The Medicago truncatula hydrolase MtCHIT5b degrades Nod factors of Sinorhizobium meliloti and cooperates with MtNFH1 to regulate the nodule symbiosis

Abstract

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression 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 a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

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

“…LCOs were not cleaved by MtHEXO2, which is consistent with observations that GH20 enzymes are exo-glycosidases that release terminal GlcNAc or GalNAc from the nonreducing end of oligosaccharides (Liu et al, 2018;Drula et al, 2022). In contrast to chitobiose, sulfated chitobiose, that is, the hydrophilic cleavage product of the LCO-cleaving hydrolases MtNFH1/MtCHIT5b (Tian et al, 2013;Cai et al, 2018;Li et al, 2022), was not hydrolyzed by MtHEXO2 under our test conditions, suggesting that sulfated chitobiose accumulates in the rhizosphere of M. truncatula or is hydrolyzed by an unknown enzyme. In this context, it is worth mentioning that certain rhizobia and fungi produce LCOs without reducing end modifications (Spaink et al, 1991;Maillet et al, 2011;Rush et al, 2020).…”

“…6(a–c) indicate co‐localization of MtHEXO2:GFP and mCherry‐expressing S. meliloti bacteria in the infection pocket of three independent curled root hairs. Focal exocytosis of proteins into the infection pocket of curled root hairs was previously reported for various symbiosis‐related proteins, including MtNFH1/MtCHIT5b (Fournier et al ., 2015; Cai et al ., 2018; Li et al ., 2022). In conclusion, our subcellular localization analysis indicates that MtHEXO2 is an extracellular enzyme which comes into direct contact with molecules secreted by AM fungi and rhizobia.…”

“…Too strong and continuous stimulation of the CSSP could eventually result in autoregulation responses that negatively affect the AM symbiosis (Staehelin et al ., 2011). In this view, the signal‐inactivating function of MtHEXO2 during the AM symbiosis would be a feedback response, which is similar to the rhizobial LCO inactivation by MtNFH1/MtCHIT5b in the nodule symbiosis (Cai et al ., 2018; Li et al ., 2022). MtHEXO2 can hydrolyze long‐chain COs such as chitohexaose and chitoheptaose, thereby transiently producing short‐chain COs (Figs 3, S14).…”

“…Overexpression of the LCO‐cleaving hydrolase gene MtCHIT5b resulted in reduced nodulation of M. truncatula roots (Li et al ., 2022). However, overexpression of MtHEXO2 did not affect root colonization by AM fungi in this study.…”

“…Plant chitinases (glycoside hydrolase families 18 and 19) not only hydrolyze chitin and COs (Drula et al ., 2022) but also may have the capacity to degrade and inactivate LCOs (Staehelin et al ., 1994a,b; Goormachtig et al ., 1998; Schultze et al ., 1998; Ovtsyna et al ., 2000; Tian et al ., 2013; Cai et al ., 2018; Li et al ., 2022). Mutant analysis revealed a symbiotic role of the LCO‐cleaving family 18 glycoside hydrolases MtNFH1/MtCHIT5b in M. truncatula and LjCHIT5 in Lotus japonicus , indicating that concentration and distribution of rhizobial LCOs are under precise control during nodulation (Cai et al ., 2018; Malolepszy et al ., 2018; Li et al ., 2022). COs/LCOs of AM fungi are believed to be inactivated by host enzymes to maintain optimal signal concentration and to avoid chitin‐triggered induction of defense responses.…”