Exploiting eIF4E-based and associated broad-spectrum recessive resistance to potyviruses in dicots and monocots
Lead Research Organisation:
Rothamsted Research
Department Name: Plant Biology & Crop Science
Abstract
The project is a collaboration between Warwick HRI working on the dicotyledonous brassica plants and Rothamsted Research working on the monocotylonenous plants barley. The main aim is to: exploit sources of recessive eIF4E-based resistance to potyviruses in these plants. The involvement of the plant gene eIF4E in the life-cycle of viruses was first discovered in experiments on Turnip mosaic virus (TuMV, a potyvirus) and Arabidopsis. Subsequently it was found that the genes eIF4E or its isoform (eIF(iso)4E) are responsible for natural resistance to potyviruses in a range of crop types. The basis of the resistance is that potyviruses require one of these genes in order to complete their life-cycle. It is known that plants with specific alterations in these genes that affect the binding between the plant protein it codes and a viral protein (VPg) are resistant to infection. We have shown that eIF4E is associated with resistance to potyviruses in brassica and barley. The resistances described so far in other plant types involving eIF4E alone are not effective against all strains of the particular potyvirus. However, we have shown that in a brassica, eIF4E in combination with another gene (retr01), confers resistance against all strains of the potyvirus (TuMV). Other genes are involved in the interaction between the viral VPg protein and eIF4E, these include a the very similar gene eIF(iso)4E and other associated genes (eIF4G, eIF4A and isoforms of these). By studying the natural variation in the eIF4E and eIF(iso)4E genes from resistant and susceptible Brassica rapa and barley lines we aim to find new sources of resistance and understand the mechanism of the resistance. We will screen a collection of plants representing 95% of the genetic variation available within the brassica species and a barley diversity panel consisting of up to 800 landraces. Those plants that differ within the eIF4E gene will be tested with informative isolates of the potyviruses to check for resistance/susceptibility. Any eIF4E or eIF(iso)4E variants conferring resistance will be sequenced. This will be supported by looking at plants that have been mutated with chemicals to identify other novel changes in eIF4E and eIF(iso)4E alleles. Lines with such changes will be tested for resistance to a panel of BaMMV and BaYMV isolates. The aim is to identify forms of eIF4E that confer resistance that is durable to all (or most) virus isolates. Using the crystal structures available for eIF4E a model will be generated for eIF4E from barley and B. rapa. Natural and chemically-induced changes in the building blocks (amino acids) of the eIF4E associated with virus resistance in barley and B. rapa will be mapped on the 3D model of the normal eIF4E proteins. The effect of the changes on the potential ability of barley and B. rapa eIF4E to bind to the viral VPg protein will then be investigated. The outcome of this work will be the prediction of superior resistance genes of barley and B. rapa eIF4E for deployment in breeding. A gene retr01 which in combination with eIF4E confers the broad-spectrum resistance to TuMV in B. rapa will be fine mapped to identify the gene's position in the plant genome. The sequence of the gene will be used to search databases to predict its function, understand the mechanism of the broad-spectrum resistance and how retr01 might interact with eIF4E. Based on the best sources of virus resistance in brassicas and barley, changes associated with the genes conferring resistance will be used to design assays which can be used by breeders to incorporate the resistances into their elite breeding stocks by normal breeding methods (crossing). The project will provide an understanding of the scientific basis of the broad-spectrum resistance. Once the mechanism is understood it should be possible to deploy such resistance in a wide range of economically important crop species.
Technical Summary
The Potyviridae is the largest family of plant viruses in the world and cause some of the most important virus diseases of crops in the world. Plant resistance to viruses is the only reliable and sustainable means of control. Potyviruses seem to require the eukaryotic translation initiation factor 4E (eIF4E plant gene) for successful multiplication in the host. Incompatibility between variants of eIF4E genes and the potyvirus VPg has been found to be the basis of natural resistance to potyviruses in several species. eIF4E-based resistance is frequently strain-specific. This project aims to look for natural allelic variation in eIF4Ee within brassica and barley. Chemically induced allelic variants of eIF4E identified using barley TILLING populations currently available will increase the likelihood of finding novel variants capable of conferring resistance to potyviruses. Using these approaches and through modelling the likely effect of the different alleles on the known binding sites within the protein from both monocots and dicots we aim to identify and predict those likely to confer resistance to resistance breaking strains of the potyviruses. Other genes have been identified that have a role in the eukaryotic initiation complex (EIC) including eIF(iso)4E which has also been shown to be capable of conferring resistance. Allelic variation and possible associated resistance in this gene will also be investigated. In B. rapa we have already mapped a second gene (retr01) which with eIF4Ee confers broad-spectrum resistance to TuMV. This presents the opportunity to develop more durable resistance. By identifying this gene we will be able to determine if it is involved in the EIC and whether it interacts with eIF4Ee to confer broad-spectrum resistance. Discovery of the gene/s underlying broad-spectrum potyvirus resistance associated with eIF4E and markers for these provides the exciting possibility of developing durable potyvirus resistance in a wide range of crops.
Organisations
Publications
Hofinger BJ
(2010)
Identification and characterization of novel natural alleles of eIF4E in barley providing broad-spectrum resistance to bymoviruses
in Biology of Plant-Microbe Interactions. Antoun H, Avis T, Brisson L, Prevost D, Trepanier M (Eds). International Society for Molecular Plant-Microbe Interactions, St. Paul, MN, USA
Hofinger BJ
(2009)
High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.
in TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik
Perovic D
(2014)
Genetic analyses of BaMMV/BaYMV resistance in barley accession HOR4224 result in the identification of an allele of the translation initiation factor 4e (Hv-eIF4E) exclusively effective against Barley mild mosaic virus (BaMMV).
in TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik
| Description | • Developed of a new methodology based on High-resolution melting technology (HRM) for rapid and cost-effective identification of variant alleles in plants. • Identified 42 previously undescribed, functionally active eIF4E alleles, extending the previously known eIF4E allelic series in cultivated barley to 47. Thus the eIF4E alleles now represent the largest allelic series of a gene conferring resistance to pathogens in plants. • We have also achieved the ultimate goal of this project - ten eIF4E alleles have been identified in barley conferring high level of resistance to all known European BaMMV and BaYMV isolates. This broad spectrum resistance will enrich the genetic basis for bymovirus resistance breeding in barley globally. |
| Exploitation Route | New eIF4E alleles conferring high level of resistance to all known European bymoviruses BaMMV and BaYMV isolates could be deployed in barley breeding programs especially in regions with long history of the viral yellow mosaic disease. This project was in consultation and informal collaboration with KWS UK Ltd (formerly CPB Twyford) based near Cambridge. All the key results, sequences of novel barley eIF4E alleles providing resistance to bymoviruses, and seeds of barley accessions carrying novel barley eIF4E alleles providing resistance to bymoviruses, have been provided by us to the UK barley breeders, Dr. Peter Werner and Mr. David Harrap (KWS UK Ltd.), ahead of results publication or public data release. In return, KWS UK Ltd. helped us to create 17 test crosses between exotic barley genotypes carrying novel barley eIF4E alleles providing broad-spectrum resistance to bymoviruses and the susceptible UK barley cv. Saffron for our future genetic studies. Also, aliquots of these materials will be stored and then deployed for bymovirus resistance breeding by KWS UK Ltd. when needed, e.g. if the new aggressive rym4- and / or rym5-resistance breaking bymovirus strain emerges. KWS is represented in about 70 countries with more than 40 subsidiaries and affiliated companies worldwide. KWS has cereals breeding stations in several countries with a long history of soil-borne mosaic disease including Germany, France and UK. New emerging strains of bymoviruses breaking resistance in elite barley cultivars are already evident in Germany and France. If these new strains or any other strains that may evolve become a recognised problem the genetic resources generated in this project will be rapidly deployed and introduced to regional barley breeding programmes in Europe. Barley yellow mosaic virus is considered a serious problem of winter barley in China. Hence, we also provided a number of barley genotypes identified to carry novel barley eIF4E alleles providing broad spectrum bymovirus resistance to Dr. Jinghuan Zhu and Prof. Jianming Yang, barley breeders at the National Barley Improvement Centre, Zhejiang Academy of Agricultural Sciences in Hangzhou, China, for introducing these superior resistance alleles into Chinese barley breeding programmes. |
| Sectors | Agriculture, Food and Drink |
| Description | This project was in consultation and informal collaboration with KWS UK Ltd (formerly CPB Twyford) based near Cambridge. All the key results, sequences of novel barley eIF4E alleles providing resistance to bymoviruses, and seeds of barley accessions carrying novel barley eIF4E alleles providing resistance to bymoviruses, have been provided by us to the UK barley breeders, Dr. Peter Werner and Mr. David Harrap (KWS UK Ltd.), ahead of results publication or public data release. In return, KWS UK Ltd. helped us to create 17 test crosses between exotic barley genotypes carrying novel barley eIF4E alleles providing broad-spectrum resistance to bymoviruses and the susceptible UK barley cv. Saffron for our future genetic studies. Also, aliquots of these F1 materials will be stored and then deployed for bymovirus resistance breeding by KWS UK Ltd. when needed, e.g. if the new aggressive rym4- and / or rym5-resistance breaking bymovirus strain emerges. KWS is represented in about 70 countries with more than 40 subsidiaries and affiliated companies worldwide. KWS has cereals breeding stations in several countries with a long history of soil-borne mosaic disease including Germany, France and UK. New emerging strains of bymoviruses breaking resistance in elite barley cultivars are already evident in Germany and France. If these new strains or any other strains that may evolve become a recognised problem the genetic resources generated in this project will be rapidly deployed and introduced to regional barley breeding programmes in Europe. Barley yellow mosaic virus is considered a serious problem of winter barley in China. Hence, we also provided a number of barley genotypes identified to carry novel barley eIF4E alleles providing broad-spectrum bymovirus resistance to Dr. Jinghuan Zhu and Prof. Jianming Yang, barley breeders at the National Barley Improvement Centre, Zhejiang Academy of Agricultural Sciences in Hangzhou, China, for introducing these superior resistance alleles into Chinese barley breeding programmes. During the lifetime of this project Yi-dan Li, a postdoctoral scientist from the Biotechnology Research Centre, Jilin Academy of Agricultural Sciences in China, has been trained for 3 months on the use of HRM for identification of variant alleles in crop plants. On two dates in October 2009 K. Kanyuka was involved in the traditional Rothamsted Fungus Foray activity with local primary schools. He helped forty year-1 school pupils (5-6 year olds) to explore what is happening in the fungi world. Each activity lasted approximately 1.5 hours and involved a foray around Rothamsted Manor House, Gardens and Woods. The fungus foray is an excellent way of introducing the key scientific skills of observation, obtaining and presenting evidence. K. Kanyuka prepared a poster, a live bymovirus-infected plants display, and a live display of various insects, known vectors of plant viruses and their natural enemy, for the Rothamsted Research Open Weekend Preview Day held on Friday 21 May 2010. This event was attended by guests from BBSRC, Defra, NFU, other academic institutions and the local community. In addition, Rothamsted Research presented its science to around 180 students from nine local schools. K. Kanyuka also prepared a poster and a live bymovirus-infected plants display linked to the display plots of resistant barley Retriever and susceptible barley Saffron for the Cereals 2010 show (http://www.cerealsevent.co.uk/) which took place near Royston, Cambridgeshire on 9-10 June. These exhibits aimed to help the farmers, their agricultural advisers and other sectors of the Agricultural Industry and the Agricultural media to know the soil-borne barley mosaic virus symptoms, which some of the farmers confuse for something else. For example, some may think that symptoms are caused by the nitrogen or trace element deficiency. This misdiagnosis may lead to farmers spending money on fixing problems they do not have and not dealing with barley mosaic viruses, which is a persistent disease. Another key message of the exhibit was that the best approach to tackle barley mosaic viruses is to grow resistant varieties, which are available in the UK. Farmers were also advised to be careful when taking machinery into and from field which contain mosaic virus contaminated soil and ensuring that de-contamination is effective, thereby preventing this persistent disease problem from spreading to additional fields. In addition, in 2007 Kim Hammond-Kosack and K. Kanyuka prepared and published a review article on resistance genes in plants in the on-line ENCYCLOPEDIA OF LIFE SCIENCES (ELS). Among other key topics this article specifically reviewed the current scientific knowledge on recessive resistances to viruses in cereal and non-cereal species. ELS is available in most of the larger public libraries and used often by undergraduate and graduate biology students as well the general public. |
| First Year Of Impact | 2010 |
| Sector | Agriculture, Food and Drink |
| Impact Types | Economic |
| Title | A new method for rapid and cost-effective identification of variant alleles in barley |
| Description | We developed a new method for rapid and cost-effective identification of variant alleles in barley. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which are predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which had previously only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allows significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The developed method involves an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover coding sequence of the gene of interest. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2009 |
| Provided To Others? | Yes |
| Impact | This strategy further reduces the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in barley eIF4E approached 100%. These data suggest that this new method could also potentially be applied to the discovery of superior alleles of genes (especially those containing large introns or a large number of introns) controlling other important traits in barley as well in other model and crop plant species. This technique will be particularly useful to laboratories where the funding and equipment set aside for the genotyping of germplasm is relatively modest. It should be readily applicable to any diploid plant species. |
| Title | The sequences of 45 novel eIF4E alleles and 4 novel MCT-1 alleles of barley |
| Description | The sequences of 45 novel eIF4E alleles and 4 novel MCT-1 alleles of barley have been deposited to the EMBL Nucleotide Sequence database (accession numbers FM244870 - FM244906, FN646082 - FN646088, FN646408 - FN646411, and FN600114). |
| Type Of Material | Model of mechanisms or symptoms - in vitro |
| Year Produced | 2012 |
| Provided To Others? | Yes |
| Impact | The novel barley eIF4E alleles provide broad-spectrum resistance to the two aconomically important viruses Barley mild mosaic virus and Barley yeallow mosaic virus. Introduction of these alleles into commercial barley breeding will provide effective and possibly durable resistance to these viruses. |
| Title | A new method for rapid and cost-effective identification of variant alleles in barley |
| Description | We developed a new method for rapid and cost-effective identification of variant alleles in barley. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which are predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which had previously only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allows significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The developed method involves an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover coding sequence of the gene of interest. This strategy further reduces the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in barley eIF4E approached 100%. These data suggest that this new method could also potentially be applied to the discovery of superior alleles of genes (especially those containing large introns or a large number of introns) controlling other important traits in barley as well in other model and crop plant species. This technique will be particularly useful to laboratories where the funding and equipment set aside for the genotyping of germplasm is relatively modest. It should be readily applicable to any diploid plant species. |
| Type Of Technology | New/Improved Technique/Technology |
| Year Produced | 2009 |
| Impact | No actual Impacts realised to date |
| Description | A review article on resistance genes in plants in the on-line ENCYCLOPEDIA OF LIFE SCIENCES (ELS) |
| Form Of Engagement Activity | A magazine, newsletter or online publication |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | We prepared and published a review article on resistance genes in plants in the on-line ENCYCLOPEDIA OF LIFE SCIENCES (ELS). Among other key topics this article specifically reviewed the current scientific knowledge on recessive resistances to viruses in cereal and non-cereal species. ELS is available in most of the larger public libraries and used often by undergraduate and graduate biology students as well the general public. no actual impacts realised to date |
| Year(s) Of Engagement Activity | 2007 |
| Description | Cereals 2010 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Type Of Presentation | Poster Presentation |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | We prepared a poster and a live bymovirus-infected plants display linked to the display plots of resistant barley Retriever and susceptible barley Saffron for the Cereals 2010 show (http://www.cerealsevent.co.uk/) which took place near Royston, Cambridgeshire on 9-10 June 2010. These exhibits aimed to help the farmers, their agricultural advisers and other sectors of the Agricultural Industry and the Agricultural media to know the soil-borne barley mosaic virus symptoms, which some of the farmers confuse for something else. For example, some may think that symptoms are caused by the nitrogen or trace element deficiency. This misdiagnosis may lead to farmers spending money on fixing problems they do not have and not dealing with barley mosaic viruses, which is a persistent disease. Another key message of the exhibit was that the best approach to tackle barley mosaic viruses is to grow resistant varieties, which are available in the UK. Farmers were also advised to be careful when taking machinery into and from field which contain mosaic virus contaminated soil and ensuring that de-contamination is effective, thereby preventing this persistent disease problem from spreading to additional fields. no actual impacts realised to date |
| Year(s) Of Engagement Activity | 2010 |
| URL | http://www.cerealsevent.co.uk/ |
| Description | Rothamsted Research Open Weekend Preview Day |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Schools |
| Results and Impact | We prepared a poster, a live bymovirus-infected plants display, and a live display of various insects, known vectors of plant viruses and their natural enemy, for the Rothamsted Research Open Weekend Preview Day held on Friday 21 May 2010. This event was attended by guests from BBSRC, Defra, NFU, other academic institutions and the local community. In addition, Rothamsted Research presented its science to around 180 students from nine local schools. no actual impacts realised to date |
| Year(s) Of Engagement Activity | 2010 |
| Description | Website publicising our barley diversity collection |
| Form Of Engagement Activity | A magazine, newsletter or online publication |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Participants in your research or patient groups |
| Results and Impact | We assembled a Rothamsted Barley Diversity (RBD) collection comprising 1090 landraces and non-current cultivars (i.e. those acquired or released prior to the 1940s), differing in the growth habits and morphological characteristics, originating from 84 countries spread over six continents. The high number of novel variant eIF4E alleles identified in the current study suggests the assembled barley collection captures a high proportion of the genetic diversity stored in large seed banks. Therefore, our barley diversity collection represents a valuable resource for the identification of useful novel alleles of other agronomically important genes. We are willing to provide access to this population and related resources (i.e. total RNA extracted from all 1090 accessions, and small amount of 1st strand cDNA prepared using oligo(dT) priming for most accessions in the diversity collection) for any interested researcher in the UK or overseas. Availability of this collection and associated resources has been advertised to the research community through the MONOGRAM website (http://www.monogram.ac.uk/) as well as the project's and PI's no actual impacts realised to date |
| Year(s) Of Engagement Activity | 2010 |
| URL | http://www.monogram.ac.uk/ |