MtERF75/74 regulates nodule number and size and nitrogen fixation
MtERF74/75 RNAi transgenic roots were analyzed for their ability to establish a symbiotic interaction. Transformed composite plants were grown in vitro and nodulation efficiency was analyzed after inoculation with S. meliloti . At two weeks post-infection, the nodule number per plants in the MtERF74/75 double knock-down roots was 30% reduced compared to the GUS RNAi control (Fig. 4a). At 3 weeks post-infection, furthermore, the N2 fixation capacity of mature nodules from MtERF74/75 RNAi transgenic roots was 52% reduced compared to the control (Fig. 4b), and a 17.6 % reduction in nodule size was observed in the MtERF74/75 RNAi transgenic roots (Fig. 4c).
To get the respective role of MtERF74 and MtERF75 in symbiotic interaction, we looked at the Medicago Tnt1 -insertion mutant population at the Samuel Roberts Noble Foundation. We identified one Tnt1 -insertion line for the MtERF74 . Unfortunately, no tnt1 insertion was available in MtERF75 gene. After validation by qPCR of the invalidation of the gene (Fig. S4A), the mutant lineMtERF74-1ko was backcrossed 4 times to clean the genetic background of this mutant. However, thisMtERF74-1ko mutant did not show any phenotype on root growth, nodulation, biomass production or nitrogen fixation (Fig. S4BCD). This absence of phenotype could be explained by a functional redundancy between the MtERF74 and MtERF75, even if the higher level of MtERF75 expression could also point for a more important role of MtERF75 in the process of nodulation (Fig. S2).
The strong impairment of N2 fixation by the double knockdown of MtERF74/75 (Fig. 4b) argues for a key role of these two TFs in the proper functioning of the nodule. In this context, the expression of different HRG genes was analyzed in 3 weeks post-inoculation control and MtERF74/75 transgenic nodules (Fig. 5). As already observed in the hypoxic roots (Fig. 3), the expression ofADH1 , PDC1 , Pgb1.1 , and ERF73 was affected in the nodules of the double knockdown root with a reduction of 77%, 40%, 39%, and 35%, respectively, compared with the control. Expression of VPE and CP6 , which are markers of nodule senescence (Pierre et al., 2014), and leghemoglobin4(Lb4 ), which is a marker of N2 fixation (Berger et al., 2020b), was unaffected in the transgenic MtERF74/75 RNAi nodules.
The expression of NR1 , but not NR2 , was greatly reduced in the double knockdown nodules, by almost 80%. Knowing the importance of the activity of NR for the production of NO (Berger et al., 2020a), we also examined the expression of marker genes that are regulated in function of NO level in M. truncatula . The expression of a glutathione S-transferase (GST ), whose expression is associated with the defense response during nodule organogenesis (Boscari et al., 2013), was reduced by 40% in MtERF74/75 RNAi transgenic nodules (Fig. 5). Analysis of NO production showed a 32% reduction in NO emission by transgenic nodules compared with the control (Fig. 6).