Inhibitors of ethylene synthesis or its physiological function enhanced nodulation in Lotus japonicus and Macroptilium atropurpureum. In contrast, the application of 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, reduced the nodule number in these legumes. These results suggest that an ethylene-mediated signaling pathway is involved in the nodulation process even in the determinate nodulators.

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Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.The symbiotic interactions between a legume and (brady) rhizobia result in a unique, nitrogen-fixing plant organ, the nodule. Recent studies have shown that the phytohormone ethylene inhibits nodule formation in some legumes (8,9,16,24,25). Application of 1-aminoocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, inhibits nodulation in Medicago truncatula (24).Rhizobitoxine [2-amino-4-(2-amino-3-hydropropoxy)-transbut-3-enoic acid] is an ethylene synthesis inhibitor that is produced by the legume symbiont Bradyrhizobium elkanii (15, 17-19, 22, 39). It is thought that production of this compound enhances nodulation of the host legume because of its inhibitory effect on ethylene synthesis. However, some reports have shown that there is not a significant difference in nodule number between plants inoculated with B. elkanii USDA61 and plants inoculated with rhizobitoxine-deficient mutants during nodulation of Glycine max, Glycine soja, Vigna unguiculata, and Macroptilium atropurpureum (26,39). Recently, Duodu et al. observed a significant difference in nodule number between plants inoculated with isogenic variants of USDA61 during nodulation of Vigna radiata (7). Although these findings do not seem to be consistent with the hypothesis that rhizobitoxine has a positive effect on nodulation, the inconsistency can be explained by differences in the ethylene sensitivity of nodulation among leguminous species; nodulation of G. max is generally not sensitive to ethylene (10, 31, 38), while nodulation of V. radiata is sensitive (7). The inconsistency could also result from differences in the abilities of the strains used in the experiments to produce rhizobitoxine; strain USDA61 is a weak producer of rhizobitoxine (39).In addition to G. max, the leguminous plant M. atropurpureum is a nodulating host for B. elkanii and Bradyrhizobium japonicum (12,15). Although the effect of ethylene on nodulation has been studied in many leguminous host plants so far, the effect of ethylene in M. atropurpureum is not known. B. elkanii was found to be more competitive than B. japonicum for nodulation of M. atropurpureum in a multistrain environment when a field soil was inoculated with a mixture of several strains isolated from the field soil (21). In general, B. elkanii accumulates rhizobitoxine in cultures and in nodules, while B. japonicum does not (5,15,18,19). These results led us to investigate the role of rhizobitoxine production on the nodulation and competitiveness of B. elkanii on M. atropurpureum by using a B. elkanii strain that produces...

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A largedeletion mutant of B. elkanii, USDA94⌬rtx::⍀1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94⌬rtx::⍀1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.Rhizobitoxine [2-amino-4-(2-amino-3-hydropropoxy)-transbut-3-enoic acid] is synthesized by the legume symbiont Bradyrhizobium elkanii (37) and the plant pathogen Burkholderia andropogonis (29). Because it induces foliar chlorosis of soybeans, rhizobitoxine has been regarded as a plant toxin (18,36,57). In terms of biochemical functions, rhizobitoxine inhibits ␤-cystathionase in the methionine biosynthesis pathway (39, 57) and 1-aminocyclopropane-1-carboxylate (ACC) synthase in the ethylene biosynthesis pathway (59).Recently, a beneficial role for rhizobitoxine in Rhizobiumlegume symbiosis has been revealed. Using a rhizobitoxine mutant, Yuhashi et al. (60) found that rhizobitoxine production by B. elkanii enhances nodulation and competitiveness in the legume Macroptilium atropurpureum (siratro), probably via the inhibition of endogenous ethylene production in the host plant. Duodu et al. (7) reported that rhizobitoxine mutants formed fewer mature nodules on Vigna radiata (mung bean) than the wild-type strain. In addition, application of ethylene inhibitors to the rhizobitoxine mutants partly restored the nodulation phenotype. Therefore, rhizobitoxine is a nodulation enhancer rather than a phytotoxin for siratro and mung bean, although it is unlikely that rhizobitoxine exerts this positive effect in nodulation of soybean cultivars (28,43,60).The biosynthetic pathway for rhizobitoxine ...

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