Performing Department
Plant Pathology
Non Technical Summary
The overall goal of this project is to discover genetic features of arbuscular mycorrhizal fungi (AMF) that are responsible for the quality of outcomes in mycorrhizal symbioses formed by these fungi with crop plants. AMF are soil fungi colonizing roots of most terrestrial plants. Because of role of AMF in plant uptake of mineral nutrients from the soil, phosphorus (P) in particular, AMF have the potential to be alternatives to non-renewable mineral fertilizers in sustainable agriculture. However, despite decades of research, understanding of the factors that determine the quality of mycorrhizal outcomes in terms of agronomic yield is limited. Previous ecological studies of AMF in natural systems indicate that the best plant productivity outcomes are achieved in interactions between locally co-adapted plants and AMF that co-exist in low fertility environments. Based on these observations, we formulated the hypothesis that populations making up individual AMF species differ in the quality of benefits provided to agronomic hosts and these benefits reflect the history of local adaptation experienced by AMF populations under conditions of low or high P availability. To test this hypothesis, we will conduct a greenhouse experiment in which we will pair experimental populations of one AMF species Rhizophagus clarus sourced from a range of soil environments differing in P availability, from high to low P, with a model host plant Medicago truncatula. Based on the outcome of this experiment, we will identify a subset of AMF populations that produce contrasting plant yield outcomes. These AMF populations will be subjected to a comparative analysis of gene activity patterns to identify fungal genetic features responsible for the contrasting outcomes of mycorrhizal symbiosis.This proposed work is prompted by a continuing depletion of global deposits of fossil phosphorus resources, which, in turn, drives the pressure to implement sustainable agronomic practices. Moreover, there is a growing public interest in commercial products that contain AMF and offer sustainable horticultural and landscaping solutions. Rational formulation and deployment of such products depends on our ability to identify AMF that reliably provide superior yield outcomes.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to discover genetic features of arbuscular mycorrhizal fungi (AMF) that are responsible for the quality of outcomes in symbioses formed by these fungi with crop plants. AMF (subphylum Glomeromycotina) are obligate biotrophs colonizing roots of most terrestrial plants. Translocation of minerals, phosphorus (P) in particular, from the soil to plant roots is a universal attribute of AMF, which have the potential to be alternatives to non-renewable mineral fertilizers in sustainable agriculture. Yet, AMF are not dependable as substitutes for chemical fertilizer. This lack of dependability is a consequence of varying photosynthate costs incurred by plants in return for mineral nutrients, as different AMF species and isolates exact different costs. Recent ecological studies suggest that the best plant productivity outcomes are achieved in interactions between locally co-adapted hosts and AMF that co-exist in low fertility environments. However, due to the nature of agronomic practices, crop plants and soil AMF have no chance to co-adapt in typical agricultural environments. Therefore, we need to discover specific genetic features of AMF that control their suitability for agronomic applications. Specifically, we hypothesize that asexual populations making up individual AMF species differ in the quality of benefits provided to agronomic hosts and these benefits reflect the history of local adaptation experienced by AMF populations under conditions of low or high P availability.To meet the overall goal of our study, we will focus on the ability of AMF to provision plants with P translocated from the soil. Our study system will consist with 12 experimental populations of one AMF species Rhizophagus clarus sourced from a range of soil environments differing in P availability, from high to low P. These populations will be paired with a mycorrhizal model host Medicago truncatula and grown under low- and high-P fertility conditions to assess plant and AMF fitness outcomes as well as functional properties of the symbiosis. A subset of AMF populations that produce contrasting plant fitness outcomes will be selected for transcriptional profiling at two key time points in the symbiosis progression to start unraveling genetic underpinnings of intraspecific AMF variability in mediating plant P nutrition. The experimental approach we propose is dictated by the asexual nature of AMF that makes conventional association mapping to link phenotypes with genotypes impractical.OBJECTIVE 1. Measure the effects that different isolates of AMF Rhizophagus clarus derived from soils with varying P fertility levels have on yield of Medicago truncatula under low- and high-P fertility conditions.In the process, we will test the hypotheses that: (1) asexual populations of AMF differ in the quality of benefits provided to plant hosts and (2) low-P-adapted AMF are superior to high-P-adapted AMF. We selected R. clarus because of its genomic resources and our experience with this species (NYC-153434). Medicago was chosen because it is a model crop used in mycorrhizal research and has excellent genomic resources. Addressing OBJ1 will allow us to examine the range of symbiotic/yield outcomes produced by populations of a single AMF species over the symbiosis progression and select the isolates and sampling times for further study in OBJ2.OBJECTIVE 2. Conduct global transcriptome comparisons between selected low- and high-P-adapted R. clarus isolates across treatments examined in OBJ1. Addressing this objective will allow us to identify genomic features that underlie the ability of AMF to deliver superior symbiotic outcomes in the form of P nutrition.
Project Methods
OBJECTIVE 1. Seedlings of Medicago truncatula Jemalong A17 will be inoculated with 25 spores of R. clarus per seedling one week after germination and planted in sterilized sand-soil mix. We will test 12 populations originating from a range of P-fertility soil conditions sourced through INVAM (https://invam.wvu.edu/). Non-mycorrhizal plants will be used as controls. Each treatment will consist of 18 plants. Plants will be maintained in the greenhouse, watered and fertilized to maintain either a low- or high-P fertility regime. Every two weeks, we will harvest 3 plants per treatment and measure plant and AMF response variables. The final harvest will occur after the first set of seed pods is generated, 7 to 9 weeks after germination [1]. Plant response variables will include shoot and root biomass, seed set, and tissue mineral content. Fungal response variable will be sporulation, quantified after spore extraction [2]. We will also measure symbiotic activity of the system by assessing AMF root colonization, quantified after staining roots with trypan blue [3] and estimated by RT-PCR of fungal ribosomal RNA (rRNA) coding genes [4]. In addition, we will determine by RT-PCR the expression of the Medicago gene encoding the arbuscule-specific phosphate transporter MtPT4, which is considered to be a marker of mycorrhizal activity [4]. All data will be analyzed by ANOVA followed by post-hoc tests. The results will allow us to: (1) identify R. clarus populations exhibiting contrasting symbiotic outcomes to be further examined in OBJ2, and (2) pinpoint key stages in the progression of mycorrhizal symbiosis to be sampled in OBJ2. We anticipate that the examined AMF populations will differ in the symbiotic outcomes in terms of plant fitness/yield as well as in AMF sporulation and mycorrhizal activity. We also expect that, in terms of timing, expression of MtPT4 will peak at the onset of Medicago flowering before maximum arbuscular root colonization that will correlate with the onset of seed development [4].OBJECTIVE 2. For gene expression profiling of AMF, we will select 4 AMF isolates that produced contrasting yield outcomes in OBJ1. Medicago plants will be inoculated as in OBJ1 and grown until harvested at two time points, which we will monitor by periodical non-destructive sampling of roots: (1) the peak of MtPT4 gene expression, corresponding with the peak of mycorrhizal activity and (2) the peak of mycorrhizal root colonization [4]. Total RNA will be extracted from roots, processed and analyzed using standard methods. In particular, RNA sequencing libraries will be prepared in 5 replicates per AMF isolate using the NEBNext mRNA Library Prep Reagent Set for Illumina and sequenced at the CU Biotechnology Resource Center. Illumina data will be quality-controlled using the FASTX-Toolkit [5]. AMF reads will be mapped onto the R. clarus genome [6] using TopHat [7], transcript abundances will be quantified with CuffDiff [8], and differential gene expression analysis performed with EdgeR [9]. In addition, we will collect plant and fungal fitness data to be able to correlate transcriptomic responses with fitness/yield outcomes. In an unlikely case of low AMF root colonization outcomes in Medicago, we will use excised Ri T-DNA transformed carrot or chicory roots to form mycorrhizae with selected isolates of R. clarus, and conduct transcriptomic analyses. These mycorrhizal organ cultures are a popular tool for mycorrhizal research [10]. When conducting transcriptional profiling experiments and data analyses, we will rely on our previous experiences with such activities [11,12]. We expect that gene expression profiles will differ between superior and inferior AMF symbionts, and these differences will be concentrated in pathways related to mineral nutrient uptake and carbon metabolism. In particular, we expect amplification in copy number of genes involved in key steps of these processes.REFERENCES1. Chabaud M, Lichtenzveig J, Ellwood S, Pfaff T, Journet E-P (2006) Vernalization, crossings and testing for pollen viability. Medicago truncatula Handbook. Ardmore, OK Noble Research Institute.2. Daniels BA, Skipper HD (1982) Methods for the recovery and quantitative estimation of propagules from soil. In: Schenck NC, editor. Methods and Principles of Mycorrhizal Research. St. Paul, MN: The American Phytopathological Society. pp. 29-35.3. Koske RE, Gemma JN (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res 92: 486-505.4. Isayenkov S, Fester T, Hause B (2004) Rapid determination of fungal colonization and arbuscule formation in roots of Medicago truncatula using real-time (RT) PCR. J Plant Physiol 161: 1379-1383.5. FASTX-TOOLKIT http://hannonlabcshledu/fastx_toolkit/indexhtml.6. S?dzielewska Toro K, Brachmann A (2016) The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus. BMC Genomics 17.7. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, et al. (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14: R36.8. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, et al. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7: 562-578.9. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139-140.10. Cranenbrouck S, Voets L, Bivort C, Renard L, Strullu DG, et al. (2005) Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root organs. In: Declerck S, Strullu DG, Fortin A, editors. In Vitro Culture of Mycorrhizas. Berlin, Heidelberg: Springer-Verlag. pp. 341-375.11. Lastovetsky OA, Gaspar ML, Mondo SJ, LaButti KM, Sandor L, et al. (2016) Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria. P Natl Acad Sci USA 113: 15102-15107.12. Mondo SJ, Lastovetsky OA, Gaspar ML, Schwardt NH, Barber CC, et al. (2017) Bacterial endosymbionts influence host sexuality and reveal reproductive genes of early divergent fungi. Nat Commun 8: 1843.