Performing Department
Restoration Ecology and Environmental Horticulture
Non Technical Summary
Large areas of formerly forested and agricultural lands in Washington State have been negatively impacted by contamination with toxic metals. Phytoremediation is an inexpensive technology that takes advantage of the natural ability of some plants to accumulate environmental pollutants in their tissues. This proposed project seeks to investigate ways to improve phytoremediation efficiency by exploiting beneficial microbe-plant partnerships to reduce the phytotoxicity of the toxic metals to plants. Phytoremediation with fast growing tree species, e.g. willows and poplars, could expand the reach of remediation efforts by state and federal agencies and return ecosystem productivity to thousands of acres of land in Washington State.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The goal of this proposed project is to investigate the mechanisms of reduced phytotoxicity of arsenic by endophytes in order to ultimately guide the optimization of bacterial-plant partnerships for phytoremediation. Specifically, this project will investigate the potential for endophytic extracellular polymeric substances (EPS) to act as a direct arsenic-tolerance mechanism in plants used in the phytoremediation of arsenic-contaminated soils. We are hypothesizing that the arsenic-induced synthesis of EPS by endophytes may benefit the host plant by reducing the in planta arsenic levels. First, we are proposing to investigate the role of EPS in endophytic arsenic-tolerance by comparing changes in tolerance and arsenic-detoxification gene expression in EPS-deficient mutant and wild type (WT) phenotypes in vitro. Secondly, we will investigate the regulation of endophytic EPS genes in plants under arsenic stress.The proposed project will focus on two endophyte strains currently being studied by the Doty lab. The first, R. aquatilis strain PD12R, was isolated from red huckleberry (Vaccinium parvifolium) growing in arsenic contaminated soils within the TSP. PD12R has a high tolerance to arsenic and produces prodigious amounts of EPS under arsenic stress. The second, Enterobacter sp. strain PDN3, was isolated from hybrid poplar (Populus deltoides x nigra) grown at an active phytoremediation site contaminated primarily with TCE. Genome sequencing of PDN3 revealed arsenic-tolerance gene sequences, and subsequent culturing in the presence of arsenic showed high tolerance and EPS production. When discussing arsenic in the following sections, we will be referring to both arsenate (AsV) and arsenite (AsIII) as both arsenic species are likely to be encountered in planta. We will screen AsV and AsIII separately, and at concentrations determinedin our prior experiments.We are proposing the following objectives:Objective 1: To quantify the sequestration of arsenic in the EPS synthesized by PD12R and PDN3. We are hypothesizing that EPS synthesis is a mechanism for reducing exposure to arsenic through sequestration in the EPS matrix. Host plants would similarly benefit from sequestration of free arsenic in planta by endophytic EPS.Objective 2: To identify key genes involved in EPS production in PD12R and PDN3 under arsenic stress, and test the role of those genes on arsenic tolerance. Arsenic stress in endophytes can be assessed via changes in tolerance and expression levels of arsenic-detoxification genes. Accordingly, if EPS lowers arsenic exposure in endophytes through sequestration then we would expect to see reduced tolerance and increased gene expression in the EPS-deficient mutants as compared to the WT.Objective 3: To quantify the expression levels of EPS and arsenic-detoxification genes by PD12R and PDN3 in a host plant under arsenic stress. Endophytes colonizing plants used for thephytoremediation of arsenic-contaminated soils would be exposed to arsenic taken up by the host plant. The synthesis of EPS by endophytes in response to this arsenic could directly benefit the host plant by reducing free arsenic levels in planta. Accordingly, we would expect to see increased levels of endophytic EPS gene expression in plants exposed to arsenic as compared to the controls. Alternatively, if EPS genes are not highly expressed in response to arsenic in the host plants then we would expect to see higher levels of arsenic-detoxification gene expression by the endophytes.
Project Methods
Objective 1: The extracellular polymeric substances (EPS) of PD12R and PDN3 will be quantitatively assayed for arsenic. To test the ability ofEPS to sequester arsenic we will measure arsenic concentration in the EPS of both strains after exposure to arsenic. Inoculums of PD12R or PDN3 be cultured in liquid media with or without arsenic. After a period of growth the EPS, cells, and media will beseparated, and the resulting fractions will be analyzed for arsenic via ICP-AES.Objective 2: The EPS genes of PD12R and PDN3 will be investigated for their role in arsenic tolerance. We will quantify this in vitro by comparing changes to arsenic tolerance and arsenic-detoxification gene expression between EPS-deficient mutant and WT phenotypes under arsenic stress.Arsenic-detoxification gene expression:We will measure changes in expression levels of the arsenic-detoxification genes in PD12R and PDN3 via ddPCR. Microbial arsenic-detoxification genes are well described in the literature, and we will evaluate the ars genes in the presence of arsenate, and the aox genes in the presence of arsenite. The genome of PDN3 has been sequenced and the arsenic-detoxification gene sequences needed for PCR primer design have been identified. The endophyte PD12R has been putatively identified as R. aquatilis, and the genome sequence of another R aquatilis strain (WP5) has already been completed by our lab. In addition, several other strains of this species have been previously published in open-source genomic databases; e.g. National Center for Biotechnology Information. Drawing on the literature, we will probe the available R. aquatilis genomes for homologous sequences of known ars and aox genes. Using alignments of these sequences we will design PCR primers to target and copy these genes from PD12R for sequencing.Generation of EPS-deficient mutants: The endophyte PDN3 is being subjected to random bar-code transposon sequencing (RB-TnSeq) under an ongoing collaboration between the Doty lab, the Department of Energy's Joint Genome Institute (JGI), and several other institutions. That project will generate a library of PDN3 mutants that can be efficiently screened for loss of function under experimental conditions. For PD12R we will identify and target the EPS genes by employing the methods described above for targeting the ars/aox genes. If successful, we will use the derived sequence alignments to perform directed mutagenesis on the candidate genes. While we feel that there is sufficient sequencing data available to identify the EPS genes in PD12R, we can as an alternative perform random Tn5 transposon mutagenesis and screen for EPS-deficient mutants using replicate plating. We will use the resulting mutant libraries for PD12R and PDN3 to screen for EPS-deficient mutants (mEPS).Inoculums of the mEPS and WT phenotypes of PD12R and PDN3 will be transferred to sterile membranes placed onto solid media plates with or without arsenic. The membranes permit diffusion of nutrients and arsenic but not the cells, and allow efficient testing of the resulting EPS and cell matrix. After a period of growth, the samples will be analyzed using a ddPCR technique called multiplexing. Multiplexing ddPCR simultaneously targets multiple DNA/RNA sequences of interest in a single environmental sample with different fluorescent reporters, which can then be used to precisely quantify multiple sequences of interest based on intensity of fluorescence.To compare changes in arsenic tolerance between the mEPS and WT phenotypes we will report colony forming units (CFUs) and minimum inhibitory concentrations (MICs). We will estimate CFUs in the EPS matrix by targeting the 16S rRNA sequences with fluorescently labeled primers that are specific to the PD12R and PDN3 strains, and then compare fluorescent signal strength against standardized CFU curves. The highly conserved 16S rRNA sequences are a standard method of species and strain identification, and were previously used to identify both strains. CFU will be reported as the relative abundance of the16S rRNA reporter, and MIC will be reported as the highest arsenic concentration at which growth no longer occurs. Concurrently, the PCR primers targeting the mRNA from the ars and aox genes will be labeled with distinct fluorescent reporters, and the relative expression levels of the ars and aox genes will be reported as the ratio of mRNA copies to 16S rRNA copies.Because they possess arsenic-detoxification genes, we expect the EPS-deficient mutants to retain arsenic tolerance. Instead, we are hypothesizing that EPS increases arsenic tolerance in endophytes through sequestration and/or reduced rates diffusion, effectively limiting the arsenic exposure of the cells. A similar phenomenon has been widely studied in antibiotic resistance. Accordingly, we expect to see reduced CFUs and MICs and increased expression of ars/aox genes in mEPS as compared to the WT phenotype.Objective 3: The regulation of EPS and arsenic-detoxification genes of PD12R and PDN3 will be quantified in planta using the model plant Arabidopsis thaliana. Using the principles described in Obj. 2, we will use multiplexing ddPCR to quantify expression levels of the key EPS and arsenic-detoxification genes in the mEPS and WT phenotypes. Finally, we will compare the gene expression levels with indicators of increased plant tolerance to arsenic; i.e. biomass, root/shoot length, and arsenic uptake and accumulation.First, we will screen the PD12R and PDN3 EPS-deficient mutants for the ability to colonize A. thaliana. EPS synthesis has been associated with promoting successful host plant colonization by endophytes. If colonization ability has been lost, then the EPS-deficient mutants will be excluded from Obj. 3. Otherwise, the experiment will include the mEPS and WT phenotypes.Surface sterilized A. thaliana seeds will be germinated in hydroponic solution under standard growth chamber conditions. The seedlings will be inoculated with either PD12R or PDN3 per Doty lab protocols, and then grown in media with or without arsenic for a period of two weeks. Uninoculated and killed-inoculation (i.e. autoclaved cells in media) plants will serve as controls. For each treatment, three hydroponic tanks (n=3) containing six plants will be used. At the conclusion of the experiment, half of the plants will be selected randomly from each tank to be analyzed for total arsenic uptake and accumulation via ICP-AES, while the remaining plants will be used for gene expression analysis. The biomass, the root length, and shoot length will be recorded, and the roots and shoots will be analyzed separately for either total arsenic or gene expression. Arsenic will be reported in ppm (mg kg-1, dry wt.) and the CFU and the relative EPS and arsenic-detoxification gene expression levels will be reported as noted in Obj. 2.Many factors influence EPS synthesis in bacteria, and internal plant tissues present a complex and highly variable environment. Additionally, endophytes can promote arsenic-tolerance in plants in multiple, non-mutually exclusive ways that will not be distinguishable from our findings. Other considerations would be EPS synthesis and arsenic-detoxification activity by indigenous endophytes, which we will control for by germinating A. thaliana from seeds and conducting the experiment in hydroponics under sterile lab conditions. Finally, because we are not quantifying arsenic in the EPS, we will not be able to conclusively demonstrate a direct interaction between EPS and arsenic in planta. However, to the best of our knowledge this proposal represents the first attempt to identify a mechanism by which endophytes might directly promote arsenic tolerance in their host plants.