ANALYSIS OF THE GENOME OF ERWINIA AMYLOVORA, IDENTIFICATION OF NOVEL VIRULENCE FACTORS USING BIOINFORMATICS AND MOLECULAR STUDIES ON THE TRANSLOCATION INTO PLANT CELLS OF DSPA/E, A PATHOGENICITY FACTOR IN E. AMYLOVORA

Abstract

Fire blight is a devastating disease of rosaceous plants, especially apple and pears. The Gram-negative bacterium Erwinia amylovora is the causal agent of fire blight and is phylogenetically related to important animal and plant pathogenic enterobacteria. The genome of E. amylovora, strain Ea273, was sequenced at the Sanger Institute, and the sequence is publicly available at their web page. In Chapter 2 a computational approach on the genome sequence at the various stages of assembly was used to produce a preliminary annotation of virulence factors by similarity with known factors. Two novel type-three secretion system (T3SS) islands, which seemed more closely related to those of animal pathogens, were identified in the genome. A comparative analysis revealed loss of genes by the genome of Ea273 when compared with closely related Escherichia coli and Salmonella sp. at the core genome level, and acquisition of plant-pathogenic virulence factors. In Chapter 3, a combination of computational and targeted genetic methods were applied to predict genetic signatures of promoter regions associated with the HrpL regulon (hrp boxes), producing a list of candidate genes possibly expressed during infection. Two of the candidate genes were characterized and hopX1 was shown to have a function related to avirulence and HR suppression. The number of genes or operons related to virulence reported previously to the availability of the genome sequence was limited, and molecular genetic studies of E. amylovora typically focused on individual systems, such as the translocation of DspA/E work presented in Chapter 4. Using a CyaA reporter system, I demonstrated that DspA/E is translocated into plant cells, and that HrpN and HrpJ are necessary for its translocation. The results presented in this Chapter suggest that HrpN might be part of the translocation apparatus of DspA/E, and HrpJ might regulate or stabilize the secretion of HrpN in vitro. The availability of the genome sequence of E. amylovora contributes to the formulation of new hypothesis, offers an important source of information for evolutive and physiological studies, and opens an era of post-genomics analysis with opportunities for the use of high throughput approaches to study this important pathogen.