RNAi Knock-Down of ENOD40 s Leads to Significant Suppression of Nodule Formation in Lotus japonicus

Abstract

ENOD40 is one of the most intriguing early nodulin genes that is known to be induced very early in response to interaction of legume plants with symbiotic Rhizobium bacteria, but its function in the nodulation process is still not known. Lotus japonicus has two ENOD40 genes: LjENOD40-1 is abundantly induced in very early stages of bacterial infection or Nod factor application, whereas LjENOD40-2 is abundantly expressed only in mature nodules. We generated transgenic lines of L. japonicus with an RNAi (RNA interference) construct that expresses hairpin double-stranded RNA for LjENOD40-1 to induce sequence-specific RNA silencing. In the transgenic plants, expression of both LjENOD40-1 and -2 was significantly reduced, and no accumulation of ENOD40 transcripts was detected upon Mesorhizobium loti inoculation. The transgenic plants exhibited very poor nodulation (only 0-2 nodules per plant) and could not grow well without additional nitrogen supply. Analysis of segregation in the T(2) progeny indicated that the suppression of nodulation is perfectly linked with the presence of the transgene. Microscopic observation of the infection process using lacZ-labeled M. loti, together with expression analysis of infection-related nodulin genes, demonstrated that ENOD40 knock-down did not inhibit the initiation of the bacterial infection process. In contrast, nodule primordium initiation and subsequent nodule development were significantly suppressed in the transgenic plants. These results clearly indicate that ENOD40 is required for nodule initiation and subsequent organogenesis, but is not involved in early infection events.

ENOD40Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant,Masym3, ofMelilotus albaDesr.

“…32 RNAi experiments in Lotus japonicus demonstrated that ENOD40 knockdown inhibited nodule primordium development, but not rhizobial infection. 33 A similar reduction in nodule formation was found for alfalfa transgenic plants with reduced ENOD40 levels brought about by cosuppression. 28 Earlier, we showed that when an alfalfa Nod -mutant (mutated in MsNORK/MtDMi2) was treated with the cytokinin 6-benzylaminopurine, ENOD40 gene expression was up-regulated, demonstrating that application of this phytohormone could bypass the Nod factor-triggered signal transduction pathway.…”

A Ubiquitin Ligase of Symbiosis Receptor Kinase Involved in Nodule Organogenesis

“…To investigate if nodulin gene expression was affected by SIE3 RNAi, we examined the expression levels of NIN and ENOD40-2, two early nodulin genes implicated in the processes of rhizobial entrance, nodule initiation, and subsequent organogenesis (Schauser et al, 1999;Kumagai et al, 2006), as well as the expression of Lb (for leghemoglobin), a typical nodulin gene required for nitrogen fixation (Ott et al, 2005). Real-time PCR analysis showed that the expression levels of the two early nodulin genes (NIN and ENOD40-2), but not Lb, were reduced drastically in the SIE3 RNAi hairy roots, as compared with those in the control hairy roots ( Fig.…”

“…We scored IT numbers in SIE3 RNAi hairy roots 8 d after rhizobial inoculation. ITs were visible as blue threads under a microscope after staining with 5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside acid (X-gal) that reacted with the lacZ-labeled M. loti cells (Tansengco et al, 2003;Kumagai et al, 2006). ITs were divided into four groups according to the cellular locations of their growing tips: 1) those emerging from curled root hairs, 2) those growing from elongated root hairs to the root epidermis, 3) those penetrating through the root cortex, and 4) those reaching to the nodule primordia, as shown in Supplemental Figure S7.…”

A MYB-related transcription factor affects nodule formation in <i>Lotus japonicus</i>

“…Primers for the Enod40-1, Enod40-2, and Nin primers were described previously (Kumagai et al 2006). The Ljmate1 primers were described by Takanashi et al (2013).…”