Biofilms on a Bead: A Novel Model System to Probe Plant-Microbe Interactions Restricted; Files Only

Abstract

The rhizosphere is a complex ecosystem where various plants, fungi, viruses, and bacteria interact. Striga asiatica has been shown to have a spatiotemporal dependence on hydroquinone for germination. However, the precise reactions of different bacterial species to root exudate in the rhizosphere are not yet fully understood. Biofilms are architecturally complex communities of bacteria that provide mutualistic benefits. The defined diffusion range of hydroquinone, a major component of root exudate, makes it difficult to study the interaction between bacterial biofilms and root exudate using simple streaking techniques. In addition, spatiotemporally controlling a biofilm akin to manipulating a Striga seed would be near impossible without a new way of utilizing biofilms. In this study, we developed a novel biofilm model system of Pseudomonas chlororaphis and Agrobacterium tumefaciens on a bead to investigate how they react to root exudate. This biofilm model system successfully propagated and showed robustness and adaptability to extreme environmental stress. We used P. chlororaphis' phenazine production and A. tumefaciens' GFP fluorescence in reaction to reactive oxidative species to observe the biofilm's and individual species' reactions. Our results showed that P. chlororaphis wild-type grown with A. tumefaciens with a mexE gene deletion (more sensitive to pyocyanin: P. chlororaphis’s phenazine) exhibited higher fluorescence intensity than the combination with P. chlororaphis without phenazine production, demonstrating the potential of the biofilm-on-a-bead model. While conducting the studies for the model, we learned of the redundancy of the phenazine pathway in Pseudomonas chlororaphis, emphasizing its importance in the bacteria and encouraging us in the future to take a different approach to its pathway deletion. To expand our findings, we plan to perform additional assays to gather more definitive data and test the bead with sorghum roots to investigate how the bacteria react to reactive oxidative species caused by the presence of both hydroquinone and phenazines. Successful completion of the biofilm-on-a-bead model will aid in understanding the complex interactions that occur between bacterial biofilms and root exudate in the rhizosphere.

Table of Contents

Chapter 1: Introduction......................................................................................................1

1.1 Biofilms and Their Nature........................................................................................1

1.2 Biofilms as a Model for Natural Coexisting Bacteria in the Rhizosphere and Related Plants................................................................................................................2

1.3 Exploring Germination and Semagenesis to Model the Reaction-Diffusion Gradient........................................................................................................................ 5

1.4 The History of Agrobacterium tumefaciens and Pseudomonas chlororaphis and Their Scientific Uses...................................................................................................... 6

1.5 Forming Biofilms With Co-cultivated Species..........................................................8

1.6 Hypothesis...............................................................................................................8

Chapter 2: Materials and Methods...................................................................................10

2.1 Agrobacterium’s Plasmid Construction and Pseudomonas’s Construct Design.... 10

2.2 Making a Biofilm on a Bead...................................................................................10

2.3 Imaging the Biofilm Beads.....................................................................................11

Chapter 3: Results and Discussion.................................................................................... 11

Chapter 4: Conclusions.....................................................................................................19

References.......................................................................................................................21

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Microbiology
Keyword	Plant-Microbe Interaction Plant-Microbe Agrobacterium Agrobacterium tumefaciens Rhizosphere Biofilm Pseudomonas chlororaphis Pseudomonas
Committee Chair / Thesis Advisor	David Lynn, Emory University
Committee Members	Richard Himes, Emory University Debjani Sihi, Emory University

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