HARROLD A. VAN DEN B · Identifying targets of ring finger and scf complex e3 ligase proteins in the plant defence response

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Project objective

Plant disease resistance (R) genes are key components in pathogen perception; R genes activate a battery of responses, and the recognition by R genes is hypothesized to be upstream of a signal transduction pathway. A cDNA-AFLP analysis resulted in the identification of ~ 150 Avr9/Cf-9 rapidly elicited (ACRE) genes. These ACRE genes became rapidly induced or repressed within 30' upon elicitation of a Cf-9 (R) tobacco cell culture. In addition to known signal transduction components ACRE genes code for putative components of the E3 ligase complexes, such as RING-H2 finger proteins, U box, and F box proteins components of the SCF complexes. E3 ligases complexes play a critical role in the selection of substrates for proteolysis. Selected substrates become 'tagged' with Ubiquitin, which targets them to the 26S proteasome. The specific substrates for each E3 ligase are expected to be negative regulators of the plant defense responses. The aim of this project will be to identify the substrates of the identified F-boxes, which seem to specifically recognise phosporylated substrates. Ubiquitination is a novel aspect in plant defense responses and it probablyplays a larger role in plants than in other organisms. Epitipe-tagged versions of the U boxes will facilitate in the identification of the putative interactors. Subsequently, an in-depth biochemical study will reconfirm the putative interactions. Finally, we will obtain a protein-protein interaction map for the identified E3 ligase complexes. The aim is to unravel the role of ubiquitination in plant defense responses. The proposed technique for the purification of protein complexes will require a substantial biochemical optimization to obtain intact E3 ligase protein complexes. Putative interactors will be identified by mass spectrometry, which is a novel aspect in the Jones Lab. Interactions need to be reconfirmed by yeast-to-hybrid screens and immuno-precipitation. This proposal is imbedded in a larger initiative on the role of ubiquitination in plants defence signalling at the Sainsbury laboratory. The Sainsbury laboratory is an excellent environment for this project as it hosts an extensive group of experienced researchers. A wide experience is available with most proposed techniques and model systems. Conclusively, I will get excellent biochemical training while I will be involved in a project at the frontier of biochemical possibilities and novel aspects of plant defence signalling.

Original text from CORDIS.

Participants

• THE SAINSBURY LABORATORY · NORWICHCoordinatorUnited Kingdom

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