Title

In vivo dynamics of the quorum sensing-related interplay during symbiotic interaction between the nitrogen fixing bacterium, Sinorhizobium meliloti, and its eukaryotic host, Medicago truncatula

Date of Award

2012

Degree Name

Ph.D. in Biology

Department

Department of Biology

Advisor/Chair

Advisor: Jayne B. Robinson

Abstract

Quorum sensing has been shown to play a role in the symbiosis between Sinorhizobium meliloti and its host plant Medicago truncatula. Quorum sensing (QS)-mediated gene regulation is central to the interactions of bacteria with their eukaryotic hosts. The ExpR/Sin quorum-sensing system of the Gram-negative bacterium Sinorhizobium meliloti plays a significant role in the establishment of symbiotic interaction with its host plant Medicago truncatula. This bacterial signaling mechanism and plant responses to bacterial QS signals are important to the success of plant-bacteria interaction. Eukaryotes, in turn, can recognize and manipulate bacterial QS. While hundreds of Sinorhizobium genes are now known to be QS-regulated in laboratory shake cultures, less is known about QS regulation in natural environments or during symbiosis. In this study, we investigated the role of quorum sensing in expression of two important Sinorhizobium meliloti genes: the AHL synthase gene sinI and the bacterial cell division gene ftsZ2 in free-living bacteria and nodules of its host plant, Medicago truncatula and the role of cell-to-cell signaling (Quorum Sensing) during these interactions in vivo. Red-fluorescent protein reporters (Rfp) were constructed in promoters of these genes and by using sinI::tdRfp and ftsZ2::tdRfp promoter reporter fusions we were able to track the expression of these genes in wild-type S. meliloti cells and QS mutant backgrounds in planta using Confocal Laser Scanning Microscopy (CLSM). The overall goal of this work was to characterize the in vivo dynamics of the QS-related interplay during symbiotic interactions between the nitrogen fixing bacterium, Sinorhizobium meliloti, and its legume host, Medicago truncatula. We have shown that the Rfp reporter genes survived infection and were expressed in the host, further they were differentially expressed in vitro in contrast to the in vivo. The production of AHL mimics by plants suggests that bacteria and plants utilize this method of bacterial communication as a control point for influencing the outcome of their interactions. Our results revealed that mexpression of ftsZ2 in free-living cells was changed or reduced in sinI mutant cells in contrast to the wild type cells in vitro while its expression in planta was substantially higher in the absence of sinI. The addition of ethyl acetate extracts prepared from bacteroids of nodules infected with wild type or sinI mutant cells to free-living cells had no effect on ftsZ2 expression in wild-type or sinI background. For sinI expression in vitro study, it was found that the addition of AHLs did no affect the levels of sinI expression. In addition, it was determined that sinI expression in vivo was the highest in wild type background and there was a high expression of sinI in sinI mutant cells.

Keywords

Quorum sensing (Microbiology) Research, Microbial genetics Research, Legumes Microbiology Research

Rights Statement

Copyright 2012, author

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