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

Abstract

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.

Lipo‐chitooligosaccharide signalling blocks a rapid pathogen‐induced ROS burst without impeding immunity

“…Regulation of LCO perception involves their degradation by host hydrolases in the rhizosphere and symbiotic tissues (Tian et al, 2013;Cai et al, 2018;Malolepszy et al, 2018). This might explain why diverse doses of LCOs trigger similar symbiotic responses and Fig.…”

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

“…As discussed below, induced genes involved in early auxin responses and SL and GA biosynthesis were all conserved between soybean and M. truncatula. Also conserved was the induction of the SWEET13 sugar transporter and the Nod factor hydrolase1 (NFH1), the latter encoding an enzyme involved in specifically inactivating Nod factors in the rhizosphere (Tian et al, 2013). Other genes identified as being induced in both species include those encoding an expansin, peroxidases, proteases, pectinesterases, and pectinesterase inhibitors.…”

“…The BCP1 promoter induces expression in cells containing arbuscules (Hohnjec et al, 2005), but our data suggest that BCP1 may not be specific to mycorrhizal infection. Other genes induced were NFH1 (Tian et al, 2013), a vestitone reductase homolog (designated Vestitone Reductase Cluster2 [VRC2]), two ABC transporters (ABC1 and ABC2), and the ortholog of Nucleolar/ Mitochondrial protein involved in Nodulation (NMN1; Libault et al, 2011). Further details on these genes are provided in Supplemental Data Set 4 (Infection Genes).…”

“…NFH1 is predicted to be secreted and has been shown to specifically degrade Nod factors (Tian et al, 2013) and so NFH1 induction could limit or interrupt the flavonoid-Nod factor positive feedback loop described above. Similar induction of NFH1 occurs in soybean (Supplemental Data Set 3, Conserved Gm).…”

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…meliloti strain 1021 (pEK327) (Schultze et al, 1992) was used for HPLC purification of NodSm-IV(C16:2, S) as described (Tian et al, 2013).…”