s) Of Engagement Activity 2018
 
Description Invited research seminar at the Max Planck Institute, Cologne, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was invited to give a research seminar at the Max Planck Institute for Plant Breeding, Cologne, Germany. Approximately 50 people, including PhD students, attended.
Year(s) Of Engagement Activity 2018
 
Description Invited research seminar at the annual Life Sciences conference, Beijing, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a talk in a paralell session focused on insect pests at the Life Sciences Conference, Beijing, China, 28-31 Oct '19.
Year(s) Of Engagement Activity 2018
 
Description Participated in conference at Syngenta 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Attended Syngenta external collaboration event. Three postdoctoral lab members in my group presented research talks and updates with their project progress.
Year(s) Of Engagement Activity 2019
 
Description Participated in visit of Syngenta to the Norwich Research Park 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussed progress on current funded projects and possibilities of future projects with colleagues from Syngenta.
Year(s) Of Engagement Activity 2019
 
Description Participated summerschool, Pwani University, Kenya 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Co-organized a two-day course as part of the two-week AfriPlantSci summerschool for ±25 professionals and students from research institutes and university in Kenya and several other countries in sub-saharan Africa. Two PhD students of my team participated in the organization of the summerschool. Protocols we taught in the course were shared and are being used in current projects of the course participants.
Year(s) Of Engagement Activity 2019
 
Description Participation in autumn workshop of The Sainsbury Laboratory 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Participated in the autumn workshop of The Sainsbury Laboratory. A PhD student in my group gave a talk and received substantial feedback.
Year(s) Of Engagement Activity 2019
 
Description Visit of Syngenta 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Visited Syngenta to give update on project progress. Three team members presented and engaged with Syngenta colleagues.
Year(s) Of Engagement Activity 2019