Defective Infection and Nodulation of Clovers by Exopolysaccharide Mutants of Rhizobium leguminosarum bv. trifolii

Abstract

The biological and symbiotic properties of two types of exopolysaccharide deficient (Exo- phenotype) mutants of Rhizobium leguminosarum were investigated. One mutant, a pss1 derivative (ANU437) of R. l. bv. trifolii, produces very low levels of acidic exopolysaccharide (EPS) and of the acidic oligosaccharide (AOS) repeating unit lacking its O-acetyl substituent. The second strain, originally a R. I. bv. viciae derivative, but containing a clover pSym plasmid (Exo-1 mutant ANU54(pBRIAN)), did not produce any EPS polymer or detectable oligosaccharide repeating unit. The Exo- mutants formed no capsules and their growth in laboratory media was comparable to their parent strains but was inhibited by the addition of the phytoalexins medicarpin and kievitone, but not by the phytoalexin pisatin. Both Exo- mutants were slow to induce clover root hair curling (Hac+), and formed growing infection threads (Inf-) only on white clovers. However, the extent of these early symbiotic defects depended on the plant growth conditions used. Both mutants induced small non-nitrogen-fixing (Fix-) nodules on white and subterranean clovers. Within the white clover nodules induced by mutant ANU437, bacteria were released into plant cells. On white clovers at elevated light and temperature conditions, the Exo-1 mutant ANU54(pBRIAN) usually failed to initiate infection threads and thus formed empty nodules which do not contain any bacteria. In addition, this mutant formed only empty nodules without infection threads on subterranean clovers under all growth conditions tested. The outermost layers of plant cells in these empty nodules had thickened cell walls and deposited materials. These experiments show that EPS synthesis is essential for the induction of rapid root hair curling, proper infection thread formation and the ability to sustain the growth of induced nodules on various clovers.

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…However, the EPS charge in the first (physicochemical) step of adhesion seems to have minor contribution, whereas the hydrophobic properties of this polysaccharide play a more dominant role in this process. In several papers, the role of EPS in early stages of symbiosis (infection of host plant roots) is well documented (Rolfe et al 1996;Cheng and Walker 1998;Abreu et al 2012;Janczarek and Rachwał 2013). Other authors confirmed that rhizobial EPS was required for attachment to inert substrates, and additional bacterial components (adhesins, glucomannan, cellulose fibrils) are engaged in the further steps of this process (Laus et al 2006;Williams et al 2008).…”

“…Besides flavonoids and Nod factors, rhizobial acidic exopolysaccharides (EPS) play a significant role in the establishment of an effective symbiosis especially with legumes that form indeterminate-type nodules (e.g. clover, pea, vetch, and alfalfa) (Rolfe et al 1996;Becker and Pühler 1998;Janczarek and Urbanik-Sypniewska 2013). This polysaccharide is indispensable for protection against host plant defence reactions and initiation and elongation of infection threads, special tubular structures via which rhizobia colonize root nodules (Cheng and Walker 1998;Jaszek et al 2014).…”

Bacterial community assembly based on functional genes rather than species

“…Functions include homologs of the OmpA protein (COG2197), which is required for adhesion to both mammalian and fish epithelial cells in a range of Proteobacteria (23,24), Listeria internalin-like proteins, which enhance attachment and biofilm formation (25,26), and the widespread colonization island, which is essential for biofilm formation, colonization, and pathogenesis in a range of bacteria (27). Proteins related to the production and excretion of galactoglycan, or exopolysaccharide II, were more abundant in the U. australis community, and apart from forming part of the biofilm matrix, is also essential for the establishment and maintenance of symbiosis in several Rhizobium strains (28)(29)(30)(31)(32). GGDEF and EAL domain proteins, which are involved in the production and degradation, respectively, of bis-(3′-5′)-cyclic dimeric GMP (cyclic-di-GMP), were also detected at a higher abundance (33,34).…”

Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharide-deficient mutant

“…Studies using GFP-marked S. meliloti revealed that succinoglycan is required early in the invasion process, as mutants unable to synthesize succinoglycan are enclosed in CCRH, but do not enable infection thread formation. Acidic exopolysaccharides now are known to be required for a number of other rhizobia to establish a symbiosis (4)(5)(6).…”