CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions

Abstract

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume-rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

Keywords: CLE peptides; autoregulation of nodulation (AON); nitrate; nodulation; rhizobia.

Figure 1

Alleles of the MtCLE35 gene identified in crispr-1, crispr-2, crispr-6 and cripr-20 plants due to the CRISPR/Cas9-mediated editing of the MtCLE35 gene.. In nucleotides sequences, each color represents a different nucleotide (A, T, C or G). PAM (protospacer adjacent motif) is shown with a red box. The target sequence is shown with a bracket. Low panel, genotypes of T0 and T1 plants: “+1” indicates 1-bp insertion within the MtCLE35 CDS, whereas −1, −5 and −15 correspond to deletions of 1, 5 and 15 nucleotides, respectively. For T1 plants, fractional numbers indicate the fraction of each genotype observed among the total number of progenies derived from the same T0 plant. “Hetero” and “homo” correspond to heterozygotes and homozygotes, respectively.

Figure 2

Box plots showing the number of nodules at 28 dpi (days post inoculation) in wild-type (R108) and MtCLE35 knock-out (crispr-1 and crispr-6) plants grown without nitrate addition. No statistical difference was found in the nodule numbers of these three genotypes (Wilcoxon test, n = 10–16).

Figure 3

Box plots showing the number of nodules at 28 dpi in wild-type (R108) and MtCLE35 knock-out (crispr-1 and crispr-6) plants grown in the presence of 10 mM KNO₃. ** p < 0.01, *** p < 0.001 (Wilcoxon test, n = 15–19).

Figure 4

Expression levels of MtCLEs in the roots after inoculation with rhizobia at 7 dpi; plants were grown in nitrogen-free (no nitrate) media or in the presence of 10 mM KNO₃. Relative expression levels were normalized to 1, relative to the control plants grown without N (no nitrate), as indicated by the dotted lines. Results are mean ± SD of 5 biological repeats. Asterisks mark statistically significant differences (ns—not significant, ** p < 0.01; *** p < 0.001, Student t-test, n = 5). p-values are indicated above the respective comparison.

Figure 5

A model explaining the increased number of nodules found in MtCLE35 knock-out plants in comparison to wild-type plants under high-nitrate conditions. The dotted line indicates an indirect effect. In the absence of nitrate (left panels), legume–rhizobia interaction results in the formation of symbiotic nodules. Rhizobia induce a signaling cascade leading to the activation of key regulators of symbiosis. Among them, the expression of the NIN gene is induced, which encodes a key transcription factor regulating both rhizobia infection and nodule primordium development. The NIN transcription factor activates the expression of the CLE genes in response to rhizobia inoculation, including MtCLE12, MtCLE13 and MtCLE35. As a result, the AON system is activated by root-to-shoot transported MtCLE peptides, activating the MtSUNN receptor kinase, which operates in the phloem cells of leaves. In turn, a shoot-derived signaling pathway is induced to inhibit nodulation via a negative feedback mechanism. Knock-out of the MtCLE35 gene does not significantly reduce the induction of AON since MtCLE35 acts redundantly with MtCLE12 and MtCLE13 to inhibit nodulation, and, therefore, the nodule number is not significantly increased in MtCLE35 knock-out plants. The presence of nitrate (left panels) downregulates symbiotic nodulation. One of the known mechanisms of such inhibition is mediated by the nitrate-activated NLP1 transcription factor, which has been suggested to inhibit nodulation by interfering with the NIN action. As a result, the expression levels of NIN-target genes, including MtCLE12 and MtCLE13, are decreased. However, the expression of the MtCLE35 gene is not significantly reduced in developing nodules under high-nitrate conditions since the nitrate-activated NLP1 transcription factor is able to induce MtCLE35 expression. Therefore, in the presence of nitrate, MtCLE35 acts as the key inductor of AON since the expression levels of two other MtCLE genes, MtCLE12 and MtCLE13, are significantly decreased. Thereby, knock-out of the MtCLE35 gene results in a significantly increased nodule number in the presence of nitrate.