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ISHS Acta Horticulturae 896: XII International Workshop on Fire Blight

DELETION OF ERWINIA AMYLOVORA FLAGELLAR MOTOR PROTEIN GENES MOTAB ALTERS BIOFILM FORMATION AND VIRULENCE IN APPLE

Authors:   J.M. Koczan, G.W. Sundin
Keywords:   amylovoran, fire blight, levan, swarming motility, xylem
Abstract:
Biofilm formation, an aggregated network that allows single-celled organisms to act in a multi-cellular fashion, plays a critical role in bacterial ecology and pathogenesis, ultimately conferring protection from environmental stress, promoting nutrient acquisition, and enabling gene exchange and acquisition at a faster rate. The Erwinia amylovora exopolysaccharides amylovoran (a pathogenicity factor) and levan (a virulence factor) are necessary for fully developed biofilm formation, implying that biofilm formation plays a critical role in pathogenesis. Additional genes with putative roles in biofilm formation were identified using bioinformatics, and single and double gene deletions were generated in amy2031 and amy2032. These genes are homologs of motA and motB which encode flagellar motor stator genes with a known role in swarming motility and biofilm formation. In vitro biofilm formation assays revealed a significant reduction in biofilm formation capabilities of the putative flagellar stator mutants. Motility assays demonstrated that single gene deletions of flagellar stators inhibited swarming motility, while a double gene deletion significantly impaired motility, confirming that flagellar stators not only function in biofilm formation, but also in the movement of the pathogen. In planta disease assays were performed and revealed that single gene deletion mutants showed reduced virulence in immature pear fruits and in shoots of seedlings and trees. In contrast, double gene deletion mutants exhibited similar virulence to the wild type. Scanning electron microscopy of inoculated tissue revealed single gene deletion mutants at the site of inoculation and in mesophyll tissue, however no sign of biofilm is present and bacteria were not found in vascular tissue. Double gene deletion mutants were primarily localized to the mesophyll tissue and the tissue surrounding the vascular tissue, though isolated aggregates could be seen in vascular tissue. These results further demonstrate the crucial role that biofilm formation plays in E. amylovora pathogenesis and identifies other key determinants that bridge biofilm formation and pathogenicity.
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