1.7.6S
ISOLATING PLANT-INDUCED GENES FROM PSEUDOMONAS FLUORESCENS

G PRESTON, N BERTRAND, M GAL, B NEISH and P RAINEY

Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK

Background and objectives
The rhizosphere-colonizing bacterium Pseudomonas fluorescens is a member of a group of bacteria known as plant-growth promoting bacteria (PGPR) and has been studied as a potential biocontrol agent. However, the nature and function of many of the gene products that facilitate its colonization of the rhizosphere and its interaction with plants and other microorganisms are unknown. Genes that contribute significantly to these traits are likely to be expressed in response to in vivo signals present in the rhizosphere. We have used a molecular approach known as in vivo expression technology (IVET) to isolate genes that display elevated levels of expression in the rhizosphere, in order to gain a deeper understanding of the ecology and plant-microbe interactions of P. fluorescens.

Materials and methods
IVET is essentially a promoter-trapping strategy which selects in vivo-induced genes through their ability to drive the expression of a gene that is essential for survival in vivo. Random chromosomal fusions from P. fluorescens SBW25 are generated upstream of a promoterless 'in vivo-selected' marker in an autonomously replicating plasmid that is then integrated into the chromosome of the bacterium. Following in vivo selection, putative in vivo-induced genes are recovered from the bacterial chromosome for detailed molecular analysis [1].

Results and conclusions
We have developed two P. fluorescens-specific IVET strategies and have used the IVET approach to isolate a number of rhizosphere-induced fusions from P. fluorescens SBW25. Sequence analysis of these fusions suggests that they represent a wide range of rhizosphere-induced genes, with several fusions exhibiting no similarity to previously characterized genes. Intriguingly, some of the isolated fusions exhibited similarity to hrc genes, which encode components of a type III secretion pathway found in many plant pathogenic bacteria. We discuss the implications of our results with regard to the molecular understanding of P. fluorescens ecology and plant-microbe interactions.

References
1. Rainey PB, Heithoff DM, Mahan MJ, 1997. Molecular and General Genetics 256, 84-87.