Identification and characterization of genes specifically up
Transkrypt
Identification and characterization of genes specifically up
Intercollegiate Faculty of Biotechnology UG & MUG. PhD seminar 09.05.2014 Identification and characterization of genes specifically up-regulated in Dickeya solani at different growth temperatures Natalia Kaczyńska, Laboratory of Plant Protection and Biotechnology, Department of Biotechnology, IFB UG-MUG. Supervisor: Prof. dr hab. Ewa Łojkowska (IFB UG-MUG). Co-supervisors: Prof. dr hab. Grzegorz Węgrzyn (FB UG), dr Robert Czajkowski (IFB UG-MUG) Pectolytic bacteria Pectobacterium spp. and Dickeya spp. that cause blackleg and soft rot disease are among the most economically important bacterial pathogens of a large number of crops [1]. P. atrosepticum typically causes blackleg and soft rot on potato plants in temperate climate, whereas Dickeya spp. are responsible for these diseases in warm climate. However in last years, new species Dickeya solani has emerged in Europe and has been isolated in Poland, the Netherlands, Finland, Norway, France, Germany, Sweden, Spain and Belgium [2,3,4,5]. D. solani is more virulent and aggressive than other Dickeya spp. and P. atrosepticum isolated from potato in Europe so far. Global warming leading to climate change and is partly responsible for faster spreading of this pathogen through Europe. Temperature may act as a signal that activates expression of specific factors in plant pathogens during infection. The main aim of the project was to identify and characterize genes up-regulated in D. solani at different growth temperatures (18, 28 and 37 °C). We have chosen a representative, isolated in Poland, D. solani strain IFB0099. In total, 7300 mutants of IFB0099 were generated by random transposon mutagenesis based on insertions of the miniTn5 transposon containing a reporter gene gusA, coding for β-glucoronidase (GUS) enzyme. Out of these mutants, 49 showed thermoregulated gusA expression on X-Gluc-containing M9 minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. 24 mutants showed an increased reporter gene expression at 37 °C, and 5 – at 18 °C. Genomic DNA of the selected temperature-induced D. solani mutants was isolated for direct genomic DNA sequencing. Insertions of the mini-Tn5 transposon were found in genes encoding oligogalacturonate-specific porin kdgM, chitinase class I family protein, multiple stress resistance protein BhsA, regulatory protein PecM, and proteins of unknown function. Acknowledgement: The work was financially supported by the Ministry of Science and Higher Education, Poland via research grant Iuventus Plus 2013 (IP2012 024172) to R. C. References: [1] Czajkowski R., Pérombelon M.C.M., van Veen J. A., van der Wolf J. M. (2011). Plant Pathology, 60, 999. [2] Laurila J., Ahola V., Lehtinen A., Joutsjoki T., Hannukkala A., Rahkonen A., Pirhonen M. (2008). European Journal of Plant Pathology, 122, 213. [3] Sławiak M., Łojkowska E., van der Wolf J.M. (2009). Plant Pathology, 58, 794. [4] Toth I.K., van der Wolf J.M., Saddler G., Łojkowska E., Hélias V., Pirhonen M., Tsror (Lahkim) L., Elphinstone J.G. (2011). Plant Pathology, 60, 385. [5] Czajkowski R., Grabe G.J., van der Wolf J.M. (2009). European Journal of Plant Pathology, 125, 263. KSZTAŁCIMY NAJLEPSZYCH – kompleksowy program rozwoju doktorantów, młodych doktorów oraz akademickiej kadry dydaktycznej Uniwersytetu Gdańskiego. Zad. 2. Life Sciences and Mathematics Interdisciplinary Doctoral Studies (LiSMIDoS) Project co-funded by European Union under the European Social Fund