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).