Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

Abstract

One of the largest contributions to biologically available nitrogen comes from the reduction of N(2) to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N(2) fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N(2) fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N(2) fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.

Fig. 1.

The symbiotic phenotype of aap bra mutants is dependent on LIV transport. (A) Amino acid transport of Rlv3841 and aap bra mutants. (B) Shoot dry weights of pea plants inoculated with Rlv3841 and aap bra mutants (n ≥ 13). (C) Six-week-old pea plants inoculated with Rlv3841 and aap bra mutants. (D) Shoot dry weights of pea plants inoculated with RlvA34 and an aap bra null mutant (RU1357) complemented with heterologous narrow-solute specificity ABC transport systems from P. aeroginosa, E. coli, and P. fluorescens (n ≥ 14). Different letters above bars indicate significant differences (P < 0.01).

Fig. 2.

Amino acid transport of RlvA34 and an aap bra null mutant (RU1357) complemented with heterologous narrow-solute specificity ABC transport systems from P. aeroginosa and E. coli (A) and P. fluorescens (B).

Fig. 3.

The biosynthetic pathway for branched-chain amino acids is down-regulated in bacteroids. The down-regulation of the Rlv3841 putative biosynthetic ilv and leu genes is shown in green (dark green, microarray; pale green, qRT-PCR; Table S1). The reduction in detectable ketol-acid reductoisomerase activity (IlvC) is given. Abbreviations are: threonine (thr), 2-oxobutanoate (2-ob), pyruvate (pyr), isoleucine (ile), valine (val), and leucine (leu).

Fig. 4.

The symbiotic phenotype of leuD mutants. (A) Acetylene reduction of pea plants inoculated with Rlv3841 and RU2267 and the respective leuD mutants LMB66 and LMB69 (n = 12); nodulation medium was supplemented with LIV at 1 mM. (B) Shoot dry weights of pea plants inoculated with the same strains (n = 8). Nodulation media were supplemented with LIV at 1 mM. Different letters above bars indicate significant differences (P < 0.01). Uninoculated control plants were the only ones for which the dry weight significantly increased on supplementing the growth medium with LIV.

Fig. 5.

Bacteroid development of aap bra and leuD mutants. (A) Light micrograph of a pea nodule formed by Rlv3841. (B) Transmission electron micrograph of Rlv3841 bacteroids. (C) Light micrograph of nodule formed by LMB66. (D) Transmission electron micrograph of LMB66 bacteroids. (E) Light micrograph of nodule formed by RU2267. (F) Transmission electron micrograph of RU2267 bacteroids. (G) Light micrograph of nodule formed by LMB69. (H) Transmission electron micrograph of LMB69-infected cortical nodule cells.