Application of Transposon Insertion Sequencing to Agricultural Science

Abstract

Many plant-associated bacteria have the ability to positively affect plant growth and there is growing interest in utilizing such bacteria in agricultural settings to reduce reliance on pesticides and fertilizers. However, our capacity to utilize microbes in this way is currently limited due to patchy understanding of bacterial-plant interactions at a molecular level. Traditional methods of studying molecular interactions have sought to characterize the function of one gene at a time, but the slow pace of this work means the functions of the vast majority of bacterial genes remain unknown or poorly understood. New approaches to improve and speed up investigations into the functions of bacterial genes in agricultural systems will facilitate efforts to optimize microbial communities and develop microbe-based products. Techniques enabling high-throughput gene functional analysis, such as transposon insertion sequencing analyses, have great potential to be widely applied to determine key aspects of plant-bacterial interactions. Transposon insertion sequencing combines saturation transposon mutagenesis and high-throughput sequencing to simultaneously investigate the function of all the non-essential genes in a bacterial genome. This technique can be used for both in vitro and in vivo studies to identify genes involved in microbe-plant interactions, stress tolerance and pathogen virulence. The information provided by such investigations will rapidly accelerate the rate of bacterial gene functional determination and provide insights into the genes and pathways that underlie biotic interactions, metabolism, and survival of agriculturally relevant bacteria. This knowledge could be used to select the most appropriate plant growth promoting bacteria for a specific set of conditions, formulating crop inoculants, or developing crop protection products. This review provides an overview of transposon insertion sequencing, outlines how this approach has been applied to study plant-associated bacteria, and proposes new applications of these techniques for the benefit of agriculture.

Keywords: biocontrol; fertilizer; microbiome; pesticide; plant growth promoting bacteria; transposon insertion sequencing; transposon mutagenesis.

FIGURE 1

Mechanisms of plant growth promotion by beneficial bacteria. Phytohormone production, abiotic stress tolerance, rhizoremediation and biofertilization are direct methods of plant growth promotion. In contrast, biocontrol is an indirect method of plant growth promotion operating through competition with pathogens, priming plant defenses and producing antimicrobial compounds.

FIGURE 2

Transposon insertion sequencing methodology for agriculturally relevant bacteria. High throughput sequencing of the starting population of bacterial mutants in the saturated transposon insertion library determines the locations of the transposon insertions in the genome. An aliquot of the mutant library is grown with a selective pressure (for example, persistence on root surfaces in either sterilized or natural soil, pesticide tolerance, abiotic stress), the surviving cells are recovered, and the transposon-chromosome junctions are sequenced. Changes in the abundances of each mutant in the starting pool compared with the challenge pool indicate which genes increase or decrease bacterial fitness under the challenge conditions. This provides information on the possible functions of these genes that can then be investigated by targeted follow up experiments.