Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora

Abstract

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...

Cellulose production, activated by cyclic di‐GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

“…We have reported previously the activation of amylovoran biosynthesis and a hyper-biofilm-forming phenotype in E. amylovora when high intracellular concentrations of c-di-GMP are induced with the overexpression of the DGC-encoding genes edcC and edcE (Edmunds et al, 2013). In an attempt to dissect the role of individual DGCs on the complete set of c-di-GMP biosynthetic proteins in this pathogen, the modulation of EPS biosynthesis and biofilm formation of two additional DGC genes, edcB and edcD, was evaluated.…”

“…This pathogen produces at least two EPSs, amylovoran and levan, each of which plays a critical role in the formation of biofilms inside the host xylem, resulting in vessel blockage and water transport restriction and, consequently, wilting and necrosis of the infected tissues (Koczan et al, 2009). Moreover, biofilm formation and amylovoran biosynthesis have been demostrated to be under the regulation of c-di-GMP, as high intracellular levels of this secondary messenger molecule induce the activation of these two cellular processes (Edmunds et al, 2013).…”

Regulation of type III secretion system in Pseudomonas syringae

“…Another well-known bacterial secondary messenger, cdi-GMP, regulates multiple important pathways including biofilm formation and virulence in a wide spectrum of bacterial species, such as Escherichia coli, Xanthomonas oryzae pv. oryzae, Dickeya dadantii, Bacillus amyloliquefaciens and Erwinia amylovora (Romling et al, 2005;Yang et al, 2012;Edmunds et al, 2013;Yang et al, 2014Yang et al, , 2015Yuan et al, 2015;Jenal et al, 2017;Sarenko et al, 2017;Yang et al, 2018;Kharadi and Sundin, 2019;Yuan et al, 2019;Li et al, 2019a). The biosynthesis of c-di-GMP in vivo depends on diguanylate cyclases, whilst its degradation is catalysed by phosphodiesterases (Jenal et al, 2017).…”

Flagella-induced transitions in the collective behavior of confined microswimmers

“…Collective motion emerges in a wide range of natural systems, from fish schools [1,2] to bacterial colonies [3][4][5][6][7][8], and is believed to play important roles in the functioning and survival of the group. For example, many species of bacteria cyclically transition from a free-swimming planktonic state into a sessile biofilm state in response to environmental conditions [9,10]. It is well known that the free-swimming state is characterized by flagella-driven motility, which is suppressed in the biofilm state.…”