Effector vector design in the Phytophthora infestans-potato pathosystem
Sammanfattning: The oomycete pathogen Phytophthora infestans is the causal agent of the devastating plant disease late blight on potato. Diverse type of transposons and many gene families are present in the genome which encodes the effector proteins involved in causing the pathogenicity. This plantpathogen is predicted to secret hundreds of effector proteins inside the host plant cells to promoteinfection. These proteins are sensed by the plant immune system in order to prevent pathogen growth. The effector proteins are divided into two main types, cytoplasmic effectors and apoplastic effectors based on their translocated status in the plant cell. In this study, the effectorencoding genes Avr3a, Epi1, Epi10, Inf1 and CRN8 were selected to monitor the potential in planta function of the effectors and to develop a stable transformation procedure for reporter gene constructs with effector gene promoters. The putative promoter sequences were derivedfrom the 5´ regions of the oomycete genes. Primers were designed to amplify the promoter regions and the amplification was confirmed by gel electrophoresis. The reporter gene GFP (encoding green fluorescent protein) was chosen for analysis of their promoter activities and tofacilitate studies on spatial and dynamic alteration of gene expression. Cloning was performed using the vector pTOR-eGFP containing a ham34 promoter and a GFP gene. The ham34 promoter was removed and the effector promoters were inserted in its place. A stable transformation procedure was examined using three vectors for the GFP-constructs and the five effector gene promoters. Transformants were obtained at similar frequencies with each combination of effector promoter and GFP; which were confirmed by gel electrophoresis.Subsequently Agrobacterium tumefaciens (C58) mediated transformation was tried for an Avr3a promoter construct. The construct was ligated into the binary vector, but the transformation of Agrobacterium was not successful.
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