Cloning, transformation and wheat infiltration assay of a puccinia striiformis f. sp. tritici effector candidate

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2013

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Özketen, Ahmet Çağlar

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The stripe rust pathogen, Puccinia striiformis f. sp. tritici is the causative agent of the yellow (stripe) rust which is globally one of the most devastating and economically significant diseases in wheat. Although using fungicide can present a practical solution against the disease, an efficient, better and environmentally safer approach is needed to grow disease free wheat. To achieve this, better comprehension of the plant-pathogen interaction at the molecular and cellular level is required. The pathogen effectors are the key to discover this molecular maze of the plant-microbe interactions. With the advances in Puccinia genome sequencing projects and development of new strategies, researchers now have a chance to predict and test the candidate effectors on various biological assays. In this thesis, one of the predicted effector candidates from Puccinia striiformis f. sp. tritici is cloned into three different vectors to test for avirulence, to determine subcellular localization and to pull-down the interacting host factors. PSTha15N21 pathogen gene is predicted as an effector candidate using bioinformatic tools and expression data (Yin et al., 2009a). The pathogen effector gene was cloned into a gateway destination vector, pK7FWG2 to study subcellular localization, using agroinfiltration method. The gene construct was also cloned into a pEDV6 vector for avirulence testing by searching the presence of hypersensitive (HR) response using Type III secretion system of P. fluorescens (EtHAn). Lastly, the gene construct with FLAG-tag fusion in its N-terminus site was cloned into pJL48-TRBO expression vector for expressing FLAG-fused protein to perform tagged protein co-immunoprecipitation experiment toward identification of host interacting factors in future studies.

Subject Keywords

Wheat rusts., Stripe rust., Agrobacterium., Puccinia striiformis.

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http://etd.lib.metu.edu.tr/upload/12616554/index.pdf
https://hdl.handle.net/11511/22928

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

A. Ç. Özketen, “Cloning, transformation and wheat infiltration assay of a puccinia striiformis f. sp. tritici effector candidate,” M.S. - Master of Science, Middle East Technical University, 2013.