A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation

The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1 -insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs 1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M . truncatula Cystathionine - β - Synthase-like1 ( MtCBS 1 ), using a promoter-β -glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca 2+ responses were observed in the cbs 1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS 1 expression occurred exclusively during Medicago -rhizobium symbiosis. Induction of MtCBS 1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS 1 , MtCBS 2 , was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall.