Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.     

Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.)

Plant root development can be largely affected through the association of roots with plant growth-promoting rhizobacteria (PGPR). However, little is known about the identity of plant genes enabling such PGPR-plant root associations. Differences in the responsiveness to PGPR among cultivars suggest genetic variation for this trait within germplasm. In this study, two genotypes of common bean (Phaseolus vulgaris L.), BAT477 and DOR364, were identified showing contrasting responsiveness in root development to inoculation with the PGPR Azospirillum brasilense Sp245. Inoculation with an A. brasilense Sp245 mutant strain strongly reduced in auxin biosynthesis or addition of increasing concentrations of exogenous auxin to the plant growth medium, indicated that the differential response to A. brasilense Sp245 among the bean genotypes is related to a differential response to the bacterial produced auxin. To further assess the role of the plant host in root responsiveness, a population of Recombinant Inbred Lines (RILs) of the DOR364×BAT477 cross was used to evaluate the efficacy of exogenous auxin on root development. We detected significant phenotypic variation among the RILs for basal root formation during germination upon addition of auxin to the growth medium. Genetic analysis revealed two quantitative trait loci (QTLs) associated with basal root responsiveness to auxin of which one explained 36% of the phenotypic variation among the RILs. This latter QTL mapped to the same location as a QTL for root tip formation at low P, suggesting that the host effect on root responsiveness to IAA interacts with specific root development. Also, significant correlations between basal root responsiveness to auxin and growth, root tips and root dry weight at low P were identified. To our knowledge, this is the first report on QTL detection for root responsiveness to auxin.     

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.     

Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction. This revised version was published online in June 2006 with corrections to the Cover Date.