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

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

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

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

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

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