Nod Factors Induce Nod Factor Cleaving Enzymes in Pea Roots. Genetic and Pharmacological Approaches Indicate Different Activation Mechanisms

Abstract

Establishment of symbiosis between legumes and rhizobia requires bacterial Nod factors (NFs). The concentration of these lipochitooligosaccharides in the rhizosphere is influenced by plant enzymes. NFs induce on pea (Pisum sativum) a particular extracellular NF hydrolase that releases lipodisaccharides from NFs from Sinorhizobium meliloti. Here, we investigated the ability of non-nodulating pea mutants to respond to NodRlv factors (NFs from Rhizobium leguminosarum bv viciae) with enhanced NF hydrolase activity. Mutants defective in the symbiotic genes sym10, sym8, sym19, and sym9/sym30 did not exhibit any stimulation of the NF hydrolase, indicating that the enzyme is induced via an NF signal transduction pathway that includes calcium spiking (transient increases in intracellular Ca 21 levels). Interestingly, the NF hydrolase activity in these sym mutants was even lower than in wild-type peas, which were not pretreated with NodRlv factors. Activation of the NF hydrolase in wild-type plants was a specific response to NodRlv factors. The induction of the NF hydrolase was blocked by a-amanitin, cycloheximide, tunicamycin, EGTA, U73122, and calyculin A. Inhibitory effects, albeit weaker, were also found for brefeldin A, BHQ and ethephon. In addition to this NF hydrolase, NFs and stress-related signals (ethylene and salicylic acid) stimulated a pea chitinase that released lipotrisaccharides from pentameric NFs from S. meliloti. NodRlv factors failed to stimulate the chitinase in mutants defective in the sym10 and sym8 genes, whereas other mutants (e.g. mutated in the sym19 gene) retained their ability to increase the chitinase activity. These findings indicate that calcium spiking is not implicated in stimulation of the chitinase. We suggest that downstream of Sym8, a stress-related signal transduction pathway branches off from the NF signal transduction pathway.Establishment of symbiosis between legumes and nitrogen-fixing rhizobia results in the formation of a new plant organ, the root nodule. Rhizobia enter the host plant usually through infection of root hairs. Bacteria within root hairs induce the formation of an infection thread that grows toward the dividing cortical cells. In a later symbiotic stage, rhizobia are released from branched infection threads into the developing nodule tissue and differentiate into nitrogen-fixing bacteroids. Nodule formation is controlled by perception of rhizobial nodulation signals: the Nod factors (NFs). Flavonoids from the host plant, in conjugation with the rhizobial activator protein NodD, induce rhizobial nodulation genes (nod, nol, and noe) that are required for NF synthesis. NFs are modified lipochitooligosaccharides, i.e. chitin oligomers linked with a fatty acid replacing the N-acetyl group on their nonreducing end (Perret et al., 2000;Ovtsyna and Staehelin, 2003). On host plants, NFs induce various responses at picomolar to nanomolar concentrations. These rapid changes include root hair curling (Hac; e.g. Heidstra et al., 1994), depolarization of plasma membranes (Ehrha...

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

“…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).…”

“…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).…”

Chitinase Gene Expression in Response to Environmental Stresses inArabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

“…Experimental evidence for chitinases acting as defense proteins was obtained using transgenic plants that overexpressed chitinases and exhibited higher resistance to pathogens. [5][6][7] Recently, however, chitinases have been recognized as acting in other plant physiological processes, such as growth and developmental regulation, [8][9][10] programmed cell death, 11) symbiosis, 12) and tolerance of various environmental stresses. [13][14][15][16] The diversity in the physiological functions of chitinases might be correlated with structural diversity.…”

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.…”

“…In another study, Nod factor cleaving pea enzymes were analyzed using Sinorhizobium meliloti Nod factors (NodSm) as substrates [59]. When intact roots, or protein extracts, were incubated with pentameric NodSm, two hydrolytic activities were observed.…”