Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots

Abstract

The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.nitrogen fixation ͉ nodulation ͉ clover ͉ root hair ͉ cellulose A central event in development of the Rhizobium-legume root-nodule symbiosis is the localized erosion of a cellulosic plant wall through which the bacterial symbiont passes to establish a nitrogen-fixing, intracellular endosymbiotic state within its legume host. Plant cell wall-degrading enzymes are predicted to participate in two steps of this infection process: during primary infection of host root hairs leading to infection thread formation (Inf) and later during bacterial release (Bar) from infection threads within host nodule cells. This process of plant cell wall degradation must be delicately balanced to allow the localized penetration of the bacterial symbiont into the host cell without its overt lysis and destruction.Several studies indicate that rhizobia produce enzymes capable of degrading plant cell wall polymers (1-14), but little is known about their molecular properties, none have previously been purified to homogeneity, and their specific role (if any) in symbiosis is undefined. The relatively low activities of these rhizobial enzymes have hampered research progress in this area. Using improved assays with increased sensitivity that reliably detect these enzyme activities, we established that cellulases are produced by wild-type strains of Rhizobium leguminosarum (biovars trifolii, phaseoli, and viciae), Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti (7, 9). Further studies using R. leguminosarum bv. trifolii ANU843 indic...

The Nodule Microbiome: N2-Fixing Rhizobia Do Not Live Alone

“…Knockout mutants of the gene encoding the cellulase CelC2 in Rhizobium leguminosarum bv. trifolii ANU843 abolished infection thread formation (Robledo et al 2008). Ibáñez et al (2009) performed coinoculation experiments on peanut with nodule-associated species of Pseudomonas, Enterobacter, and Klebsiella together with Bradyrhizobium.…”

Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

“…Bacterial enzymes capable of degrading plant cell wall components have long been suggested to play a crucial role in establishing the nitrogen-fixing Rhizobium-legume symbiosis. Recently, Robledo et al (2008) were the first to support this hypothesis with molecular and genetic evidence by identifying a cellulase gene from R. leguminosarum. CelC2 can erode the cell wall of host root hair tips and is essential during the initial stages of symbiosis.…”

“…However, bacterial enzymes capable of breaking down plant cell wall material have long eluded identification, and induction of degradative host enzymes for remodelling the plant cell wall seemed just as likely. Only recently, Robledo et al (2008) succeeded in purifying and characterizing a cell-bound cellulase from Rhizobium leguminosarum. This CelC2 enzyme can erode the cell wall of host root hair tips and is essential for establishing a symbiosis with the R. leguminosarum host plant, white clover (Trifolium repens).…”

The Temperature-Sensitive brush Mutant of the Legume Lotus japonicus Reveals a Link between Root Development and Nodule Infection by Rhizobia

“…This resulted in a similar phenotype as described for MOR1. Interestingly, Robledo and colleagues demonstrated that the cell-bound bacterial cellulase CelC2 is essential for the primary infection process in Rhizobium leguminosarum (Robledo et al, 2008). These data suggest that cellulytic activity is required for both proper root development and rhizobial infection.…”