A positive regulatory role for LjERF1 in the nodulation process is revealed by systematic analysis of nodule-associated transcription factors of Lotus japonicus

Abstract

We have used reverse genetics to identify genes involved in legume-rhizobium symbiosis in Lotus japonicus. We obtained the sequences of 20 putative transcription factors from previously reported large-scale transcriptome data. The transcription factors were classified according to their DNA binding domains and patterns of expression during the nodulation process. We identified two homologues of Medicago truncatula MtHAP2-1, which encodes a CCAAT-binding protein and has been shown to play a role in nodulation. The functions of the remaining genes in the nodulation process have not been reported. Seven genes were found to encode proteins with AP2-EREBP domains, six of which were similar to proteins that have been implicated in ethylene and/or jasmonic acid signal transduction and defense gene regulation in Arabidopsis (Arabidopsis thaliana). We identified a gene, LjERF1, that is most similar to Arabidopsis ERF1, which is up-regulated by ethylene and jasmonic acid and activates downstream defense genes. LjERF1 showed the same pattern of up-regulation in roots as Arabidopsis ERF1. The nodulation phenotype of roots that overexpressed LjERF1 or inhibited LjERF1 expression using an RNA interference construct indicated that this gene functions as a positive regulator of nodulation. We propose that LjERF1 functions as a key regulator of successful infection of L. japonicus by Mesorhizobium loti.

Figure 1.

Phylogenetic tree of nodulation-associated L. japonicus AP2-EREBP genes. Phylogenetic analysis was carried out using the sequences of the AP2-EREBP domains of the L. japonicus (Lj) genes and their Arabidopsis (At) homologues. Locus identifiers (such as At1g22190) are shown for the Arabidopsis genes. Arabidopsis RAP2.11 and M. truncatula MtERN1 were included as an outgroup.

Figure 2.

Effects of ethylene (ET) and/or JA on the expression of AP2-EREBP genes in shoots and root. The expression levels of the indicated L. japonicus AP2-EREBP genes in shoots (light gray) and root (dark gray) are expressed relative to controls. Controls are either nontreated shoots or root. Data represent averages ± se of three independent experiments. The y axis in each bar graph indicates the relative transcript level.

Figure 3.

Nodulation phenotype of LjERF1-specific RNAi-transformed hairy roots. A, Two constructs targeting different regions of LjERF1 (RNAi-1 and RNAi-2) were used to transform plants. B, Plants were inoculated with M. loti and grown in the absence of nitrogen. Photographs were taken 3 weeks after infection and show the growth phenotype of plants that were transformed with an empty vector (control), RNAi-1, or RNAi-2. C, Fluorescence microscopy of transformed hairy roots. The formation of nodules on control hairy roots was evident, but nodules were observed only in nontransformed hairy roots in the RNAi plants, as distinguished by GFP fluorescence. D, The growth of plants in which the expression of LjERF1 was suppressed by RNAi was similar to that of control plants in the presence of a nitrogen source.

Figure 4.

LjERF1 promoter∷GUS assay after M. loti inoculation. A, Histochemical GUS staining was observed in an epidermal region of the LjERF1 promoter∷GUS transgenic hairy root at 3 h after M. loti inoculation. B and C, GUS staining at 10 d after M. loti inoculation indicated that LjERF1 is induced in epidermal and cortical regions where no morphological changes for nodulation were observed (B) but not in the nodule primordia or mature nodules (C). Bars = 0.1 mm.

Figure 5.

Expression of LjPR10-1 examined by RT-Q-PCR. A, Expression of LjERF1 and LjPR10-1 in response to infection by wild-type and noninfectious mutant strains of M. loti. B, Expression of LjPR10-1 in nodule-containing roots that overexpress LjERF1 (overexpressor) or nonnodulating roots in which the expression of LjERF1 was suppressed by RNAi at 3 weeks after M. loti inoculation. The y axes indicate relative expression levels compared with those in control plants.