Exploitation of genomic knowledge for sustainable resistance to the crop pest Globodera pallida

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences

Abstract

Plant parasitic nematodes cause estimated annual losses to global agriculture of $125b . Potato cyst nematode costs the UK potato industry an estimated £50 m/year. Control often depends on nematicides that are both harmful to the environment and possibly human health. They are often essential for economic potato cropping in the UK. The new technology to be used in this work can control plant parasitic nematodes without harming the environment. A protein and the role it plays in a cell can be abolished if the gene that provides the code specifying it becomes inactive. This is a natural effect that probably protecting cells from mobile genetic elements such as certain viruses. Its basis is that the some viruses make a double not the normal single strand of the intermediary molecule (RNA). It is involved in the reading of the genetic code when each new protein molecule is made. The natural gene silencing effect can be induced experimentally by introducing the double stranded form of RNA to a cell. This can cause an organism to silence one of its own genes. This is experimental manipulation is achieved by introducing to a cell a sequence that matches precisely that of the gene to be silenced. This is termed RNA interference (RNAi) and the effect was first demonstrated for the free-living nematode Caenorhabditis elegans after it swallowed dsRNA. The same effect induced by introducing dsRNA to a range of organisms. Plants can be modified to make the dsRNA of interest. RNAi provides a powerful experimental tool. Individual genes of interest can be specifically silenced and the effects of this studied. We established the use of RNAi for analyses to function of particular genes of plant parasitic nematodes. We have identified genes the nematode must express to feed in plants effectively. Others have used the approach to silence the function of individual genes in plants. In parallel we have used other methods to define which plant genes must express locally in roots for nematode to feed successfully. In this proposal we will express in the potato plant a small number of dsRNA molecules known to silence particular nematode genes. Silencing will occur after the animal has swallowed the dsRNA when feeding from the plant. We will also target a few plant genes that must be active for the nematode to feed normally. The key advantage of the approach is that no novel protein is made. There is no known basis for any particular dsRNA to be harmful in either the human diet or to the environment. It is therefore highly preferable to current use of pesticides or control procedures that are less cost effective. The dsRNA will be driven from a gene switch (promoter) that is naturally active just where the nematode feeds in the root and not in its tubers or leaves. The safe dsRNA will not even be in the potato tubers we eat. The silencing of these genes will prevent the development of the nematode and so stop the damage it causes to the crop. The work will provide an approach that helps meet the urgent need for UK potato growers to reduce pesticide use. It will allow cultivars favoured by consumers but currently damaged by nematode to be widely grown without any risk to food safety. The approach will have no environmental harm and allow natural control of insects to flourish by eliminating pesticides that currently harm them. The approach has wider potential than to be developed in the approach. The current application represents a basis for competitive and sustainable UK potato cropping in an improved environment for invertebrates and those that feed on them in UK potato fields.

Technical Summary

Plant parasitic nematodes cause losses to global agriculture of $125b annually. Potato cyst nematodes cost the UK potato industry an estimated £50 m/year. Control often depends on nematicides that are both harmful to the environment and possibly human health. A new biosafe technology will be developed to control plant parasitic nematodes that will have minimum environmental impact. The approach is based on post-translational gene silencing triggered by double stranded RNA, termed RNA interference (RNAi). We established the use of RNAi for functional analysis of plant parasitic nematodes genes, to identify genes nematodes must express to be a successful plant parasites. In parallel work, we have used microarray analysis to identify plant genes upregulated where a female cyst nematode modifies plant cells to form a syncytium. The nematode feeds at this one site and is developmentally committed to it. In this project, we will apply our new knowledge to direct expression of dsRNA in planta. We will use the approach to target nematode and plant genes that are crucial for a compatible host/pathogen interaction. The choice of plant genes has ensured that unwanted effects outside the feeding site are unlikely. Ingestion by the nematode of dsRNA targeted at one of its genes will induce a silencing effect with the detrimental consequences for the parasite. An RNAi effect has already been demonstrated after targeting nematodes genes both in vitro and in planta. We will use the same promoter to restrict expression of dsRNA targeted at either nematode or plant genes. The promoters to be used are up regulated in the feeding cell but show little activity elsewhere in vegetative plant tissues. The dsRNA approach lacks inherent food or environmental hazard and involves no transgenic protein expression for nematode control. This eliminates the perceived consumer or environmental risk associated with transgenic expression of proteins.

Publications

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Description Plant parasitic nematodes cause estimated annual losses to global agriculture of $125b . Potato cyst nematode costs the UK potato industry an estimated £50 m/year. Control often depends on nematicides that are both harmful to the environment and possibly human health. They are often essential for economic potato cropping in the UK. The new technology developed in this work can control plant parasitic nematodes without harming the environment.

We have made transgenic plants that when parasitised by a nematode induce silencing of specific nematode genes and therefore stop the parasitic process. The mechanism that silences the genes is termed RNA interference (RNAi) and is dependent on the delivery of double stranded RNA (dsRNA) that matches the sequence of the gene to be silenced.

In vitro experiments: A series of RNAi experiments were performed in vitro to silence cysteine proteinase genes of the potato cyst nematode Globodera and the root-knot nematode Meloidogyne. Globodera and Meloidogyne juvenile (J2) nematodes were exposed to dsRNA corresponding only to their innate proteinase in addition to a dsRNA molecule that contained the two sequences in tandem. When the Globodera was subsequently allowed to infect plants a lower proportion of the treated nematodes exposed to dsRNA designed to Globodera sequence (47.2%) or the dsRNA containing the dual sequence (41.8%) successfully established on the plants, in comparison to the controls. Similarly, when the Meloidogyne was subsequently used to infect plants a lower proportion of the treated nematodes exposed to dsRNA designed to Meloidogyne sequence (43.7%) or the dsRNA containing the dual sequence (41.3%) successfully established on the plants, in comparison to the controls. Transcript abundance of the targeted genes for both Globodera and Meloidogyne was also assessed and shown to be markedly reduced in comparison to controls.

The mechanism of RNAi is sequence dependent. Our work evaluated the level of sequence specificity required to effect gene knock down in plant parasitic nematodes. The proteinase of the Globodera species, G. rostochiensis was silenced using dsRNA designed from G. pallida (98% identity) but not by dsRNA designed to match the proteinase of the Meloidogyne spp. (70% identity). The proteinases from the Heterodera species H. glycines and H. schachtii were not silenced by either dsRNA designed to G. pallida (~85% identity) or Meloidogyne (~69%).

This was the first demonstration of multiple genes being silenced by a single dsRNA containing multiple sequences.

Nematode challenge of transgenic hairy root cultures expressing dsRNA: Globodera and Meloidogyne J2s were used to challenge hairy root cultures that expressed constructs designed to produce dsRNA in planta. Three transgenic lines for each construct were selected based on the presence of high levels of transgene RNA. Those transgenic root cultures that produced dsRNA corresponding to Globodera sequence or both the Globodera and Meloidogyne sequence showed a significant reduction (in comparison to the controls) in Globodera that established on the plant, with the most effective lines showing a reduction of 63.2% and 72% respectively. There was no significant reduction in the Globodera burden on root cultures producing dsRNA that corresponded only to sequence from Meloidogyne. Analogous results were obtained for Meloidogyne infections.

Nematode challenge of whole plants expressing inverted repeat transgenes: Transgenic potato plants, expressing the same constructs that were used to generate root cultures, were selected by PCR and confirmed by RT-PCR. These plants were tested initially in a growth system that allows rapid evaluation of nematode infection prior to full evaluation in containment. The results of both studies were similar to those recorded in the screen using root cultures. Mean nematode numbers on all four lines expressing dsRNA corresponding to Globodera sequence were significantly reduced compared to those on the WT controls. These experiments gave confidence to advance the plants for evaluation in the field.

In addition to plants that generated dsRNA corresponding to nematode cysteine proteinases we also generated plants that targeted the potato myo-inositol oxygenase (MIOX) gene that is highly expressed in the syncytial feeding site of Globodera. The expression of this dsRNA was under the control of the root-specific ARSK-1 promoter. These plants also showed resistance (64.6 %) to the nematode in comparison to the controls.

We had shown in other work that a ribosome inactivating protein (b32RIP) was functional against plant cells. A construct was developed in which b32RIP was driven by the promoter of the MIOX gene. This gene is only expressed in the nematode feeding site and in floral organs. The b32RIP gene causes cell death by inactivating the plant ribosome, and restriction of expression is required to parts of the plant (e.g. flowers)that are not necessary for potato production and the nematode feeding cells. Plants expressing this construct revealed resistance of 60 % when tested in bioassay in containment glasshouses.

This was the first demonstration that potato cyst nematodes are susceptible to such an in planta RNAi approach.

Field trial evaluation of plants expressing inverted repeat transgenes: The three best performing transgenic lines that targeted a Globodera cysteine proteinase, or the plant MIOX gene through the RNAi route, were advanced to evaluation in the field. Those plants expressing the b32RIP from the MIOX promoter were also tested in the field. Of those lines targeting a Globodera cysteine proteinase, the one that performed best in containment also delivered the highest level of resistance (66 %) in the field.
This was the first demonstration in the field of RNAi technology working against a plant pest.

Analysis of combinatorial RNAi in Arabidopsis plants expressing inverted repeats to target multiple nematode and plant genes: Arabidopsis has been transformed with constructs designed to deliver RNAi to target either a nematode cysteine proteinase, a parasitism gene expressed in the nematode oesophageal glands or a plant hexose transporter. Additionally, lines have been generated to target all three nematode genes simultaneously. Preliminary data suggests that 80% resistance has been achieved against a cyst nematode using this strategy.
Exploitation Route This project was focussed on developing strategic applications of previous BBSRC-funded fundamental research. The outputs have a clear utility for the implementation of transgenic resistance strategies against plant parasitic nematodes both via the private sector and through public sector involvement in technology transfer to subsistence farmers in developing world countries The RNAi technology for nematode resistant plants developed and evaluated within this project has attracted interest from the private and public sector both in the UK and abroad.
Sectors Agriculture, Food and Drink

 
Description The RNAi technology has been donated, together with that already provided by our laboratory, to the developing world through a USAID-funded initiative. Plant transformation constructs developed as a result of this work are being used within our collaboration on nematode-resistant East African Highland banana as part of a USAID ABSPII project, based in Uganda. The technology is also being used to produce transgenic egg-plants in India as part of a RCUK-funded project on sustainable agriculture.
First Year Of Impact 2010
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description Sinochem research funding
Amount £500,000 (GBP)
Organisation Sinochem Group 
Sector Private
Country China
Start 09/2012 
End 08/2015
 
Description Wain Fellowship
Amount £43,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2009 
End 08/2009
 
Description BBC Countryfile 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact PU appeared in the BBC1 programme Countryfile, describing the GM crop research carried out as part of this and other BBSRC grants. He was filmed at the field trial site where trials were carried out as part of this project. A BBC1 programme was broadcast in September 2009.

Increased interest in the work of the research group
Year(s) Of Engagement Activity 2009
 
Description Cafe Scientifique (Halifax) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The talk was followed by a question and answer session and there was general discussion about the pros/cons of GM technology and, in particular, its benefits for the developing world.

Members of the audience reported that their views on GM crops had been changed after participating in the event
Year(s) Of Engagement Activity 2013
 
Description Cafe Scientifique (Hull) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The talk was followed by a question and answer session and there was general discussion about the pros/cons of GM technology and, in particular, its benefits for the developing world.

Members of the audience reported that their views on GM crops had been changed after participating in the event
Year(s) Of Engagement Activity 2009
 
Description Frontier Potato Group Presentation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk sparked questions and discussion afterwards: possibilities of commercial direction.

The audience were aware of GM technology that could provide a solution to nematode pathogens
Year(s) Of Engagement Activity 2012,2015
 
Description Interview for BBC news 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact PU was interviewed by BBC news to provide comment about GM field trials

None reported
Year(s) Of Engagement Activity 2009
 
Description Parliamentary and Scientific Committee 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact HJA gave a presentation to a meeting of the Parliamentary and Scientific Committee on the topic of "Can GM help to feed 9 billion people by 2050?"
The talk sparked questions and discussion amongst the attendees.

Impacts will be long term in influencing decision making concerning the support for GM crops
Year(s) Of Engagement Activity 2009
 
Description Potato Conference 2010 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact HJA led a workshop on potato cyst nematodes at the Potato Growers Research Association: 21st Annual Potato Conference. The focus of the workshop was "How will consumers affect potato production in the future?" This stimulated interesting discussion between growers/industry/researchers.

The workshop led to useful sharing of ideas and experiences. Direct dialogue with growers informs future research directions for nematode control
Year(s) Of Engagement Activity 2010
 
Description US Embassy Food Security 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Discussion of Food security issues with particular emphasis on its potential for the developing world

None reported
Year(s) Of Engagement Activity 2009
 
Description Yorkshire Post 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact An interview with the Yorkshire Post about receiving Defra's approval for further research into genetically modified potatoes.

A large numeber of new contacts regarding our GM field trials.
Year(s) Of Engagement Activity 2010
 
Description cafe scientifique (York) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk sparked a lively discussion about the development and use of GM crops

A number of audience members reported that their views on GM crops had changed after hearing the presentation and ensuing discussion
Year(s) Of Engagement Activity 2009