Colonisation of Wheat Roots by Azospirillum Brasilense Wild Type and by Mutant Strains Impaired in Flocculation and Motility

International audienceSurface colonisation of wheat roots by Azospirillum involves the polar flagellum and the production of exo-polysaccharides (Vande Broek,Vanderleyden, 1995). Bacteria on the root surface are ovoid in shape, resembling differentiated cyst-like cells

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data

Molecular Tools to Study Azospirillum sp. and Other Related Plant Growth Promoting Rhizobacteria

Molecular methods have been used in the study of 'Azospirillum' and other related PGPRs to carry out gene functional analysis, create gene knockouts, generate genetically engineered strains, and carry out gene expression studies. Genetic transformation has routinely been carried out using conjugation, while chromosomal modifi cations have been performed using unstable, suicide plasmids, or more stable, broad host-range vectors. Gene expression studies are often carried out using promoter-bound reporter genes; however, quantitative methods such as reverse transcribed polymerase chain reaction can now be used to directly study gene expression. In this chapter we describe the common types of vectors used in 'Azospirillum', as well as methods for transformation and mutagenesis. We also describe the use of promoter-bound reporter genes and the applications of quantitative RT-PCR for 'Azospirillum' gene expression studies. Methods for the isolation of DNA and RNA from 'Azospirillum' for use in molecular and gene expression studies are also described

Azospirillum Cell Aggregation, Attachment, and Plant Interaction

'Azospirillum' cellular and morphological transformation in culture as well as cyst formation, aggregation, and flocculation in response to nutritional limitations and increasing oxygen levels are discussed and typical protocols for flocculation and aggregation are presented. An overview of the mechanisms of attachment to plant roots and other surfaces is followed by protocols for labeling 'Azospirillum' cells with reporter genes and using such genetically labelled cells in qualitative and quantitative assays of 'Azospirillum'-plant associations. The potential of 'Azospirillum' in plant pathogen and disease suppression is discussed