Kamal Kumar Marmath, Priyanka Giri, Gohartaj, Sugandha Sharma, Dinesh Pandey, Anil Kumar
Molecular Biology and Genetic Engineering, College of Basic Science and Humanities, G.B. Pant University of Agriculture & Technology, Pantnagar 263145, Dist. US Nagar (Uttrakhand), India.

ABSTRACT

In India rapeseed mustard are cultivated on 6.68 mha mainly in northern plains. Under Indian conditions White rust and Alternaria blight are two most important diseases that lead to major yield losses as well as deterioration in quality. Alternaria blight is caused by a fungus Alternaria brassicae which infects the plant leaves and pods and the economic losses rendered, depends upon the severity of the disease incidence. Plant responds toward fungal attack through intricate signal transduction pathways involving MAPKs. The objectives of this study were to identify the role of MAPK-4 in plant defense against Alternaria blight and In -silico prediction of its potential upstream Kinases. In the present study four host plants were taken. RNA was isolated from Brassica spp. namely Brassica juncea cv Varuna, Brassica juncea cv Divya, Sinapsis alba and transgenic Brassica0 leaves of 45 days old plants which were treated with three different kinds of treatments at different time intervals. In the first treatment 10 μl volume of Alternaria brassicae spore along with 10 μl of 1ppm zeatin, was applied on to plants. In second and third treatment Alternaria brassicae spore suspension (10μl) and zeatin (10μl) were applied individually respectively. Gene specific primers for MAPK-4 were designed and RT-PCR amplification was done. Various bioinformatics studies were conducted with different in-silico tools after sequencing of RT-PCR product. To find out the upstream MAPKKs, MAPK-4 was docked with all known MAPKKs by the patch dock on line server which is based on shape complementarity principles and results were refined using Fire dock on- line server. In order to correlate the tertiary structures with function superimposition of MAPKKs protein structures were performed by MOE. The expression of MAPK4 in all the treatments showed the highest expression at 6hrs. Expression of MAPK4 at 9hr was observed in Divya and transgenic Brassica where as S. alba and Varuna did not show the expression. On the basis of superimposition result MAPKK could be categorized into three groups- A, B and C. Group A have MAPKK1 and MAPKK4, group B have MAPKK 2, MAPKK 3, MAPKK 6, MAPKK7, MAPKK 8 and MAPKK 10 and group C contains MAPKK 5 and MAPKK 9. Docking results of all 10 MAPKKs with MAPK4 showed that MAPKK5, MAPKK8, MAPKK3 and MAPKK9 have better interaction with MAPK4 The results of the present study clearly indicate the role of MAPK4 in plant defense against Alternaria blight. Moreover according to the superimposition results MAPKK9 and MAPKK5 also belong to the same group C. So it could be hypothesized that MAPKK 9 might be the upstream kinase not only for MAPK3/MAPK6 but also for MAPK4. Read more…