Site directed mutations of one of the plant pathogen effector gene for functional analysis

Mustafa, Zemran
Wheat (Triticum aestivum L.) is one of the most important crops planted in the world and used as major food resource. Yet, there are major losses of productivity due to different biotic and abiotic destructive agents. Among the biotic factors especially the yellow rust is one of the most frequent. Yellow rust or also called “stripe rust”, due to stripe like appearances on the leaves, disease is caused by Puccinia striiformis f. sp. tritici, which is an obligate plant fungal pathogen. To cope with high yield losses caused by these pathogens, investigation of host-pathogen interaction at the molecular level can empower the scientists in fight against the pathogen. In this thesis research, one of the Puccinia striiformis f. sp. tritici candidate effector genes (PstHa2a5 / Yin et al., 2009) was studied for functional analysis by conducting site directed mutagenesis (SDM) on the gene. The amino acid sequence of this effector vi indicates the presence of a conserved FXC motif. The function of this motif is currently unknown but it is suspected that it is needed for intracellular targeting of effector proteins. Mutated versions of the gene were cloned into pK7WGF2 and pK7FWG2 gateway destination vectors to be transformed first into Agrobacterium tumefaciens by electroporation and then delivered into Nicotiana benthamiana leaves via agrobacterium-mediated gene transformation. The distorted motif of PstHa2a5 by SDM was tested for its role in intracellular localization of the protein. By expressing the mutant protein in N. benthamiana leave cells and tracking green fluorescent protein (GFP) expressed as a fusion to the modified gene, under microscope functional analysis were conducted.
Citation Formats
Z. Mustafa, “Site directed mutations of one of the plant pathogen effector gene for functional analysis,” M.S. - Master of Science, Middle East Technical University, 2015.