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Summary Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo‐chitooligosaccharides (LCOs). The latter are also produced by nitrogen‐fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown.Here, we analyzed the expression of a β‐N‐acetylhexosaminidase gene ofMedicago truncatula(MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role ofMtHEXO2during symbiosis.We found that expression ofMtHEXO2is associated with AM symbiosis and nodulation.MtHEXO2expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs.M. truncatulamutants deficient in symbiotic signaling did not show induction ofMtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs intoN‐acetylglucosamine (GlcNAc).Hexo2mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected inhexo2mutants.In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal. 

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. InMedicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors ofSinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b ofM. truncatulaexpressed inEscherichia colican release lipodisaccharides from Nod factors. Analysis ofM. truncatulamutant plants indicated that MtCHIT5b, together with MtNFH1, degradesS. melilotiNod factors in the rhizosphere.MtCHIT5bexpression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of aMtCHIT5bknockout mutant was not significantly altered whereas overexpression ofMtCHIT5bresulted in fewer nodules. Reduced nodulation was observed whenMtCHIT5bandMtNFH1were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation ofM. truncatulais regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.