Expression of the CLE-RS3 gene suppresses root nodulation in Lotus japonicus

Abstract

Cell-to-cell communication, principally mediated by short- or long-range mobile signals, is involved in many plant developmental processes. In root nodule symbiosis, a mutual relationship between leguminous plants and nitrogen-fixing rhizobia, the mechanism for the autoregulation of nodulation (AON) plays a key role in preventing the production of an excess number of nodules. AON is based on long-distance cell-to-cell communication between roots and shoots. In Lotus japonicus, two CLAVATA3/ESR-related (CLE) peptides, encoded by CLE-ROOT SIGNAL 1 (CLE-RS1) and -RS2, act as putative root-derived signals that transmit signals inhibiting further nodule development through interaction with a shoot-acting receptor-like kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1). Here, an in silico search and subsequent expression analyses enabled us to identify two new L. japonicus CLE genes that are potentially involved in nodulation, designated as CLE-RS3 and LjCLE40. Time-course expression patterns showed that CLE-RS1/2/3 and LjCLE40 expression is induced during nodulation with different activation patterns. Furthermore, constitutive expression of CLE-RS3 significantly suppressed nodule formation in a HAR1-dependent manner. TOO MUCH LOVE, a root-acting regulator of AON, is also required for the CLE-RS3 action. These results suggest that CLE-RS3 is a new component of AON in L. japonicus that may act as a potential root-derived signal through interaction with HAR1. Because CLE-RS2, CLE-RS3 and LjCLE40 are located in tandem in the genome and their expression is induced not only by rhizobial infection but also by nitrate, these genes may have duplicated from a common gene.

Legume nodulation: The host controls the party

“…Regulation of the Lotus japonicus genes occurs via the transcription factor, nodule inception (NIN; Soyano, Hirakawa, Sato, Hayashi, & Kawaguchi, ), although it is not clear whether NIN is responsible for the subtle temporal differences in the expression patterns between the rhizobia‐induced genes (Okamoto et al, ; Mortier et al, ; Reid, Ferguson, & Gresshoff, ; Ferguson et al, ). In L. japonicus , a third gene encoding for a rhizobia‐induced CLE peptide has also been identified ( LjCLE‐RS3 ; Nishida, Handa, Tanaka, Suzaki, & Kawaguchi, ), with LjCLE5 , MtCLE34 , and MtCLE35 also potentially having a role in nodulation control (although the former two are likely pseudogenes, rendering them non‐functional; Hastwell, de Bang, Gresshoff, & Ferguson, ).…”

“…LjNRSYM1 acts locally in the root and not only controls nodule numbers but also regulates nitrogen fixation activity and nodule size in pathways that are independent of LjHAR1 (Nishida et al, ). Other CLE peptides of L. japonicus are also reported to be induced by nitrate (e.g., LjCLE40 ; Nishida et al, ), but their role, if any, in nodulation is unknown. Although no specific nitrate‐induced CLE peptide has been characterized in M. truncatula , an orthologue of NIC1 was recently identified bioinformatically (Hastwell et al, ).…”

Hormone modulation of legume‐rhizobial symbiosis

“…The expression of CLE peptides or prepropeptides genes, such as LjCLE‐RS1 and 2 in L. japonicus (Okamoto et al ; Magori and Kawaguchi ), MtCLE12 and 13 in M. truncatula (Mortier et al ; Saur et al ), and GmRIC1 and 2 in soybean (Lim et al ) and PvRIC1 and 2 in P. vulgaris (Ferguson and Mathesius ), was induced by NFs or rhizobia in roots. Over‐expression of MtCLE12 and MtCLE13 in M. truncatula (Mortier et al ), LjCLE‐RS3 in L. japonicus (Nishida et al ) and GmRIC1 in soybean (Lim et al ) inhibited or even abolished nodule formation, which indicate that CLE peptides serves as negative regulators of nodule formation.…”

Triarabinosylation is required for nodulation‐suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum

“…The AON pathway begins in response to initial rhizobia infection events, with the production of CLAVATA3/endosperm surrounding region‐related (CLE) peptides. In soybean, these peptides are GmRIC1 and GmRIC2 (Reid, Ferguson, & Gresshoff, ), with orthologues in other legumes having also been identified (Ferguson et al, ; Mortier et al, ; Nishida, Handa, Tanaka, Suzaki, & Kawaguchi, ; Okamoto et al, ; Reid, Ferguson, & Gresshoff, ). Although there is no clear distinction between the biological role of GmRIC1 and GmRIC2, there is some temporal separation in their expression patterns (Reid, Ferguson, & Gresshoff, ).…”

“…This nitrate regulation of nodulation pathway begins with the production of nitrate‐induced CLE peptides (called GmNIC1a and its duplicate GmNIC1b in soybean) that are perceived by the GmNARK receptor located in the root (Lim, Lee, Lee, & Hwang, ; Reid, Ferguson, & Gresshoff, ). CLE peptides induced by nitrate to regulate nodulation have not been reported in most other legumes, with the exception of Lotus japonicus where the rhizobia‐induced CLE peptides LjCLE‐RS2 , LjCLE‐RS3 , and LjCLE40 are reported to exhibit increased expression with nitrate application (Nishida et al, ; Okamoto et al, ).…”