Ken-Ichi Yuhashi

Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum

ABSTRACT Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkaniiproduces 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.

DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii

ABSTRACT 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 rtxAshowed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxAand these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxAand rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions ofrtxA 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, thertxC 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 thatrtxC 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.

1-Aminocyclopropane-1-Carboxylate Deaminase Enhances Agrobacterium tumefaciens-Mediated Gene Transfer into Plant Cells

ABSTRACT Agrobacterium-mediated gene transfer is widely used for plant molecular genetics, and efficient techniques are required. Recent studies show that ethylene inhibits the gene transfer. To suppress ethylene evolution, we introduced 1-aminocyclopropane-1-carboxylate (ACC) deaminase into Agrobacterium tumefaciens. The ACC deaminase enhanced A. tumefaciens-mediated gene transfer into plants.

Quantitative and time-course evaluation of nodulation competitiveness of rhizobitoxine-producing Bradyrhizobium elkanii

Abstract Regression analysis of results from a mathematical competition model showed that rhizobitoxine production by Bradyrhizobium elkanii USDA94 gave this strain a nodulation competitiveness about 10 times greater than that of a non-rhizobitoxine-producing mutant strain on Macroptilium atropurpureum (Siratro). Rhizobitoxine enhancement of competitive nodulation occurred at a late stage in the time-course of nodulation. All other known rhizobial factors that affect nodulation competitiveness act in the rhizosphere and during the initial interaction with legumes. This unique late action of rhizobitoxine could prove advantageous in inoculant production, because inoculum often fails to nodulate in the latter stages of nodulation kinetics.