NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula

Abstract

Summary Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen‐fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen‐reducing activities remains elusive. Here, through mutant screen, map‐based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA‐Seq assay shows that many stress‐responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response‐related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

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

“…Selecting of NCR genes to be targeted by CRISPR/Cas9 editing The M. truncatula genome contains a large gene family coding for more than 700 NCR peptides but only few of them, NCR169, NCR211, NCR247, NCR343 and NCR-new35, have been proved to be crucial for symbiosis [10][11][12][13][14][15] . The sequences of the essential NCR genes are highly variable, and their common feature is that they contain four cysteine residues in conserved positions.…”

“…NCRs compose a large gene family with more than 700 members 9 in M. truncatula and because this large number of NCRs, their function was thought to be redundant. The identi cation of few individual NCRs crucial for nitrogen-xing symbiosis disproved this concept [10][11][12][13][14][15] . These essential NCRs except for NCR247, which was analysed by reverse genetic approach, were identi ed fortuitously using deletion mutant collections of M. truncatula.…”

“…The removal of variable number of non-transgenic roots from each plant and the potential presence of root cells with non-edited genome did not enable the reliable analysis of the nitrogen starvation phenotype (stunted growth, yellow leaves) of the shoots of transformed plants. The nodules of all the previously identi ed mutants defective in essential NCRs were white and slightly elongated and showed defects in colonization of the region corresponding to the nitrogen xation zone 10,11,[13][14][15] . Therefore, we assessed the symbiotic phenotype of genome edited nodules mostly based on their size, colour, and the colonization of the nitrogen xation zone.…”

“…Although these high number of peptides show remarkable amino acid sequence variation, their function was initially presumed redundant. Nevertheless, recent studies have identi ed 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-xing symbiosis [10][11][12][13][14][15] . The identi cation of most of these essential peptides was random during the forward genetic analyses of mutagenized M. truncatula populations.…”

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

“…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.…”