Holistic view of interactions between biological control and pathogenic bacteria on floral surfaces
Our long-term goal is to improve efficacy and reliability of biocontrol of plant diseases, such as fire blight of apple flowers caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens A506 and Pantoea vagans C9-1 are commercial biocontrol agents for fire blight management, but are not used widely by growers due to unexplained variations in control in orchards. Colonization is a critical phase in pathogenesis of E. amylovora and biocontrol of fire blight, but key genes for colonization of flowers are unknown. Objective 1: Identify genes required for colonization of flowers by commercial biological control agents and the pathogen E. amylovora. We will identify and test genes that are essential for colonization of floral surfaces by the biocontrols and the pathogen using a high saturation mutagenesis method. Identification of these genes in the pathogen may reveal new targets for management of fire blight and those in A506 and C9-1 may lead to improvements in formulations of biocontrol agents to support colonization. Once established on flowers, A506 and C9-1 suppress the pathogen by pre-emptive exclusion and peptide antibiotics, respectively. Our previous work showed that interactions between A506 and C9-1 are critical to biological control: the interactions can either reduce control or make it more reliable. Combining A506 and C9-1 did not improve efficacy compared to single strains. We found that an extracellular protease of A506 inactivated the peptide antibiotics of C9-1. Biocontrol was improved by altering a single factor (protease production) mediating interactions between C9-1 and A506. Yet, we recognize that microbial interactions are complex and rarely determined by a single factor. We postulate that biological control of fire blight may be further enhanced by identifying factors shaping interactions between the biocontrol agents. Objective 2: Identify genes expressed by A506, C9-1 and E. amylovora, inoculated individually and in combination, on floral surfaces. We will use genomics-enabled approaches to characterize interactions between A506, C9-1, and E. amylovora on flower surfaces, the habitat where biocontrol operates. The transcriptional profiles of the biocontrols and pathogen during interactions on floral surfaces will be characterized with RNA-Seq. From the combined approaches, we will identify genetic elements determining interactions between the two bacterial antagonists and the target pathogen on floral surfaces that are critical to biological control. We will examine when genes identified as important in microbial interactions are expressed on stigmas and nectaries of flowers in Objective 3: Evaluate the spatial and temporal patterns of expression of genes key to bacterial interactions on floral surfaces. Through this research, we will gain an understanding of the genetic elements influencing suppression of the pathogen by biocontrol agents, which may lead to methods to improve the efficacy and consistency of disease control. Finally, the novel molecular approaches developed in this project will be applicable to other studies of plant-microbe interactions in natural habitats on plant surfaces.
Newly available genome resources and genomics technology, such as Transposon Directed Insertion-site Sequencing (TraDIS) mutagenesis and direct RNA sequencing (RNA-Seq), provide the opportunity to characterize the molecular basis of microbial interactions on plant surfaces directly. We will use TraDIS mutagenesis to identify genes required for floral colonization by the fire blight pathogen E. amylovora and two biocontrol agents, P. fluorescens A506 and P. vagans C9-1 (Obj. 1). TraDIS is a high-throughput method, coupling saturation mutagenesis with new-generation sequencing technology, to identify genes essential to microbial survival in any habitat. We will inoculate flowers with a high-density pool of transposon mutants. At 1 and 4 days after inoculation, we will recover the mutants that successfully colonized flowers. We will sequence the pool of mutants recovered from flowers and compare to the pool inoculated. Mutations present in the inoculated pool but not in the harvested pool will define genes potentially required for colonization of flowers. Selected genes identified through TraDIS will be mutagenized to evaluate their role in colonization. We will use RNA-Seq to provide a comprehensive view of the transcriptome of each of the three bacterial strains on floral stigmas (Obj. 2). RNA-Seq is a powerful method to evaluate transcriptomes of bacteria grown individually, and we propose to extend this technology to determining transcriptomes of two bacterial genera co-inhabiting floral surfaces. In a preliminary simulated in silico RNA-Seq experiment, transcriptomes of co-cultivated A506, C9-1, or E. amylovora could be distinguished sufficiently. Initially, the transcriptome of each bacterium in a culture medium that mimics the chemical environment of stigmas will be defined and validated with qRT-PCR. Then, we will analyze the transcriptomes of bacteria introduced as individual strains and in pairwise combinations onto flowers maintained in growth chambers under conditions to ensure high populations of each strain. From these data, we expect to identify genes that are expressed on floral surfaces, including those contributing to colonization, pathogenicity or biocontrol. We also will examine changes in expression profiles during intergeneric microbial interactions on stigmas. RNA-Seq is limited by the need for high population sizes for sufficient quantities of mRNA, thus reporter genes will be used to assess gene expression during early phases of interactions and on the nectary, where surface population sizes are small. For Obj. 3, we will examine the spatial and temporal expression of genes key to colonization and interactions (Obj 1 & 2) with reporter gene constructs on floral stigmas and nectaries over time. Daily, cells will be washed from stigmas and nectaries and gene expression monitored with a flow cytometer. This method will permit evaluation of genes of interest at earlier time points during colonization and interactions of the bacteria on the stigma and nectary, two floral habitats important in the biological control of fire blight.
Fire blight, resulting from floral infections by Erwinia amylovora, is the most destructive bacterial disease to pear and apple orchards. Biological control bacteria, such as Pseudomonas fluorescens A506 and Pantoea vagans C9-1, are commercially available but not used widely by growers due to variable performance in orchards. Successful biocontrol is dependent on colonization of flowers and subsequent suppression of growth of the pathogen before infection, but the array of genes mediating these processes on flowers is not known. We will use genome-enabled methods to identify and test genes that are essential for colonization of floral surfaces by the biocontrol bacteria and the pathogen. Identification of these genes in the pathogen may reveal new targets for management of bacterial diseases. Accordingly, their identification in A506 and C9-1 may lead to improvements in formulations supporting successful colonization. We postulate that biological control may be enhanced by identifying factors shaping interactions between the biocontrol agents and the pathogen. We will identify the gene expression profiles of bacteria on floral surfaces, thereby characterizing the interactions between A506, C9-1, and E. amylovora on flowers. Understanding the genes resulting in the suppression of the pathogen by biocontrol agents may lead to methods to improve the level and consistency of disease control. Finally, the novel molecular approaches developed in this project will be applicable to other studies of plant-microbe interactions in natural habitats on plant surfaces.