Isolation and characterization of a bacterial auxin gene and its promoter

Abstract

Bacteria that inhabit the rhizosphere may influence plant growth by modifying phytohormone levels in plants tissues, for example, by adding to a plant's pool of the hormone auxin. In phytopathogenic bacteria, the auxin indoleacetic acid (IAA) is produced mainly via the indoleacetamide pathway, and has been implicated in the induction of plant tumors. Beneficial bacteria synthesize IAA predominantly by the indolepyruvic acid pathway; however, the role of IAA in plant growth-promotion remains inconclusive.

The ipdc gene encoding indolepyruvate decarboxylase, which catalyzes a key step in the latter pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 by colony hybridization and PCR. The similarity of the amino acid sequences among other indolepyruvate decarboxylases, pyruvate decarboxylase, and acetolactate synthase suggests an evolutionary relationships among these proteins. Because IAA accumulate decarboxylases, pyruvate decarboxylase, and acetolactate synthase suggests an evolutionary relationship among these proteins. Because IAA accumulates in the culture medium of P. putida GR12-2 grown in the presence of exogenous tryptophan, transcription of ipdc may be activated by tryptophan. To test this hypothesis, the ipdc promoter region was isolated by inverse PCR, and inserted upstream of the bioluminescent reporter gene luxAB on a plasmid in P. putida GR12-2. Activity of the ipdc promoter, measured by quantifying light production, increased dramatically in the presence of tryptophan, confirming that ipdc expression is induced by tryptophan. In addition, ipdc is regulated by the stationary phase sigma factor RpoS: the ipdc promoter contains a sequence similar to the RpoS recognition sequence, and transformation of P. putida GR12-2 with rpoS induced promoter activity before the onset of stationary phase when RpoS is not normally produced, and prolonged a higher level of transcription at the last stages of the cell cycle.

To determine if IAA is involved in the stimulation of plant growth by P. putida GR12-2, an IAA-deficient mutant was constructed by insertional mutagenesis of ipdc. The lengths of canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35-50% longer than roots from seeds treated with the IAA-deficient mutant, and roots from uninoculated seeds. In addition, exposure of mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain, stimulated the formation of many, very small, adventitious roots. Fewer roots were initiated by the IAA-deficient mutant. These results suggest a major role for bacterial IAA in the development of the host plant root system.