MtEFD and MtEFD2: Two transcription factors with distinct neofunctionalization in symbiotic nodule development

Abstract

Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. MtEFD (Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION) encodes an AP2/ERF (APETALA2/ETHYLENE RESPONSIVE FACTOR) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the MtRR4 (RESPONSE REGULATOR 4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a non-legume homologue of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralogue MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, CRISPR-Cas9 mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with down-regulation of a set of genes, including notably MtNF-YA1 (NUCLEAR FACTOR-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We therefore conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.

The Medicago truncatula nodule‐specific cysteine‐rich peptides, NCR343 and NCR‐new35 are required for the maintenance of rhizobia in nitrogen‐fixing nodules

“…The identified deletions in mutants Mtsym19 and Mtsym20 overlapped by c. 32 kb removing three genes including the gene NCR-new35, formerly termed NCR014 (de Bang et al, 2017) but renamed according to the recent genome release of M. truncatula (Mt5.0; Figs 4b, S7b; Jardinaud et al, 2022), which made NCR-new35 the best candidate for MtSYM19 and MtSYM20. To confirm that the deletion of NCR-new35 caused the ineffective symbiotic phenotype of Mtsym19 and Mtsym20, roots of the two mutants were transformed with the WT NCR-new35 gene and inoculated with S. medicae WSM419 (pXLGD4).…”

Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes

“…4d ), this symbiotic stage also involved genes that are part of the ancestral transcriptomic response. Suppressors of plant defence in nodules, SymCRK 67 and RSD 73 , as well as the transcription regulator EFD required for both plant and bacteroid differentiation in M. truncatula 74 , 75 participate in this ancestral response. Looking specifically at the M. pudica data, we found VAPYRIN , RPG , some flotillin and remorin genes and the syntaxin SYN , which are well-known infection-associated genes 41 , 76 – 78 .…”

High endoreduplication after drought-related conditions in haploid but not diploid mosses

“…Some of these transcripts belonged to the transcription factor families AP2/ERF, MYB and HSF. Interestingly, these families have been implicated in endoreduplication and endocycle control ( Tominaga et al , 2008 ; Zhu et al , 2009 ; Dubois et al , 2013 ; Rotelli et al , 2019 ; Ding et al , 2022 ; Jardinaud et al , 2022 ; Nomoto et al , 2022 ). Thus, the association between endopolyploidy and desiccation tolerance seems obvious.…”