Tml1 and tml2 synergistically regulate nodulation but not arbuscular mycorrhiza in medicago truncatula.

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of Nodulation (AON) and Autoregulation of Mycorrhization (AOM) share multiple components - plants that make too many nodules usually have higher arbuscule density. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus. M. truncatula has two sequence homologs: MtTML1 and MtTML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in either gene produced twice the nodules of wild type plants whereas plants containing mutations in both genes displayed a synergistic effect, forming 20x more nodules and short roots compared to wild type plants. The synergistic effect on nodulation was maintained in the presence of 10mM nitrogen, but not observed in root length phenotypes. Examination of expression and heterozygote effects suggest genetic compensation may play a role in the observed synergy. However, plants with mutations in both TMLs had no detectable change in arbuscular mycorrhizal associations, suggesting that MtTMLs are specific to nodulation and nitrate signaling. The mutants created will be useful tools to dissect the mechanism of synergistic action of MtTML1 and MtTML2 in M. truncatula nodulation as well as the separation of AON from AOM.