Figure 1.
Phylogenetic tree of MKK family in A. thaliana and five Solanaceae species.
The species are A. thaliana (At), potato (Sotub), tomato (Solyc), N. benthamiana (Nb), N. attenuata (Na) and N. tabacum (Nt). Genes are grouped into 4 groups: A (green), B (red), C (blue) and D (orange) [4]. Potato genes are marked with dots. The numbers on the nodes are percentages from a bootstrap analysis of 1000 replicates. The scale bar indicates the branch length that corresponds to 0.06 substitutions per site.
Figure 2.
Gene expression pattern of MKK family in the HR response against PVY.
Cultivar Rywal (HR response, conferred by Ny-1 gene) and NahG-Rywal (impaired accumulation of SA) were analysed for whole transcriptome response 1, 3 and 6 days after PVYN-Wi infection [24]. A. thaliana and S. tuberosum PGSC orthologues were assigned to each probe. Log2 fold changes of PVY in infected vs. mock-inoculated plants are indicated for each time point (1, 3 and 6 dpi). Statistically significant differences (FDR corrected p<0.05) are in bold. Up-regulated values are in blue and down-regulated values are in yellow.
Figure 3.
Phylogenetic tree of potato MKK6 and its orthologues in different plant species, divided into classes.
The species of origin for each MKK is marked by species acronym before the protein name: Cr, Capsella rubella; Aly, Arabidopsis lyrata; At, Arabidopsis thaliana; Br, Brassica rapa; Bn, Brassica napus; Gh, Gossypium hirsutum; Pp, Prunus persica; Mt, Medicago trunculata; Gm, Glycine max; Pt, Populus trichocarpa; Rc, Ricinus communis; Vv, Vitis vinifera; Oo, Origanum onites; St, Solanum tuberosum; Le, Solanum lycopersicum (Lycopersicum esculetum); Nt, Nicotiana tabacum; Na, Nicotiana attenuata; Nb, Nicotiana benthamiana; Ma, Musa acuminata; Hv, Hordeum vulgare, Ata, Aegilops tauschii; Tu, Triticum urartu; Bd, Brachypodium distachyon; Os, Oryza sativa, Og, Oryza glaberrima; Ob, Oryza brachyantha; Si, Setaria italica; Sb, Sorghum bicolor; Zm, Zea mays, Ps, Picea sitchensis; Sm, Selaginella moellendorffii. Potato MKK6 is marked with red dot. The numbers on the nodes are percentages from a bootstrap analysis of 1000 replicates. The scale bar indicates the branch length that corresponds to 0.06 substitutions per site.
Figure 4.
StMKK6 gene expression in development and stress [40].
Data are obtained from the potato eFP browser [39]. A. Tissue and developmental gene expression pattern of StMKK6 in double monoploid S. tuberosum Group Phureja (left) and in heterozygous diploid S. tuberosum Group Tuberosum (right). B. Changes in StMKK6 gene expression under biotic stress conditions (treatments of leaves with P. infestans, SA analogues acibenzolar-S-methyl and fungal elicitor DL-β-amino-n-butyric acid and wounding) and abiotic stress conditions (treatments of whole plants with salt, heat and hormones cytokinins, 6 benzylaminopurine; gibberellins, GA3; abscisic acid; and auxin, IAA) and control treatments.
Figure 5.
Predicted regulatory domains of StMKK6 native promoter from S. tuberosum cv. Rywal, 790-bp-long.
The crucial domains that regulate stress responses and development are CCAAT-box (MYBHv1 binding site; yellow), GCN4 motif (endosperm expression; pink), HSE (heat stress; brown), P-box (gibberellins; blue), Skn-1 motif (endosperm expression; purple), TCA element (salicylic acid; green), WUN motif (wounding; red) and circadian motif (control of circadian clock; grey). The arrow indicates the predicted beginning of the 5’ UTR region and the red shaded ATG indicates the start of StMKK6 coding region. The complete list of StMKK6 promoter domains from cv. Santé and cv. Rywal with their position, strand and sequence are specified in Tables S3 and S4 in File S1.
Figure 6.
Expression of StMKK6 in control and SA-treated Rywal and NahG-Rywal potato plants.
Expression of the endogenous StMKK6 in NahG-Rywal is at least two-fold lower than in Rywal. After the SA-treatment the expression of StMKK6 in NahG-Rywal increased for at least three times, comparing to the non-treated control. Difference in the endogenous StMKK6 expression between Rywal and NahG-Rywal as well as difference in the StMKK6 expression between SA-treated and non-treated NahG-Rywal plants are statistically significant (p<0.05).
Figure 7.
Epidermal cells of N. benthamiana expressing translational fusion of StMKK6 with YFP under native promoter.
Leaves were agroinfiltrated when the virus has spread uniformly through the inoculated leaves (8 days after inoculation) and observed after 72 h in two independent experiments. Examples from two plants (left and right panels) are shown. Control of transformation (fluorescent marker without StMKK6 fusion) is in the Figure S1A. A. Localisation of StMKK6 in mock-inoculated leaves. No fluorescence was observed. B. Localisation of StMKK6 in PVY-inoculated leaves, where the protein accumulates predominantly in nucleus. Additional images of StMKK6 localisation under native promoter are in Figures S1D and S1E.
Figure 8.
Yeast two-hybrid assays screening StMKK6 interaction partners.
StMKK6 protein was fused with Gal DNA-binding domain as bait and StMAPK4_1, StMAPK4_2, StMAPK6 and StMAPK13 were used as prey fused with Gal DNA-activation domain. Interaction pairs P53/SV40 large T-antigen and Lam/SV40 large T-antigen were used as positive and negative controls respectively. StMKK6/empty vector pair was used as a control to discard auto-activation of bait protein. Serial dilutions of interaction pairs were plated on DDO media for co-transformation selection. Interactions of bait and prey proteins were examined by assessing growth on several selective media with different levels of restrictiveness i.e. SD/-Leu/-Trp/-His (TDO), SD/-Leu/-Trp/x-a-Gal/Aba (DDO/X/A) and QDO/X/A. Only co-transformed colonies growing on the most restrictive media (QDO/X/A) were considered as positive interaction transformants.
Figure 9.
Gene expression pattern of confirmed StMKK6 targets in the HR response against PVY.
The experimental setup is as in Figure 2. To each probe an A. thaliana orthologue, potato orthologue and a PGSC gene ID were assigned. Log2 of gene expression differences between PVY-infected and mock-inoculated plants are indicated for each time point (1, 3 and 6 dpi). Statistically significant differences (FDR corrected p-value <0.05) are given in bold. The star (*) for StMAPK6 PGSC gene ID indicates that the gene was not predicted in PGSC gene model. Up-regulated values are in blue and down-regulated values are in yellow.