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

Abstract

Summary In the nodules of IRLC legumes, including Medicago truncatula, nitrogen‐fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule‐specific cysteine‐rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen‐fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence‐related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray‐ or transcriptome‐based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR‐new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF‐FN9363. We found that the expression of NCR‐new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein‐tagged version of NCR343 and NCR‐new35 localized to the symbiotic compartment. 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

“…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.…”

“…To increase the e cacy of targeted mutagenesis by CRISPR/Cas9, 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 e ciency 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 identi ed NCR gene which 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 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.…”