Class III chitinases in Medicago truncatula facilitate arbuscular mycorrhizal symbiosis with Rhizophagus intraradices

Abstract Plant chitinases are important defense enzymes in plant-microbe interactions and also participate in nodule symbiosis between legumes and nitrogen-fixing rhizobia. However, the roles of chitinases in arbuscular mycorrhizal (AM) symbiosis remain incompletely defined. In this study, we characterized two class III chitinase genes in the legume Medicago truncatula (ecotype R108), namely MtCHIT3e and MtCHIT3f. Heterologous expression in Nicotiana benthamiana confirmed hydrolytic activity toward colloidal chitin, a fluorogenic chitooligosaccharide substrate, chitooligosaccharides, and lipo-chitooligosaccharides. In M. truncatula roots inoculated with AM fungi, transcripts of the two genes were significantly upregulated. Analysis of M. truncatula hairy roots transformed with promoter–β-glucuronidase constructs indicated strong induction of MtCHIT3e and MtCHIT3f promoter activity in response to applied chitooligosaccharide and lipo-chitooligosaccharide signals. Furthermore, fluorescent protein fusions localized the chitinases predominantly to the apoplast of host cells during AM colonization. Tnt1 insertion mutants in the chitinase genes exhibited reduced AM colonization, whereas nodule formation induced by Sinorhizobium meliloti was unaffected. Together, these results support a role for class III chitinases in promoting AM symbiosis and provide genetic evidence that they are required for optimal root colonization by AM fungi.