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UV‐B activates a ‘group A’ mitogen activated protein kinase in Oryza sativa
(Springer, 2016) Wankhede, Dhammaprakash Pandhari; Singh, Pallavi; Jaggi, Monika; Rao, Kudupudi Prabhakara; Raina, Susheel Kumar; Sinha, Alok Krishna
Increased level of Ultra violet-B radiation at earth’s surface has several deleterious consequences for plants and ecosystems. Higher UV-B level affects crop plants in several ways and also gives penalty in terms of crop yield. With changing climatic conditions it is crucial to elucidate signal transduction pathway involved in UV-B stress. Here, involvement of mitogen activated protein kinases (MAPKs) in UV-B stress was investigated in rice (Oryza sativa). Transcripts profiling of all 15 rice MAPKs have shown UV-B induced expressions of a few MAPK genes including OsMPK3, OsMPK6, OsMPK4, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-4 and OsMPK20-5. In-gel kinase assay as well as immuno-kinase assay showed activation of single MAPK of size ~45 kDa in response to UV-B treatment. Further UV-B responsive activity of rice MAPKs, OsMPK3, OsMPK4 and OsMPK6 was checked using antibodies for the respective orthologs in Arabidopsis which indicated activation of OsMPK3 in UV-B stress. Additionally, GST: OsMPK3 protein showed UV responsive phosphorylation when incubated with crude protein extract from UV exposed plants in an in-vitro phosphorylation assay. These results indicate activation of OsMPK3 in UV-B stress. Further, possible involvement of UV-B responsive OsWRKY89 downstream of MAPK cascade was studied. It was observed that staurosporin and other specific chemical inhibitors of MAPK could attenuate UV-B induced expression of OsWRKY89. OsMPK3 was also found to interact with OsWRKY89 in yeast two-hybrid assay, although the interaction was weak in nature. The present work gives an account of MAPKs in UV-B stress in rice.
Rice mitogen activated protein kinase kinase and mitogen activated protein kinase interaction network revealed by in-silico docking and yeast two-hybrid approaches
(PLOS, 2013) Wankhede, Dhammaprakash Pandhari; Misra, Mohit; Singh, Pallavi; Sinha, Alok Krishna
Protein-protein interaction is one of the crucial ways to decipher the functions of proteins and to understand their role in complex pathways at cellular level. Such a protein-protein interaction network in many crop plants remains poorly defined owing largely to the involvement of high costs, requirement for state of the art laboratory, time and labour intensive techniques. Here, we employed computational docking using ZDOCK and RDOCK programmes to identify interaction network between members of Oryza sativa mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK). The 3-dimentional (3-D) structures of five MAPKKs and eleven MAPKs were determined by homology modelling and were further used as input for docking studies. With the help of the results obtained from ZDOCK and RDOCK programmes, top six possible interacting MAPK proteins were predicted for each MAPKK. In order to assess the reliability of the computational prediction, yeast two-hybrid (Y2H) analyses were performed using rice MAPKKs and MAPKs. A direct comparison of Y2H assay and computational prediction of protein interaction was made. With the exception of one, all the other MAPKK-MAPK pairs identified by Y2H screens were among the top predictions by computational dockings. Although, not all the predicted interacting partners could show interaction in Y2H, yet, the harmony between the two approaches suggests that the computational predictions in the present work are reliable. Moreover, the present Y2H analyses per se provide interaction network among MAPKKs and MAPKs which would shed more light on MAPK signalling network in rice.
Involvement of mitogen activated protein kinase kinase 6 in UV induced transcripts accumulation of genes in phytoalexin biosynthesis in rice
(Springer, 2013) Wankhede, Dhammaprakash Pandhari; Kumar, Kundan; Singh, Pallavi; Sinha, Alok Krishna
BACKGROUND: Ultra violet radiation leads to accumulation of phytoalexins (PA) in rice (Oryza sativa) which are typically accumulated when the plants are infected with rice blast pathogen Magnaporthe oryzae. Although extensive works have been done in elucidating phytoalexin biosynthesis, UV stress signal transduction leading to accumulations of rice phytoalexin is largely unknown.
Arsenic stress activates MAP kinase in rice roots and leaves
(Elsevier, 2011) Rao, Kudupudi Prabhakara; Vani, Gubbala; Kumar, Kundan; Wankhede, Dhammaprakash Pandhari; Misra, Mohit; Gupta, Meetu; Sinha, Alok Krishna
The toxic metalloid arsenite has become a potential threat to rice growing regions leading to serious contamination in food chain. In the present study effect of different physiological concentration of arsenite that is toxic and triggers the molecular events were evaluated in rice seedlings. Along with severe effect on the growth of rice seedling, production of reactive oxygen species (ROS) and nitric oxide (NO) in arsenite treated rice roots was also observed. Activation of a 42kDa mitogen activated protein kinase (MAPK/MPK) by arsenite was observed in rice leaves and 42 and 44kDa in roots in dose dependent manner. The activated MAPK could be immunoprecipitated with anti-phospho-tyrosine antibody, 4G10. The kinetic of MAPK activation by arsenite was found to be dose dependent. Transcript analysis of MAPK family and immunokinase assay in arsenite treated rice seedling revealed significant level of induction in OsMPK3 transcripts in leaves and OsMPK3, OsMPK4 transcripts in roots. Among MAPK kinase (MKKs) gene family, OsMKK4 transcripts were found to be induced in arsenite treated rice leaves and roots. In-silico homology modeling and docking analysis supported OsMPK3-OsMKK4 interaction. The data indicates that arsenite stress is transduced through MAPK signaling cascade in rice.

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