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

Özketen, Ahmet Çağlar
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.


Characterization of a candidate effector of wheat yellow rust targeting chloroplast with a novel transit peptide
Andaç, Ayşe; Akkaya, Mahinur S.; Gür Dedeoğlu, Bala; Department of Biotechnology (2019)
Fungal pathogen, Puccinia striiformis f. sp. trtici, is the causative agent of stripe disease of wheat which causes disruption on wheat yield in many parts of the world. Fungal pathogens secrete effector molecules into host plant cells to suppress host immunity via virulence to colonize plants. Ongoing efforts are being made to identify and characterize effector proteins in many fungal plant pathogens. Nevertheless, the precise biological and biochemical functions of many effectors, such as their traffickin...
Bacterial expression of an effector protein of yellow rust pathogen and a resistance protein of wheat and characterization of the effector protein
Erdoğan, Sayıt Mahmut; Akkaya, Mahinur S.; Özçubukçu, Salih; Department of Biotechnology (2019)
Yellow rust is one of the most important wheat disease encountered in large parts of the world and in our country caused by a pathogen which is called as Puccinia striiformis f. sp. tritici (Pst). Rapid alterations in the pathogen virulency can make previously resistant varieties susceptible to the disease. Finding out common or species-specific genes, participated in the plant-pathogen interactions will provide an understanding of the biological mechanisms of the disease. Within the scope of this thesis fo...
Transfer of stripe rust resistance gene Yr26 to Turkish wheats using microsatellite markers
Yildirim, A; Karadag, Y; Sakin, MA; Gokmen, S; Kandemir, N; Akkaya, Mahinur; Yildirim, F (2004-01-01)
Stripe rust (yellow rust) is one of the most devastating diseases of wheat worldwide. The most effective control method of stripe rust is the use of resistance genes. A very effective stripe rust resistance gene, Yr26, originating from Triticum turgidum L., was transferred into two common wheat (Triticum aestivum L.) cultivars of Turkey through backcrossing using closely linked microsatellite markers. A 6VS/6AL translocation line (92R149) was used as the source of Yr26 because this gene was originally thoug...
Akkaya, Mahinur; Dagvadorj, Bayantes(2013-12-31)
Cloning and expression of a plasmid-linked pediocin determinant trait of Pediococcus acidilactici F
Osmanagaoglu, O; Beyatli, Y; Gündüz, Ufuk (2000-01-01)
Plasmid DNA from Pediococcus acidilactici F was prepared by lysozyme-mutanolysin method and purified by cesium chloride-ethidium bromide (CsCl-EtBr) density gradient ultracentrifugation. Agarose gel electrophoresis of plasmid DNA and plasmid-curing experiments suggested that bacteriocin activity was harboured on a small plasmid of approximately 9.1 kb (kilobasepair) in Pediococcus acidilactici F. Plasmid encoding bacteriocin production in P. acidilactici F was examined for restriction enzyme cleavage patter...
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.