Characterisation of a novel type of surface polar attachment induced in Rhizobium leguminosarum by a pea root exudate metabolite

Legumes such as peas and beans can grow well in soils which do not contain added nitrogen fertiliser. This strong growth occurs because the legumes are infected by bacteria (called rhizobia), which can capture N2 gas from the atmosphere and convert it to a form (ammonia), that can be used by the legumes. In order for rhizobia to efficiently infect legume roots, they must attach to root-hair cells, where they produce chemicals that induce changes in the legume root allowing infection to occur. The way that rhizobia attach to root hairs is unusual / they stick on in a polar manner using one of their ends. In contrast rhizobia grown in the laboratory attach side-on to glass surfaces. We have recently shown that an unknown compound that is exuded by pea roots enables Rhizobium leguminosarum to stick end-on to glass surfaces. In this work we will identify what this compound is and how it promotes this end-on attachment. This will involve identifying which genes are induced during end-on attachment, defining (by mutagenesis) the genes that are required for attachment and identifying if any of the induced proteins are targeted to the bacterial poles. We will analyse how the root exudate compound induces the gene induction required for the end-on attachment and where and when this induction occurs. By analysing legume infection by the mutant bacteria we will determine the contribution of end-on attachment to legume infection.

Interactions between rhizobia and legumes are usually considered to be initiated by plant-made flavonoids, which induce rhizobial nodulation (nod) genes. Our analysis of biofilm formation by Rhizobium leguminosarum has enabled us to identify a hitherto unrecognised aspect of rhizobial-legume communication totally independent of plant-made flavonoids and rhizobial nod genes. In static culture, R. leguminosarum cells attach to the glass surface side-on along their longitudinal axes in long chains of bacteria lying side-by-side. We have identified and purified a metabolite from pea root exudate which induces R. leguminnosarum to attach end-on (analogous to end-on attachment observed on root hairs) to glass in what appears to be haxagonal close-packed arrays). This proposal aims to chemically identify the factor inducing end-on attachment, understand its mode of action and determine the mechanism of end-on attachment that is induced. This will involve using microarrays to identify induced changes in gene expression and mutating induced genes to identify which are required for end-on attachment. The regulatory mechanism of gene induction will be identified by analysing transcript start sites and identifying mutants defective for gene induction by the factor. The timing and location of gene induction will be measured in situ and the cellular localisation of induced proteins will be analysed using translational GFP fusions. Using the mutants generated, the role of end-on attachment during nodulation, and the effects of pre-induction (and constitutive expression) of the genes will be assessed in competitive nodulation experiments.