Verification of 3D structures of rice MAPKs and MAPKKs.

<p>DOPE–Discrete Optimized Protein Energy, PDF–Probability Density Function.</p

ZDOCK and RDOCK score of OsMKK5 against each of eleven rice MAPKs.

<p>E_R – Energy RDOCK score. Lower values of ZRANK score and E_RDOCK and higher ZDOCK score indicate top/better docking of the complex. Clash ‘0′ indicates no stearic clash between the proteins after refinement by RDOCK protocol.</p

ZDOCK and RDOCK score of OsMKK4 against each of eleven rice MAPKs.

<p>E_R – Energy RDOCK score. Lower values of ZRANK score and E_RDOCK and higher ZDOCK score indicate top/better docking of the complex. Clash ‘0′ indicates no stearic clash between the proteins after refinement by RDOCK protocol.</p

Ramachandran plot analysis of theoretical 3D structure of rice MAPKKs and MAPKs.

<p>The 3D Structures of eleven rice MAP kinases (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK17-1, OsMPK20-2, OsMPK20-3, OsMPK20-5 and OsMPK21-2) and five MAP kinase kinases (OsMKK3, OsMKK4, OsMKK5, OsMKK6, OsMKK10-2) were validated using Ramachandran plot. The green dots/yellow dots show the amino acids that are in the most favoured regions and additionally allowed region while red dots show the amino acids that are in generously allowed region or disallowed regions. The regions covered by sky blue line show most favoured regions, while the regions covered by pink line show additionally allowed regions. Other regions of the plot show the generously allowed or disallowed region.</p

ZDOCK and RDOCK score of OsMKK3 against each of eleven rice MAPKs.

<p>E_R – Energy RDOCK score. Lower values of ZRANK score and E_RDOCK and higher ZDOCK score indicate top/better docking of the complex. Clash ‘0′ indicates no stearic clash between the proteins after refinement by RDOCK protocol.</p

Direct comparison of yeast two-hybrid assay and computational dockings with respect to rice MAPKK-MAPK interaction.

<p>MAPKs in bold font suggests that their interaction with respective MAPKK have been reported using both <i>in-silico</i> prediction and Y2H screen.</p>*<p>MAPKs were not included in <i>in-silico</i> docking studies.</p

Protein-protein interaction network among rice MAPKKs and MAPKs based on Y2H analyses and <i>in-silico</i> predictions.

<p>Solid lines from upper panel OsMAPKKs to middle panel OsMAPKs indicate findings from yeast two-hybrid screen while dashed line from lower panel OsMAPKKs to middle panel OsMAPKs indicate findings from <i>in-silico</i> protein-protein dockings analyses. Lines originating from specific OsMAPKK are represented in same colour.</p

Yeast two-hybrid assay involving rice MAPKKs and MAPKs.

<p>OsMKK3, OsMKK4, OsMKK6 and OsMKK10-2 were fused with GAL4 DNA-binding domain and were used as a bait against each of the fifteen rice MAPKs (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK16-2, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-2, OsMPK20-3, OsMPK20-4, OsMPK20-5 and OsMPK21-2) fused with GAL4 activation domain as preys. OsMKK1 fused with GAL4 activation domain as prey and fifteen MAPKs fused to DNA-binding domain baits were used to study protein interactions. Plasmids for bait and prey were co-transformed in AH109 yeast strain in different combinations as mentioned and selected on nutrient medium lacking Leu and Trp (upper panels). Interaction of bait and prey combinations were checked by assessing growth of co-transformed colonies streaked on selective medium lacking Ade, His, Leu and Trp (lower panels). p53 and SV40 large T-antigen are two proteins that are known to interact in yeast were used as a positive control.</p

Theoretical 3D models of rice MAPKKs and MAPKs build by homology modelling

<p>. Structure of eleven rice MAP kinases (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK17-1, OsMPK20-2, OsMPK20-3, OsMPK20-5 and OsMPK21-2) and five MAP kinase kinases (OsMKK3, OsMKK4, OsMKK5, OsMKK6, OsMKK10-2) are shown. The red region represents the alpha helices, sky blue regions the beta sheets, green coloured regions depict the turns whereas the grey colour represents the loops.</p

Top docking poses of OsMKK3 with rice MAPKs.

<p>The best docking positions of OsMKK3 with each of the eleven rice MAPKs (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK17-1, OsMPK20-2, OsMPK20-3, OsMPK20-5 and OsMPK21-2) are shown. OsMKK3 is represented as a wireframe pattern whereas all OsMAPKs as a solid ribbon form.</p