TheMedicago truncatulanodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules

Abstract

The Medicago truncatula-Sinorhizobium sp. symbiosis is one of the most effective nitrogen-fixing interaction. In the Inverted Repeat-Lacking Clade (IRLC) legumes, including M. truncatula, rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialised for nitrogen fixation. This irreversible transition of rhizobia is controlled by the host plant and mediated by nodule-specific cysteine-rich (NCR) peptides, of which about 700 are encoded by a large gene family in the M. truncatula genome. Recent studies have demonstrated that some NCR peptides, NCR169, NCR211 and NCR247, are unique and essential for nitrogen-fixing symbiosis. By analysing the symbiotic phenotype of ineffective M. truncatula mutants, Mtsym19, Mtsym20 and NF-FN9363, we found that the nodulation phenotype, the bacteroid and symbiotic host cell differentiation were defective likewise in the formerly studied ncr mutants, Mtdnf-1 (ncr211) and Mtdnf7-2 (ncr169). We demonstrated that the incomplete differentiation of bacteroids triggers premature senescence of rhizobia in the nitrogen fixation zones of mutant nodules. We proved that Mtsym19 and Mtsym20 mutants are allelic and the MtSYM19/20 gene encodes the peptide NCR-new35. We identified a deletion of four NCR genes in the symbiotic locus of NF-FN9363 and demonstrated that the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. The activity of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCR genes, NCR169, NCR211 and NCR343 which are highly expressed in the interzone and nitrogen fixation zone. The fluorescent protein-tagged version of NCR343 and NCR-new35 localize to the symbiotic compartment. We also demonstrated that the conserved first cysteine residues are required for the symbiotic function of NCR343 and NCR-new35. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

“…S2f-h, n-p). The morphology and bacterial occupancy of genome edited nodules were in agreement with the nodulation phenotype of Mtsym19 and Mtsym20 nodules defective in NCR-new35 13 . These results also revealed that NCR-new35 is crucial for the symbiotic interactions not only between M. truncatula 2HA and S. medicae WSM419 but between M. truncatula R108 and S. meliloti FSM-MA as well.…”

“…Therefore, the Arabidopsis U6-26 promoter was replaced with MtU6.6 in the pKSE401-RR resulting in the vector pKSE466-RR. We inserted the sgRNAs NCR169a and another one, targeting the NCR-new35, of which requirement for effective symbiosis has been recently demonstrated 13 , into pKSE466-RR, respectively. To validate the efficiency of the pKSE466-RR vector construct, the empty vector and pKSE466-RR-NCR169a were introduced into wild-type M. truncatula 2HA plants using A. rhizogenes-mediated hairy root transformation and plants were inoculated with S. medicae WSM419.…”

“…We also applied the developed CRISPR/Cas9 toolkit to target NCR-new35, a recently identified NCR gene that is crucial for the persistence of bacteroids in M. truncatula cv. Jemalong nodules 13 . The empty vector and the pKSE466-RR-NCRnew35 constructs were introduced into the roots of M. truncatula 2HA and M. truncatula ecotype R108 plants using A. rhizogenes-mediated hairy root transformation.…”

“…The M. truncatula genome contains a large gene family coding for more than 700 NCR peptides but only a few of them, NCR169, NCR211, NCR247, NCR343 and NCR-new35, have been proven to be crucial for symbiosis [10][11][12][13][14][15] . Apart from the fact that these peptides contained four cysteine residues in conserved positions, no other common features could be identified 10,11,13 . Therefore, it was not straightforward to ascertain which additional NCR peptides are essential and should be targeted.…”

“…Although this high number of peptides show remarkable amino acid sequence variation, their function was initially presumed redundant. Nevertheless, recent studies have identified some peptides, NCR169, NCR211, NCR247, NCR343 and NCR-new35, crucial for the bacteroid differentiation and persistence in M. truncatula nodules but also found peptides (NCR178, NCR341, NCR344 and NCR345) which are not essential for nitrogen-fixing symbiosis [10][11][12][13][14][15] . The identification of most of these essential peptides was random during the forward genetic analyses of mutagenized M. truncatula populations.…”