An extracellular β‐N‐acetylhexosaminidase of Medicago truncatula hydrolyzes chitooligosaccharides and is involved in arbuscular mycorrhizal symbiosis but not required for nodulation

Abstract

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 of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis. We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N‐acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants. 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.

CRISPR/Cas9-Mediated Generation of Mutant Lines in Medicago truncatula Indicates a Symbiotic Role of MtLYK10 during Nodule Formation

“…It is worth mentioning in this context that M. truncatula mutants carrying a Tnt1 retrotransposon in a β-N-acetylhexosaminidase gene (MtHEXO2) exhibit reduced colonization by AM fungi. MtHEXO2 is an extracellular enzyme that releases N-acetylglucosamine from fungal chitooligosaccharide signals [69]. Future research will reveal whether MtMFS1 and MtMFS2 are N-acetylglucosamine transporters and whether they are required for the establishment of AM symbiosis.…”

“…In previous studies, pISV2678, a binary vector with a Bar gene, was found to be very effective for the A. tumefaciens-mediated transformation of M. truncatula leaf explants [41,[67][68][69]. We therefore aimed to construct a CRISPR/Cas9 vector with a pISV2678 backbone and a DsRed1 expression cassette for the visual confirmation of transformants.…”