Genome-wide identification of colonisation determinants in rhizobium leguminosarum using BarSeq
Keywords: root
colonisation
legumes
rhizobium
rhizosphere

Many plant-beneficial bacteria must persist in the rhizosphere and colonise the roots of their host plant to exert their beneficial effects. Plants primarily influence bacterial colonisation of the rhizosphere and roots through modulation of their root exudate composition, whilst bacteria-bacteria interactions affect bacterial persistence and root colonisation through metabolic exchanges, secretion of antimicrobial compounds and other processes. Rhizobium-legume symbioses are one of the best characterised beneficial interactions, due to their potential to alleviate our reliance on nitrogen fertilisers. Despite this, most studies on Rhizobium have focused on its symbiotic lifestyle as an endosymbiont in root nodules and therefore the initial stages of rhizosphere growth and root colonisation remain relatively under characterised.
The multifactorial nature of bacterial growth in the rhizosphere and root colonisation means that transposon insertion sequencing techniques such as random-barcode transposon-site sequencing (RB-TnSeq) provide a unique opportunity to study gene function at the whole genome level. Through incorporation of a random DNA barcode into transposons RB-TnSeq allows barcode sequencing (BarSeq), which can be used to assay mutant fitness in a high throughput manner. The aim of the work described in this thesis was to use RB-TnSeq to characterise how plant species and bacterial competition affect rhizobial growth in the rhizosphere and root colonisation using the model rhizobial strain R. leguminosarum bv. viciae 3841 (Rlv3841).
RB-TnSeq was established in Rlv3841 and initial validation in planta demonstrated the suitability of BarSeq for identification of genes contributing to rhizosphere growth and root colonisation. Using BarSeq, phenotypes for 80% of genes encoded by the Rlv3841 genome were assessed during growth in the rhizosphere and colonisation of host legumes (pea, lentil, Lathyrus), a non-host legume (alfalfa) and a non-legume (barley). A core set of 73 genes were identified as commonly required for growth in all five plant rhizospheres and a further 58 genes contributed to root colonisation. Analysis of these genes revealed that growth in the rhizosphere and colonisation of roots required the synthesis of compounds (amino acids, ribonucleotides and cofactors), alteration of metabolic function, adaptation to various stresses (such as changes in osmolarity) and sensing of external stimuli coupled with modification of gene expression. Additionally, chemotaxis and flagella-mediated motility were common prerequisites for root colonisation. Notably, many genes showed plant-specific dependencies. In contrast to common determinants, genes implicated in transport accounted for a large proportion of these likely reflecting altered resource availability due to differences in root exudate composition between plant species.
Traditionally, bacterial colonisation has been examined under sterile laboratory conditions that mitigate the influences of bacterial interactions. To identify genes required for competitive rhizosphere growth and root colonisation, an RB-TnSeq mutant library was generated in the beneficial plant growth promoting rhizobacterium Pseudomonas fluorescens SBW25 and co-inoculated with the Rlv3841 RB-TnSeq library. This allowed, for the first time, simultaneous assessment of gene fitness contributions for two different strains by BarSeq and highlighted the need for altered transport, metabolic and regulatory requirements during conditions of competition.
Overall, this work highlights the significant adaption required to different plant rhizospheres and the presence of a competitor, thereby providing a greater understanding of factors promoting rhizosphere fitness and root colonisation in two plant-beneficial bacteria.