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Exploiting eIF4E-based and associated broad-spectrum resistance to potyviruses in dicots and monocots

Background Information

• Barley mild mosaic virus (BaMMV) and Barley yellow mosaic virus (BaYMV) (genus Bymovirus; family Potyviridae) cause an economically important yellow mosaic disease in barley (Hordeum vulgare) (Figure 1). BaMMV and BaYMV infected fields have been reported from the UK, several European countries and from East Asian countries.




Figure 1. Symptoms of yellow mosaic disease in barley caused by bymoviruses.


• Recessive allelic rym4 and rym5 genes conferring resistance to BaMMV and BaYMV are used in European barley breeding programmes. Each of these genes confers resistance only to a subset of bymovirus isolates.
• We recently demonstrated that rym4 and rym5, and another allelic resistance gene rym6 which is not yet used in European barley breeding correspond to mutant alleles of the eukaryotic translation initiation factor 4E (eIF4E) gene (Kanyuka et al., 2005). The plant eIF4E protein or its isoform, eIF(iso)4E, plays an important role in successful multiplication of many different potyviruses via an interaction with VPg, the viral protein covalently attached to the 5'-end of viral RNA. An impaired interaction between eIF4E and VPg is thought to result in resistance to potyviruses.

Project Objectives

• The aim of our study is to identify novel eIF4E alleles in barley, potentially providing broad-spectrum resistance to BaMMV and BaYMV.
• Study of the natural allelic variation of eIF4E in the barley germplasm using an allele mining approach.
• Discovery of chemically induced novel alleles of eIF4E and eIF(iso)4E in barley using a TILLING approach.
• In silico modelling of the 3D structure of novel eIF4E protein variants to predict superior resistance alleles for the future deployment in breeding programmes.
• Development of molecular markers for detection of superior resistance alleles for the use in breeding programmes.

Mining for Novel Natural elF4E alleles

• We assembled a selection of 1100 barley accessions (primarily landraces and old cultivars collected before the 1940s) from all main barley-growing regions of the world to explore the natural variation in eIF4E.
• A cDNA-based high-resolution melting (HRM) was adapted as a high-throughput and low-cost assay to screen for mutations in the coding sequence of the eIF4E gene (Figure 2). The eIF4E sequence of barley accessions predicted to carry mutations was determined by direct Sanger sequencing of cDNA amplicons.




Figure 2. Detection of mutant alleles of eIF4E using high-resolution melting (LightScanner^™ system; Idaho Technology Inc.) assay.


• Up until now we identified >30 different alleles of eIF4E gene in our barley collection. The vast majority of the mutations detected are non-synonymous point mutations resulting in amino acid changes in the encoded protein. Most of the novel alleles have one, two or three amino acid exchanges (Figure 3). Some of the novel alleles contain a small deletion or insertions of 9 or 12 nucleotides in length.




Figure 3. Haplotype network representing phylogenetic relationships of novel eIF4E alleles (Phylogenetic Network software v. 4.5.1.0; Fluxus Technology Ltd.).


• The diversity of eIF4E alleles in Europe and Asia was higher than in Australia, Africa, North and South America. Interestingly, the highest diversity of eIF4E alleles was found in East Asia (i.e. Japan, Korea, and East China). This is a geographic region with a reported history of yellow mosaic disease.

Identification of Alleles Conferring Broad-spectrum Resistance

• Barley accessions with novel eIF4E alleles are being tested for resistance to five European BaYMV and BaMMV isolates by mechanical inoculation of leaves.
• Most of the newly identified eIF4E alleles were found to direct resistance to at least one or more of these European virus isolates. Several of the tested novel eIF4E alleles confer broad-spectrum resistance to all five European bymovirus isolates including those known to overcome rym4- and rym5-mediated resistance.
• Protein 3D modelling of eIF4E indicates that the majority of the amino acid exchanges identified are located in a specific region close to the cap-binding pocket suggesting a potential role of this region in binding of viral protein VPg (Figure 4).




Figure 4. Locations of amino acid exchanges in a 3D-protein model of barley eIF4E (A: front view of the cap binding pocket; B: side view). The mutations are highlighted in pink (INSIGHT II software; Accelrys Inc.).


• This study demonstrates that the natural barley gene pool is an excellent source of novel resistance specificities.
• A selection of accessions carrying superior eIF4E alleles has been handed to breeders for improving resistance to bymoviruses of elite breeding germplasm.

Identification of Novel Chemically-Induced eIF4E and eIF(iso)4E Alleles

• Using TILLING we identified several additional alleles of eIF4E not present in the natural barley germplasm including one allele containing a premature stop codon in the coding sequence. Further examinations of these mutants are currently under way.

References

Journal Article

2005

• Kanyuka K, Druka A, Caldwell DG, Tymon A, McCallum N, Waugh R, Adams MJ. (2005) "Evidence that the recessive bymovirus resistance locus rym4 in barley corresponds to the eukaryotic translation initiation factor 4E gene.", Molecular Plant Pathology , 6: 449–458.

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