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