Abstract
Symbiotic mesorhizobia are soil bacteria which have coevolved with diverse integrative and conjugative elements that confer the capacity for nitrogen-fixing symbiosis with legumes (ICESym). The isolate Mesorhizobium japonicum R7A is a model mesorhizobial symbiont of plants in the Loteae tribe of legumes, including the model plant Lotus japonicus Gifu. Wildtype M. japonicum R7A (R7AWT) carries the symbiosis island ICEMlSymR7A, and a derivative strain cured of the ICESym, M. japonicum R7ANS (R7ANS), has previously been developed. Through acquisition by horizontal transfer of different ICESyms, R7ANS is conferred a symbiotic host-range dependent on the specific ICESym it harbours.
The rhizosphere is the zone of plant influence which surrounds the roots of a plant in the soil. The rhizosphere effect is the change in the rhizosphere affected by a growing plant on the soil and the microbiota therein. Research suggests that the rhizosphere effect of plants is a directed process that may actively shape microbial communities. Little is known about the growth of R7AWT or R7ANS in the rhizosphere of L. japonicus or Arabidopsis thaliana, or what effect the ICEMlSymR7A has on competition therein. Here I present comparative and functional genomic research on the basis of ICESym host range and R7A rhizosphere competition.
Through comparative genomics of Loteae nodulating ICESyms, I identified an evolutionary relationship between them that is correlated with their conferred host ranges. I identified that across the Loteae nodulating ICESyms compared, 155 genes were conserved representing on average 30% of a given ICESym’s genes. I also found that on average 35% of an ICESym’s genes were unique to it. A total of 53 genes and 4 nod box symbiotic regulatory motifs were uniquely conserved in the broad host range L3 lineage of Loteae-ICESyms, and I present a discussion of these genes and how they may account for the differing symbiotic host ranges.
Using the functional genomic screening method Tn-seq, I screened over 800,000 unique Tn5 mutants in both R7AWT and R7ANS, identifying 600 core essential genes for growth in vitro while also identifying that ICEMlSymR7A modulates the essential phenotype of the house-keeping genes pncB, groELS and a lysine tRNA gene. I also identified 19 genes with essential phenotypes encoded by ICEMlSymR7A which may be the result of toxic effects caused by Tn insertion. Some of these genes resembled defence systems to mobile genetic elements. The Tn-seq results indicated the Entner-Doudoroff pathway was essential for catabolism of glucose, and this was confirmed by insertional mutagenesis.
Finally, I used Tn-seq to screen symbiotic and non-symbiotic rhizosphere competition of R7AWT and R7ANS identifying 129 pan-rhizosphere genes in L. japonicus and A. thaliana rhizospheres that resulted in decreased competitiveness when mutated. In 1:1 competition assays I observed that ICEMlSymR7A conferred an increase in rhizosphere competitiveness, but that Nod factor signalling conferred a relative fitness cost. Analysis of metabolic genes with a loss of fitness in the rhizosphere Tn-seq screens indicated that metabolism of organics acids was important for rhizosphere competitiveness, which may relate to the decreased competitiveness observed for R7ANS.