All ELISA procedures followed the manufacturer’s instructions

better understand how these mj-far-1 overexpressing roots became more susceptible to M. javanica infection, expression analysis of a set of genes belonging to the fatty acid metabolic pathways and that are involved in oxylipins biosynthesis e.g. JA, were analyzed. The effect of mj-far-1 expression on plant root lipoxygenases and a-dioxygenases catalyzing the formation of fatty acid hydroperoxides was examined. Downregulation of a component of the DOX pathway, the LEa-DOX2, was observed in both root lines mj-far-1 overexpressing and vector control roots upon nematode infection. These findings illustrate that while LEa-DOX2 is not precisely affected by FAR overexpression, oxylipins initiated by plant a-dioxygenases might exerts a specific purchase AGI-5198 function in regulating plant response to nematodes in addition to their critical role in tomato plant development. Among the three 9-LOX isoforms, TomLOXA, TomLOXB and TomLOXC showed no obvious effect in their expression profile, either as function of Mj-FAR-1 or upon nematode infection. Unlike the 9-LOXs, the TomLOXD gene, acting upstream in JA biosynthesis pathway, showed a moderate up-regulation in both root lines mj-far-1 overexpressing and vector control upon nematode infection, while no direct effect could be observed as a consequent result of FAR overexpression. These results show that DOXs as well as LOXs expression are not significantly affected by the ectopic Mj-FAR-1 expression upon M. javanica infection on tomato roots. In contrast to our results, Prior et al. showed inhibition of LOX by recombinant G. pallida rGp-FAR-1 protein. Mj-FAR-1 Induces Host Susceptibility to RKN This differences might be explained by differences in substrate availability occurring in the in 10753475 vitro system compared with the in planta system 26836578 we used or it might be that FAR is involved in manipulating LOX activity but not its expression. Analyzing the interaction of FAR with JA pathway Given that Jasmonic acid acts as signal activating the expression of various genes, such as the proteinase inhibitors, 12-oxophytodienoate reductase and c-thionin their expression profile was used to reflect the induction/suppression of the JA metabolic pathway. Both tomato proteinase inhibitor 2 and c-thionin were significantly down-regulated in roots overexpressing mj-far-1, while OPR3 expression remained unstable during the independent experiments. Overall, these findings suggest that JA responsiveness pathways have been manipulated in roots overexpressing mj-far-1 to support nematode parasitism. Interestingly, cthionins appear to play diverse roles as they present; i) antibacterial and/or anti-fungal activity, ii) ability to inhibit mammalian cell growth by membrane permeabilization and iii) the ability of inhibiting insect a-amylases and proteinases. Thus, these intrinsic characteristics described for plant cthionins, along with the significant suppression of c-thionins as function of FAR, might contribute to the increased susceptibility response observed for roots overexpressing mj-far-1. Moreover, the observed suppression of proteinase inhibitor found in roots overexpressing mj-far-1 might also facilitate nematode parasitism. Similarly, tomato mutant deficient in jasmonate synthesis, def1, fails to accumulate proteinase inhibitors in response to wounding and is considerably more susceptible than WT to attack by tobacco hornworm larva. Furthermore, the implication of protease inhibitor in enhancing plant resistance to nematodes have