Nod Factors of Rhizobium leguminosarum bv. viciae and Their Fucosylated Derivatives Stimulate a Nod Factor Cleaving Activity in Pea Roots and Are Hydrolyzed In Vitro by Plant Chitinases at Different Rates

Abstract

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Nonmodified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.

The Nodulation Factor Hydrolase of Medicago truncatula: Characterization of an Enzyme Specifically Cleaving Rhizobial Nodulation Signals

“…Due to structural similarities to chitin, various NFs can be hydrolyzed by plant chitinases (Staehelin et al, 1994a(Staehelin et al, , 1994bGoormachtig et al, 1998;Minic et al, 1998;Schultze et al, 1998;Ovtsyna et al, 2000). Chitinases (EC 3.2.1.14) are defined as enzymes that cleave b-1,4 glycosidic linkages in chitin (poly-GlcNAc) and chitin oligosaccharides (Collinge et al, 1993).…”

“…When tested in root hair deformation bioassays, the purified cleavage products are at least 1,000-fold less active (Heidstra et al, 1994;Staehelin et al, 1994b). In fact, NFs produced by compatible rhizobia were found to be rapidly degraded in the rhizosphere of legumes (Heidstra et al, 1994;Staehelin et al, 1994bStaehelin et al, , 1995Ovtsyna et al, 2000Ovtsyna et al, , 2005. NFs with a shorter oligosaccharide backbone and carrying additional chemical substitutions are usually more resistant against degradation by chitinases (Staehelin et al, 1994a(Staehelin et al, , 1994bMinic et al, 1998;Schultze et al, 1998;Ovtsyna et al, 2000).…”

“…In fact, NFs produced by compatible rhizobia were found to be rapidly degraded in the rhizosphere of legumes (Heidstra et al, 1994;Staehelin et al, 1994bStaehelin et al, , 1995Ovtsyna et al, 2000Ovtsyna et al, , 2005. NFs with a shorter oligosaccharide backbone and carrying additional chemical substitutions are usually more resistant against degradation by chitinases (Staehelin et al, 1994a(Staehelin et al, , 1994bMinic et al, 1998;Schultze et al, 1998;Ovtsyna et al, 2000). NodSm-IV(C16:2, Ac, S), an O-acetylated and sulfated tetrasaccharide with a C16:2 fatty acid chain (Lerouge et al, 1990), is the most abundant NF produced by Sinorhizobium meliloti (Supplemental Fig.…”

“…Nevertheless, when added to roots of the host plant alfalfa (Medicago sativa), NodSm-IV(C16:2, Ac, S) was rapidly degraded to a lipodisaccharide, and increased NF hydrolysis was observed when roots were pretreated with NFs (Staehelin et al, 1995). NFs were also found to induce an NF-cleaving activity in the rhizosphere of pea (Pisum sativum) plants, and this induction did not happen in NF signaling-deficient pea mutants (Ovtsyna et al, 2000(Ovtsyna et al, , 2005. Similarly, chitinolytic activities of certain chitinase isoforms of legumes are induced during symbiosis with rhizobia or in response to purified NFs (Staehelin et al, 1992;Xie et al, 1999;Ovtsyna et al, 2005).…”

Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

“…One activity released the lipo-disaccharide, whereas the other activity formed the lipo-trisaccharide as it was described for a chitinase lysozyme in alfalfa roots [60]. The lipo-disaccharide-forming pea hydrolase was specifically induced by Nod factors from Rhizobium leguminosarum bv viciae [59,61], whereas the lipodisaccharide-forming enzyme from alfalfa was induced by NodSm factors [62]. In pea plants, the degradation of Nod factors correlated with a chitinase which was stimulated by high doses of Nod factors as well as stress-related signals.…”