Investigation of subcellular localization of a yellow rust pathogen effector candidate

Andaç, Ayşe
Puccinia striiformis f. sp. tritici is one of the fungal pathogens, which causes one of the most disruptive diseases of wheat called, stripe rust, in many parts of the world. Because of their highly destructive character, Puccinia species including yellow rust are responsible for considerable yield losses in worldwide wheat production. Despite its economic importance, the genomics of the pathogens and the plant-pathogen interactions have not been identified yet. For sustainable control of this plant disease, creating durable resistant crops will require accumulation of information for the long-term solution against fungal pathogens. Toward which, the main objective of this thesis is to address developing better understanding of plant-pathogen interactions. Together with, characterization of pathogenicity and secreted effector proteins can help to guide these efforts. In this thesis, one of the candidate effector genes of Puccinia striiformis f. sp. tritici Pstha12h2 is studied. The aim is to identify its function during host infection and for future investigation of the interaction between host factors of wheat Triticum aestivum L. infected with yellow rust. This effector gene was synthesized with FLAG-Tag at its N-terminus with the PacI-NotI restriction sites at the ends. This gene was cloned into pK7FWG2, which is a Gateway destination vector, and transformed into Agrobacterium tumefaciens for expression in Nicotiana benthamiana leaves. By achieving this, the candidate effector gene was expressed using a GFP fusion protein in N. benthamiana. Additionally, the gene construct was cloned into pEDV6 vector, which is also a gateway destination vector for an expression in wheat leaves, by Pseudomonas fluorescens mediated transformation. Here, the aim will be observe hypersensitive response caused by effector gene if it happens to be an Avr against any resistance (R) gene. The advantage of this bacterial system is its ability to deliver genes to wheat cells by their Type III secretion systems (TTSS). As part of a future work, wheat cultivars will be infiltrated by P. fluorescens suspensions and the delivery of effector genes can be observed by staining of the presence of hydrogen peroxide accumulation, if the complementary ‘R’ gene is present in the wheat line tested. Also, the gene construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein with FLAG-tag will be isolated and purified as a future study. By immunoprecipitation, effector protein will be used to puldown putative host interactors. Using mass spectroscopy analysis, the interactor protein will be easily identified.


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Citation Formats
A. Andaç, “Investigation of subcellular localization of a yellow rust pathogen effector candidate,” M.S. - Master of Science, Middle East Technical University, 2013.