An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses

BACKGROUND: The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function. METHODOLOGY/PRINCIPAL FINDINGS: To investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus. CONCLUSIONS/SIGNIFICANCE: Mutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study

Le TILLING : Un outil pour générer de nouveaux allèles de résistance au virus

National audienc

TILLING in tomato: a reverse genetic approach for functional analysis of eIF4E-mediated resistance and extension of the set of resistance alleles for breeding

National audienc

Primers used to amplify <i>Sl-eIF4E1</i>, <i>Sl-eIF4E2</i>, <i>Sl-eIF(iso)4E</i>, <i>Sl-eIF4G</i> and <i>Sl-eIF(iso)4G</i> genes and the size of the tilled amplicons.

<p>*F and R indicate forward and reverse primers, respectively.</p

Potyvirus resistance assays of the <i>Sl</i>-eIF4E1 G1485A splicing mutant.

<p><i>Sl</i>-eIF4E1 G1485A mutant and the corresponding Hm-WT were inoculated with PVY-LYE90, PVY-LYE84, TEV-HAT or PepMoV-Texas. (<b>A</b>) At 15 dpi, plants were assayed for potyviral coat protein accumulation by DAS-ELISA in non-inoculated leaves. (<b>B</b>) PVY-LYE90 and PepMoV RNA accumulation was assessed by RT-PCR in inoculated (il) and systemic leaves (sl). Mock indicates non inoculated M82 plants.</p

Representation of induced mutations in <i>Sl-eIF4E1</i>, <i>Sl-eIF4E2</i>, <i>Sl-eIF(iso)4E</i>, <i>Sl-eIF4G</i> and <i>Sl-eIF(iso)4G</i>.

<p>Black boxes represent the exons. Lanes linking exons indicate introns. Dashed lines indicate the genomic regions screened for mutations. Triangles pointing up indicate mutations in coding regions, whereas those pointing down indicate mutations in noncoding regions. Red, black and grey triangles represent alterations causing truncations, missense and silent mutations, respectively. Only exons 7 to 9 are shown for <i>Sl-eIF4G</i>.</p

Mutations discovered in <i>Sl-eIF4E1</i>, <i>Sl-eIF4E2</i>, <i>Sl-eIF(iso)4E</i>, <i>Sl-eIF4G</i>, and <i>Sl-eIF(iso)4G</i>.

<p>Only exonic mutations are shown. The position of the mutations are indicated relative to the first base of the GenBank sequences.</p

Tilled genes and mutation density in the M82 mutant population.

<p>M82 mutant population was screened for mutations in the listed genes. The total size of the screened amplicons, for each gene, the number of mutants identified and the mutation frequency for each amplicon are indicated. The average mutation frequency was estimated to one mutation per 574 kb and is calculated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011313#pone.0011313-Greene1" target="_blank">[21]</a>, except that the sizes of all the amplicons were summed and divided by the total number of identified mutants.</p

Protein analysis of <i>Sl</i>-eIF4E1 G1485A splicing mutant.

<p>(<b>A</b>) Western blot analysis of total soluble leaf protein of <i>N. tabacum</i>, <i>S. lycopersicum</i> Hm-WT and <i>Sl</i>-eIF4E1 G1485A mutant probed with an antibody raised against <i>N. tabacum</i> Nt-eIF4E1. (<b>B</b>) Soluble protein extracts of the Hm-WT and G1485A mutant were purified by affinity chromatography on m7G-sepharose column. Total protein extract (lane 1), the flow through (lane 2), the wash (lane 3) and the bound eIF4E proteins eluted with an m7GDP-cap analogue were analysed by Western blot, using Nt-eIF4E antibody.</p

Comparison of expected and observed types of mutations in tilled exonic regions.

<p>The percentage of expected mutations was calculated based on the CODDLE analysis of the tilled exonic regions.</p