The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis. Erwinia amylovora is the causal agent of fire blight and a devastating phytopathogen that infects plant species of the family Rosaceae, most notably apple and pear trees (1). E. amylovora can infect flowers, fruits, actively growing shoots, and rootstock crowns (2). During the primary infection via the flower (1), E. amylovora cells multiply rapidly on the stigma. Motility and free moisture are important factors in the subsequent dissemination of cells down the outside of the stigma to nectarthodes, which provide entry into the plant (3-5).Following flower infection, E. amylovora cells spread systemically through host vascular tissues and cortical parenchyma. The wilting symptoms of fire blight are the result of bacterial invasion, the secretion of extracellular polysaccharide (EPS), and the formation of biofilms within host xylem that plug these tubes, restricting water flow (6, 7). E. amylovora secretes two distinct EPSs, amylovoran and levan, both of which contribute to plant infection (6). Amylovoran is an acidic polysaccharide composed of repeating units of galactose and glucuronic acid (8-10), while levan is a homopolymer of fructose residues synthesized from sucrose by the secreted enzyme levansucrase (11). Biofilm formation by E. amylovora is required for effective colonization of host xylem tissues, the exit of pathogen cells from infected leaves into host stems, and systemic spread within trees (6, 7). The...
We concluded that the Erwinia amylovora diguanylate cyclase genes edcB and edcD did not synthesize cyclic di-GMP (c-di-GMP) or regulate biofilm formation, amylovoran production, or motility when overexpressed under the conditions examined. We have since learned that the expression vectors for these enzymes do not contain the full open reading frame for these genes. We have since made the correct expression plasmids and determined that edcB and edcD do synthesize c-di-GMP and inhibit motility while inducing biofilm formation and amylovoran production when overexpressed in E. amylovora. We apologize for this error.
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