Bharani Manoharan firstname.lastname@example.org
The use of transposon libraries to investigate the evolution of bacterial pathogenesis
Pseudomonas syringae pathovar phaseolicola (Pph) is the seed borne causative agent of halo blight disease in the common bean, Phaseolus vulgaris. Gene-for-gene interactions are widespread and a very important aspect of plant disease resistance. Pph race 6 strain 1448A contains no known avirulence genes and can cause disease on all bean cultivars in the differential series; however, Pph race 4 strain 1302A contains the avirulence gene avrPphB, and causes a rapid hypersensitive response (HR) in bean cultivar Tendergreen.
Screening transposon (Tn) mutant libraries may allow discovery of many known and novel genes necessary for many characteristics such as adaptations to the environment, to colonise and to cause disease. Two random Tn mutant libraries, Pph 1302A::Tn and Pph 1448A::Tn were created using Tn IS-Ω-Km/hah. Pph mutants were successfully produced using the optimized conditions. A total of 960 mutants in Pph strains 1302A and 1448A were tested for their phenotypic characterisation and for identification of genome mobility in Pph 1302A and competence genes in Pph 1448A.
To investigate genes involved in Pph colonisation of plants and evolution of Pph pathogenesis by HGT, the Tn mutant libraries were screened for changes in bacterial phenotypic characteristics such as colony morphology, motility, biofilm formation, growth rate in bean apoplastic fluid and conjugation. Selected mutants were assessed for in vitro and in planta growth rate and pathogenicity. Results showed that number of Pph mutants with different phenotypes caused restriction of growth in plants and/or reduced symptoms.
In colony morphology screening, 28 small (11 Pph 1302A and 17 Pph 1448A) and 29 big (four Pph 1302A and 25 Pph 1448A) colony variant mutants were obtained. Eight Pph mutants showed highly reduced colony size and were also affected in both swarming and swimming motility. One very small colony mutant had a Tn insertion in the gene for HdtS protein which is involved in quorum sensing of bacteria. Among 29 big colony mutants, a Tn insertion in the gene for outer membrane protein A (OmpA) affected colony morphology, motility and highly impaired in planta growth and pathogenicity symptoms.
Other phenotypic screening methods included motility tests, where several Pph mutants showed highly reduced swarming and swimming motility. Six Pph 1302A::Tn mutants showed some reduction in biofilm formation/attachment. Three Pph 1302A::Tn mutants had a higher growth rate in minimal media supplemented with apoplastic fluid. Two Pph 1448A::Tn mutants showed significantly reduced growth rate in vitro in Luria Bertani liquid media. Finally, eight Pph 1448A::Tn mutants showed complete absence of disease in bean pods. These results demonstrate the identification of many genes involved in key bacterial behaviour and are important for plant colonisation and therefore disease in bean plants.
Screening for genome mobility by Pph 1302A::Tn mutant library showed three Pph 1302A::Tn mutants which may have the ability to transfer from Pph 1302A to Pph 1448A. These mutations were in areas of the genome associated with antibiotic resistance and genes for the Omp family, mobile genetic elements and type III effector protein and conserved hypothetical and adhesion protein. All three mutants may be involved in HGT and contribute to the evolution of Pph pathogenesis.
Thirty five Pseudomonas strains were examined for their conjugation and transformation ability. Results showed two Pph strains Pph 882 (race 2) and Pph 1375A (race 5) have transformation ability and 28 Pseudomonas strains have conjugation ability. However, during this study transformation ability of Pph 1448A appeared to be lost.
Examination of the loss of competence in Pph 1448A showed two changes i.e. growth rate reduction in vitro in LB liquid media and plasmid genome alteration by bean pod passage.
Plants and bacteria can interact with one another in a variety of different ways. The interaction may be beneficial, harmful or neutral for the plant. This work has contributed to the understanding of these interactions between Pph 1302A and Pph 1448A in their host plants. These may allow subsequent development of sustainable disease control strategies.
|APA6 Citation||Manoharan, B. The use of transposon libraries to investigate the evolution of bacterial pathogenesis. (Thesis). University of the West of England|
|Keywords||evolution, bacterial pathogenesis|