ROS induce WAT1 in Presence of Nematodes molekulare phytomedizin Rheinische Friedrich-Wilhelms- Universität Bonn www.mpm.uni-bonn.de, mpm@uni-bonn.de ROS induce WAT1 in Presence of Nematodes Sina-Valerie Mahlitz, Shahid Siddique, Florian M. W. Grundler Rheinische Friedrich-Wilhelms Universität Bonn, INRES, Molekulare Phytomedizin, Karlrobert-Kreiten Strasse 13, 53115 Bonn, Germany The sedentary cyst and root-knot nematodes infect the roots of Arabidopsis thaliana. This process involves the release of reactive oxygen species (ROS) as part of a plant defense mechanism. The production of ROS upon nematode infection involves genes which belong to the respiratory burst oxidase homologues (Rboh) D and F. RbohD/F dependent ROS have been shown to promote the nematodes in establishment of their feeding sites, named syncytia and giant cell. Transcriptome analysis of 15dpi syncytia of Arabidopsis rbohD/F double mutant revealed a significant down regulation of the gene WALLS ARE THIN (WAT1). The mutation of WAT1 showed an altered cell wall constitution and a reduced susceptibility towards vascular pathogens. The cyst and root-knot nematodes are both endophytic parasites with a biotrophic lifestyle. The freshly hatched juveniles assemble in their migratory stage near the root where they start to have different invasion pathways. Heterodera schachtii: The cyst nematode can pierce randomly (mostly they prefer the root elongation zone) with its stylet until a suitable cell is found in vascular tissue across the endodermal barrier (approx. 10h). Meoloidogyne incognita: The root-knot nematode invades in the elongation zone (2h) and moves down to the meristematic region (8h). A U-turn guides the plant parasite trough the differentiation zone to the vascular cylinder (16h). Induction of WAT1 by Meloidogyne incognita Elongation zone Casparian strips Suberized endodermis M. Incognita H. schachtii x Root tip The expression of WAT1 after infection with M. incognita was more intense(more internse compared to what?). The staining only occurred in the lower part of the main root and lateral roots. The comparison of the root tip after 4 dpi shows an intense expression of WAT1 and the root tip is swollen. Modified from Julia Hohlbein, 2015 Induction of WAT1 by ROS Transformation of proWAT1::GUS in rbohD/F background The final confirmation whether RbohD/F dependent ROS induces WAT1 or not will be achieved by transforming proWAT1::GUS into a rbohD/F background. In theory, RbohD/F dependent ROS accumulate after a nematode established its feeding side. The accumulated ROS should then induce the expression of WAT1. The transformation of proWAT1::GUS into a rbohD/F mutant should lead to no expression of WAT1 at the cyst nematodes newly established feeding site. Expression of proWAT1::GUS in A. thaliana plants after an exogenous application of different H2O2 (ROS) concentrations. Induction of WAT1 by Heterodera schachtii Summary WAT1 is expressed in presence of H2O2 a kind of ROS Meloidogyne induces strong expression of WAT1 in all observed time points. In addition, the swollen root tip can be observed The expression of WAT1 in the presence of H. schachtii is very intense. The expression for the sedentary stage the colouring was decreased. ROS plays an important role for the cyst nematode to establish the feeding side and its maturation is completed but the expression of WAT1 stops when the feeding site is established. The transformation of proWAT1::GUS into a rbohD/F background will give us the final information whether ROS induces WAT1 and in which mechanism. The expression of WAT1 at the infection site of H. schachtii is visible at all time points. At 7dpi the expression of WAT1 is reduced.