Browsing by Author "Nissinen, Marjo"

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a plant pathogen which causes bacterial speck in the host plants, especially tomato. The genome sequence of Pst DC3000 was resolved in 2003 by Buell et al. and since then it has served as an important model organism in plant pathology. Pst DC3000 secretes proteins through so-called type III pathway (T3SS). The secreted proteins, called Hrp (Hypersensitive response and pathogenicity) or Hop (Hrp outer protein), are considered as main virulence determinants of the bacterium. Aim of the study was to find out the suitability of the phage Mu DNA transposition complex to generate a transposon insertion mutant library for Gram-negative phytopathogen. Then further screen the mutant library for Hop effector protein knock-out mutants and evaluate their virulence in Lycopersicon esculentum cv. Moneymaker tomato plants. The thesis was part of a research project that characterized virulence determinants of Gram-negative phytopathogenic bacteria, including their gene regulation and effects on the plant-bacterium interaction. Pst DC3000 transposon insertion mutant library was generated by electroporation of phage Mu DNA transposition complex and a nested PCR method was used for screening the library. Three hop transposon insertion mutants were found, and those strains were extracted from the library. The three mutant strains where then used to infect tomato plants. Virulence was evaluated through phenotypic lesions and bacterial endophytic growth. Some of the results were different from the previous studies. The ability of HopM1 mutant to form lesions was weakened and it´s endophytic growth was slowed down. In previous studies the ability to form lesions was also weakened, but the endophytic growth was not affected until avrE gene was also deleted. HopM1 deletion alone did not have an effect to bacterial growth. In HopAO1 mutant the virulence was not affected. In other studies, HopAO1 deletion weakened bacterial growth in Arabidopsis and tobacco plants, but in tomato it has not been tested. HopD1 mutant was avirulent in this study. In previous studies HopD1 was essential for Pst DC3000 bacteria in defeating plants effector-triggered immunity (ETI), but hopD1 deletion alone did not make the bacteria avirulent. Pst DC3000 has 28 different effector proteins so usually one effector protein mutation does not affect its virulence. Hence the next step of the study was to make double mutants for Hop proteins, and this included deletion of the antibiotic marker by Cre-lox mechanism, which was started in this study.