Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties

The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance\u2013a hypersensitive response in leaves challenged with P. viticola\u2013was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12+ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12+ haplotype is shared by 15 varieties, the most ancestral of which are the century-old \u2018Zarja severa\u2019 and \u2018Michurinets\u2019. Before this knowledge, the chromosome segment around Rpv12+ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12+ has an additive effect with Rpv3+ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3+ plant

Gene and protein expression levels after transfection with <i>GLI1</i> (GLI1) and additional stimulation with Shh protein (GLI1+SHH).

<p><i>ERα</i> gene expression increases in MCF-7 cells only after additional Shh stimulation (A) while <i>ERα</i> gene expression does not change in SkBr-3 cells (B). Gli1, Ptch1 and ERα protein levels in MCF-7 cells after <i>GLI1</i> transfection and additional Shh stimulation (C). Protein bands were quantified and normalized relative to actin and non-treated conditions and the relative values are denoted below each band. Relative gene expression of <i>PTCH1</i> (D, E) and <i>ERα</i> (F,G) after silencing of <i>PTCH1</i> gene in MCF-7 and SkBr-3 cell line. Efficient silencing (<30% of residual expression) was achieved 24 h post-transfection in MCF-7 cell line, and 48 h post-transfection in SkBr-3 cell line.</p

Differentially expressed proteins in MCF-7 cells treated with cyclopamine and tamoxifen compared with non-treated control cells.

<p>General Functions are obtained from the UniProt and NCBI Gene databases. Protein numbers correspond to the numbers marked on the 2-D gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114510#pone.0114510.s001" target="_blank">Figure S1</a>). Numbers in the table correspond to spot numbers denoted on the 2-D gel images; missing numbers in the table are unidentified proteins or proteins with score less than 39.</p><p>Differentially expressed proteins in MCF-7 cells treated with cyclopamine and tamoxifen compared with non-treated control cells.</p

Effects of cyclopamine (A) and tamoxifen (B) on Hh-Gli pathway gene expression in MCF-7 and SkBr-3 cells.

<p>The Hh-Gli pathway is upregulated after short-term combined treatment in MCF-7, but the effect is negated after longer treatment. On the Western blot image, band quantification relative to actin and non-treated cells is denoted below the bands. (C). The effect of combined treatment on SkBr-3 cell line is weak (D). Gene expression levels are shown on graph as relative fold change relative to non-treated conditions with reference value 1 pointed out with emboldened bar. Only combined cyclopamine and tamoxifen treatment induces migration in MCF-7 cells. Representative images of the wound healing assay at 0 and 26 h (after processing with TScratch software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114510#pone.0114510-Gebck1" target="_blank">[23]</a>) are shown for non-treated conditions (NT; N = 16), cyclopamine treatment (CYC; N = 16), tamoxifen treatment (TAM; N = 14) and combined treatment with cyclopamine and tamoxifen (C+T; N = 12) (E). Quantitative analysis of the percentage of open wound areas is shown on the graph, (*) P<0.05 (F). Transwell migration assay confirmed increased migration capacity of cells after combined cyclopamine and tamoxifen treatment. Representative images of migrated cells after 48 h are shown for non-treated conditions (NT; N = 15), cyclopamine treatment (CYC; N = 15), tamoxifen treatment (TAM; N = 15) and combined treatment (C+T; N = 15) (G). Quantitative analysis of the relative number of migrated cells (analyzed relative to non-treated cells) is shown on graph, (*) P<0.0001 (H).</p

Effect of cyclopamine and tamoxifen combination on MCF-7 cell proliferation.

<p>When tamoxifen is in higher concentrations, and cyclopamine in lower concentrations, MCF-7 cell viability is decreased. However, when cyclopamine concentration is increased (with tamoxifen concentration remaining constant) cell viability increases (A). Similar effect can be seen vice-versa, when cyclopamine concentration is constant and tamoxifen concentration is increased (B) as measured by MTT assay after 48 h.</p

Cell viability after tamoxifen (A,B), cyclopamine (C,D) or combined treatment (E,F) in MCF-7 and SkBr-3 cell lines.

<p>Tamoxifen and cyclopamine each inhibit proliferation of MCF-7 cells in a dose dependent manner (A,C). When administered simultaneously, they cause a short term survival effect in MCF-7 cells (C+T 48 h) – pointed out with arrow, whereas long term simultaneous treatment induces strong cell death in these cells (C+T 96 h). Combination treatment of cyclopamine for 48 h followed by tamoxifen for 48 h (C 48 h →T 48 h) or vice versa (T 48 h →C 48 h) showed an effect similar to tamoxifen alone (E). Tamoxifen and cyclopamine show only a mild inhibitory effect on SkBr-3 cell proliferation at longest exposures (B,D) while combined treatment has no pronounced effect (F).</p

Immunofluorescent staining of MCF-7 cell line in non-treated cells (NT) and treated with Shh protein detected by confocal microscopy.

<p>ERα is stained green (column 1), Shh is stained red (column 2), nuclei are stained blue with DAPI (column 3), and the last column shows the overlay of signals. Yellow staining shows areas of green and red signal co-localization (A). Shh-treated cells show significantly decreased nuclear staining and increased co-localization of ERα and Shh compared to non-treated cells, as determined by ImageJ software, (*) P<0.05. (B). Shh protein co-immunoprecipitates with ERα protein in MCF-7 cells, both in non-treated conditions and after treatment with exogenous Shh protein for 48 h; NT = non-treated, neg.ctrl. = negative control. Western blot of input proteins is provided as control for presence of the proteins in cell lysates (C).</p

Selective sweep at the Rpv3 locus during grapevine breeding for downy mildew resistance

The Rpv3 locus is a major determinant of downy mildew resistance in grapevine (Vitis spp.). A selective sweep at this locus was revealed by the DNA genotyping of 580 grapevines, which include a highly diverse set of 265 European varieties that predated the spread of North American mildews, 82 accessions of wild species, and 233 registered breeding lines with North American ancestry produced in the past 150 years. Artificial hybridisation and subsequent phenotypic selection favoured a few Rpv3 haplotypes that were introgressed from wild vines and retained in released varieties. Seven conserved haplotypes in five descent groups of resistant varieties were traced back to their founders: (1) 'Munson', a cross between two of Hermann Jaeger's selections of V. rupestris and V. lincecumii made in the early 1880s in Missouri, (2) V. rupestris 'Ganzin', first utilised for breeding in 1879 by Victor Ganzin in France, (3) 'Noah', selected in 1869 from intermingled accessions of V. riparia and V. labrusca by Otto Wasserzieher in Illinois, (4) 'Bayard', a V. rupestris x V. labrusca offspring generated in 1882 by George Couderc in France, and (5) a wild form closely related to V. rupestris accessions in the Midwestern United States and introgressed into 'Seibel 4614' in the 1880s by Albert Seibel in France. Persistence of these Rpv3 haplotypes across many of the varieties generated by human intervention indicates that a handful of vines with prominent resistance have laid the foundation for modern grape breeding. A rampant hot spot of NB-LRR genes at the Rpv3 locus has provided a distinctive advantage for the adaptation of native North American grapevines to withstand downy mildew. The coexistence of multiple resistance alleles or paralogues in the same chromosomal region but in different haplotypes counteracts efforts to pyramidise them in a diploid individual via conventional breeding

Effect of stimulation with Shh protein on pathway activity in MCF-7 (A,C) and SkBr-3 cells (B).

<p>Gene expression levels are shown on graph as relative fold change relative to non-treated conditions with reference value 1 pointed out with emboldened bar. Relative gene expression of <i>ERα</i> after treatment with Shh protein (D,E). Non-treated cells (NT) have a relative value 1. ERα protein expression in MCF-7 cells increases after treatment with Shh protein for 48 h (F) Protein bands were quantified and normalized relative to actin and non-treated conditions and the relative values are denoted below each band.</p