Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099

Abstract

Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the beta-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fixation regulator gene, nifA. The histochemical GUS assay showed that there was exclusive expression of acdS in mature root nodules. Two homologous nifA genes, mll5857 and mll5837, were found in the symbiosis island of M. loti and were designated nifA1 and nifA2, respectively. Quantitative reverse transcription-PCR demonstrated that nifA2 disruption resulted in considerably diminished expression of acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption slightly enhanced expression of the acdS transcripts and suppressed nifH to some extent. These results indicate that the acdS gene and other symbiotic genes are positively regulated by the NifA2 protein, but not by the NifA1 protein, in M. loti. The mode of gene expression suggests that M. loti acdS participates in the establishment and/or maintenance of mature nodules by interfering with the production of ethylene, which induces negative regulation of nodulation.

FIG. 1.

Physical and restriction enzyme map of the acdS, nifA1, and nifA2 genes and the flanking region in M. loti MAFF303099. The arrows represent open reading frames. (A) Construction of gusA reporter fused with the acdS gene. A single-crossover mutation between pPD3 containing an acdS-gusA fusion and the genomic DNA generated the ACD-GUS strain harboring an intact genomic acdS gene and an acdS-gusA fusion. (B) Generation of the nifA1 mutant. A double-crossover mutation between pKnifA1::Gm and genomic DNA generated the nifA1 mutant. (C) Generation of the nifA2 mutant. A double-crossover mutation between pKnifA2::Tc and genomic DNA generated the nifA2 mutant. B, BamHI; E, EcoRI; N, NdeI; Sa, SalI; Sm, SmaI; St, StuI.

FIG. 2.

GUS activity in M. loti MAFF303099 expressing gusA under control of the constitutively active aph promoter (ML-GUS) or the acdS promoter (ACD-GUS) in free-living conditions and during nodulation. Constitutively gusA-expressing strain ML-GUS (A, C, and E) was used as the positive control for ACD-GUS (B, D, and F). Free-living cell cultures (A and B) and root nodules on L. japonicus at 5 DAI (C and D) and 10 DAI (E and F) were incubated with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc). Invasion of cortical cells of nodule primordia by the ML-GUS strain in panel C is indicated by an arrowhead. On the other hand, ACD-GUS showed GUS activity exclusively within mature nodules (F). (C and D) Bars = 100 μm. (E and F) Bars = 300 μm.

FIG. 3.

Phenotypes of 56-day-old L. japonicus plants inoculated with ML-GUS (A), the nifA1 mutant (B), and the nifA2 mutant (C) on nitrogen-free medium. Root nodules cut in half are shown in the lower panels. Plants inoculated with the nifA1 mutant exhibited a wild-type phenotype (B), like ML-GUS-inoculated plants (A). The nifA2 mutant-inoculated plants had tiny white nodules on the roots and yellow leaves (C). Bars = 600 μm.

FIG. 4.

Quantitative real-time RT-PCR analyses for the acdS, nifH, nifA1, and nifA2 genes in M. loti MAFF303099 for roots at 10 DAI. The relative transcript level for each target gene was normalized to the gusA copy number, and the transcript levels for ML-GUS were defined as 1.0. The values are means of three independent experiments, and the error bars indicate standard errors. NT, not tested.