DUAL SPECIFIC PROTEIN KINASES MEDIATE NEEM FRUIT EXTRACT-INDUCED DEFENSE GENE EXPRESSION IN SOLANUM LYCOPERSICUM L.

Abstract

The molecular aspects of interaction between botanical extracts and plant cells through Mitogen-Activated Protein Kinase (MAPK) signaling cascades leading to the expression of defense genes are not well understood. The current study aims to better understand the role of dual-specific protein kinases in inducing defense gene expression in tomato as a result of aqueous neem fruit extract treatment.Staurosporine, Lavendustin A and K252a were used as kinase regulators which may modify the neem fruit extract induced MAPK cascades followed by defense gene expression in tomato plants. Tomato plants raised under aseptic conditions were treated either with neem fruit extract alone and pretreatment with either any one of the above kinase pathway inhibitors. Cytoplasmic defense proteins like PAL, TAL, and POX were studied for their activities and gene expression.. The differential reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) approach using mRNA extracted after treatment in all the groups was done for analyzing relative gene expression of PAL, POX and MAPKs, MAPKKs, MAPKKKs. The results demonstrated that neem fruit extract induced TAL and POX enzyme activities were inhibited by Staurosporine, Lavendustin A and K252a whereas PAL activities were inhibited by K252a only and superinduced by the pretreatment of Staurosporine or Lavendustin. Some of the neem fruit extract induced cytoplasmic and cell wall bound specific isoPOX proteins were inhibited by Lavendustin A / K252a, but Staurosporine could either inhibit / partially inhibit /superinduce them.. RT-qPCR analysis revealed that induction of gene expressions by neem treatment significantly inhibited the PAL, POX, all 3MAPKs, all 3 MAPKKKs, MAPKK1, MAPKK2, MAPKK3, MAPKK5 in plants pretreated with Lavendustin A and K252a but not by Staurosporine. Since Staurosporine can both activate and inhibit serine/threonine kinases our results indicate that staurosporine pretreatment in tomato plants treated with neem fruit extract leads to inhibition of TAL, POX activity, partial inhibition of cytoplasmic POX proteins, superinduction of PAL activity, cytoplasmic and cell wall bound POX proteins, their genes and MAPK cascade gene expression. The involvement of tyrosine kinases was confirmed by the inhibition of MAPK3, MAPKKs, defense gene and proteins by Lavendustin. Results indicate the expression of defense genes and enzymes in cytoplasm and cell wall by the neem fruit extract through the involvement of dual specific serine/threonine/Tyr (STY) kinases such as MAPK3, MAPKKs leading to establishment of signaling cascade.