Source: UNIV OF NORTH CAROLINA submitted to
MULTISPECIES INTERACTIONS IN THE MICROBIOME: DYNAMIC RESPONSES OF PARASITE INDIVIDUALS, POPULATIONS, AND COMMUNITIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1010399
Grant No.
2016-67013-25762
Project No.
NC.W-2016-07014
Proposal No.
2016-07014
Multistate No.
(N/A)
Program Code
A1222
Project Start Date
Sep 1, 2016
Project End Date
Aug 31, 2021
Grant Year
2016
Project Director
Mitchell, C.
Recipient Organization
UNIV OF NORTH CAROLINA
(N/A)
CHAPEL HILL,NC 27514
Performing Department
Biology
Non Technical Summary
Every individual human, animal and plant serves as host for a diversity of microbes that together comprise its microbiome. While the microbiome is comprised mostly of organisms that usually do no harm to the host, or even benefit the host, the microbiome also almost always includes some disease-causing organisms: pathogens and parasites. Each organism in the microbiome interacts not only with the host, but with other organisms in the microbiome. Also, each host's microbiome is different. Some hosts may have a microbiome that partially protects them against infection by pathogens transmitted from other host individuals. A current scientific issue is how transmission of pathogens across the population of hosts is influenced by the differences between hosts in their microbiomes. That is the issue that will be addressed by this project. This project will study the microbiome of leaves of the agriculturally important grass tall fescue. The microbiome of leaves is dominated by fungi, and accordingly the project will study fungal species that range from pathogens to mutualists of the plant. The project will test whether key members of the microbiome can reduce pathogen infection of host individuals, and under what conditions they can also reduce pathogen transmission across the host population. This will be done by integrating a range of methods, including high-throughput genomic sequencing, field experiments, greenhouse experiments, field surveys, and mathematical models. The ultimate goal of the project is to better understand role of the microbiome in pathogen transmission. In the long-term, this project may provide insights into sustainable management of pests. The focal pathogens are some of the most important pests of pastures, turf grass, and small grain crops, and tall fescue is one of the most important grasses for both turf and feeding livestock. Moreover, the project aims to provide general understanding that will apply across systems, potentially providing a basis for improvements in plant, animal, and human health.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240201070100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1070 - Ecology;
Goals / Objectives
The overarching goal of this project is to determinewhether and how interactions among parasites and mutualists within host individuals (i.e. within the microbiome) scale up to influence parasite transmission dynamics across the host population. To achieve this goal, we will address three major research questions.Question 1: To what degree are epidemic dynamics driven by responses of parasite community structure to microbial interactions?Question 2: To what degree are epidemic dynamics driven by population genetic responses of parasites to microbial interactions?Question 3: To what degree are epidemic dynamics driven by plastic responses of individual parasites to microbial interactions?
Project Methods
This project will leverage a growing ecological and evolutionary model system (the widespread and agriculturally important grass tall fescue and its defensive symbiont Epichloë coenophiala, as well as multiple fungal parasites) to take a mechanistic experimental approach. It will characterize the linkage between the within-host microbial interactions and the transmission dynamics of the multi-parasite assemblage. The project will integrate diverse approaches ranging from field experimental manipulations of the species composition of the microbiome to transcriptomic analysis of parasite individuals.

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:During this reporting period, we reached at least three target audiences: fellow scientists, K-12 teachers, and middle school students. Specifically, we reached fellow scientists by publishing papers in peer-reviewed journals. Additionally, via outreach, we reached other target audiences, including both K-12 teachers and middle school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this reporting period, this project trained 3 graduate students, 4 undergraduate students, 3 technical IT staff, and 7 research technicians who are recent college graduates planning to pursue graduate work in ecology or related fields. Each undergraduate and graduate student received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students additionally received training in publishing research in scientific journals. Additionally, all students and research technicians received mentoring in career development. In terms of professional development, during this reporting period, this project supported the following oral and poster presentations at conferences and universities. Invited presentations: O'Keeffe, K.R. 2018. The effect of within-host microbial interactions on disease: from parasite individuals to populations. Invited oral presentation at the UNC Department of Biology Annual Symposium (one of two invited graduate student speakers out of 100+ graduate students). Mitchell, C.E. 2018. Pathogen species interactions across scales. North Carolina State University (Department of Entomology and Plant Pathology). Mitchell, C.E. 2018. Symbiont species interactions across scales. Rice University (Department of BioSciences series in Ecology and Evolutionary Biology). O'Keeffe, K.R. 2018. The effect of within-host microbial interactions on disease: from parasite individuals to populations. Stanford University (Department of Biology, ecology group). Halliday, FW. 2017. Diversity and interactions of foliar fungal parasites, from host organs to communities. University of Virginia (Department of Biology EBio Seminar Series). Contributed presentations: O'Keeffe, K., B.T. Wheeler, and C.E. Mitchell, 2018. A defensive symbiont may decrease the spread of a fungal plant pathogen through a host population but not within hosts. Ecological Society of America, New Orleans LA (Oral presentation). Halliday, F.W., J. Umbanhowar, C.E. Mitchell, 2018. A defense hormone and viral infection modify parasite epidemics and within-host priority effects in a grass host. Ecology & Evolution of Infectious Diseases Conference (16th Annual), University of Glasgow (Poster presentation). O'Keeffe, K., B.T. Wheeler, and C.E. Mitchell, 2018. A defensive symbiont decreases the spread of a fungal plant pathogen through a host population but not within hosts. Ecology & Evolution of Infectious Diseases Conference (16th Annual), University of Glasgow (Poster presentation). This project provided professional development activities for several participants: Mitchell, Halliday, and O'Keeffe attended the 16th Ecology and Evolution of Infectious Disease (EEID) Conference in Glasgow Scotland 29 May - 1 June 2018. Mitchell, Halliday, and O'Keeffe attended Duke University Symposium on Disease and Health: ecological perspectives from individuals to ecosystems 12 April 2018. Halliday attended one-day workshop on Presenting Data and Information by Edward Tufte, Raleigh, NC. O'Keeffe participated in the 2017 UNC 3 Minute Thesis Competition, in which she had to describe her dissertation research in under 3 minutes for a broad audience. O'Keeffe took a class in Fall 2017 through UNC's SPIRE postdoc program on college teaching practices. Geyer completed a one-day UNC workshop on using QIIME2 to process and analyze prokaryotic 16S microbiome sequencing data. Geyer completed a one-day UNC workshop on using the UNC computing cluster, Longleaf. How have the results been disseminated to communities of interest?Through the SciMatch program run through the NC Science Festival, O'Keeffe was connected to a middle school teacher at York Chester Middle School in Gastonia, North Carolina. In May 2018, she visited (in person, a 2.5 hour trip each way) three of the teacher's classes and taught students about epidemics with a lesson plan she had previously designed, and also discussed her research. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, and specifically testing the roles of population and community genetics, individual plasticity, and community structure. We will continue the seasonal spray experiment and the Epichloë field experiment. To test the role of microbial interactions in parasite epidemics at a more tractable scale under field conditions, we will conduct further kiddie pool experiments that build on O'Keeffe's pool experiments with Epichloë and Rhizoctonia. To improve on the less-than-expected percent of Epichloë-inoculated plants that tested positive for Epichloë infection, we will consult with experts on Epichloë, including our seed source at the University of Kentucky. To test whether Epichloë can modulate interactions between Rhizoctonia and Colletotrichum within a leaf, including responses of Rhizoctonia individuals, we will conduct a growth chamber experiment. We will further analyze and perhaps elaborate our mathematical model to dissect the roles of leaf-level and host population-level parasite interactions in epidemics. We will extend our work characterizing the fungal leaf microbiome and its relationship with parasite infection to include the prokaryotic leaf microbiome. We will continue to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses to microbial interactions.

Impacts
What was accomplished under these goals? This project investigated how transmission of microbial parasites across a population of hosts is influenced by differences between host individuals in their microbiomes. To do this, we conducted several field experiments and field surveys with the grass tall fescue, and extended our field results using a mathematical model. To understand the genetic basis of effects on parasite transmission, we developed new ways to analyze genetic shifts across known microbial populations and species, and new ways to analyze the genetic diversity of unknown microbial populations and species. To test the effects of shifts in the level of expression of microbial genes on parasite transmission, we identified a previously overlooked problem that may have undermined previous studies, and found a way for future studies to avoid that problem. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of parasite phenology on parasite epidemics, Halliday and O'Keeffe (with Newberry, Crews, Long, and Muirhead) continued the seasonal spray experiment, surveying leaves within tillers for parasite infection and leaf relative age each month year-round. We longitudinally tracked parasite infections of each leaf on 1152 tillers weekly June-October; each tiller was harvested and is being tested for infection by the non-parasite Epichloë. B-To test effects of Epichloë on parasite epidemics, we planted our proposed field experiment manipulating Epichloë infection of host populations, at three levels: 99%, 55%, and 10% of seed from plants putatively infected by Epichloë. Forty replicates per treatment yielded 120 fully randomized plots (each 2x6m). C-To test effects of Epichloë on Rhizoctonia epidemics, O'Keeffe and Wheeler grew either Epichloë-inoculated or Epichloë-free tall fescue in kiddie pools (13 multi-tillered plants per pool, and 13 replicates per treatment), then inoculated with Rhizoctonia. The purpose of the pools was to reduce transmission from outside each pool; to check this, additional pools were left uninoculated with Rhizoctonia. Twice weekly, we surveyed seven leaves per plant for infection by each parasite. Weekly, we also surveyed one tagged tiller per plant for percent leaf area infected. D-To explore shifts in the leaf microbiome associated with fungal parasites, we Illumina-sequenced fungal ITS1 of naturally (un)infected leaf sections. Now O'Keeffe is applying a pipeline developed by Carbone's group in T-BAS (see Other Products): First BLAST to place each sequence in a fungal family, then place it within that family's phylogeny, then use DADA2 (see Other Products) to test hypotheses using all unique sequence variants (not OTUs). E-To determine the conditions under which parasites can suppress each other's epidemics, Umbanhowar revised the structure of the mathematical model to better examine seasonal dynamics. He then performed a nullcline analysis. 2-DATA A-In the seasonal spray experiment, we collected data on: (i) monthly prevalence of parasite infections, stratified by tiller and leaf relative age, (ii) weekly parasite infection histories of individual leaves, and (iii) infection of each tagged tiller by Epichloë (lab tests ongoing). B-In the Epichloë field experiment, we observed the density and growth of fescue. C-In the pool experiment, we collected data on infection prevalence, and parasite infection histories of individual leaves (including infection severity). Lab tests are ongoing for infection of each plant by Epichloë. D-In the microbiome survey, we obtained data on genetic sequences of foliar fungi at ITS1. E-In the mathematical model, we observed predicted population growth rates of each parasite. 3-RESULTS A-In the seasonal spray experiment, the monthly prevalence survey yielded 768 plot-level observations based on about 61,000 leaf-level observations (stratified by tiller and leaf relative age). We obtained about 10,000 infection histories of individual leaves. We are still testing tillers for Epichloë. B-In the Epichloë field experiment, a tall fescue population established in each plot. Populations varied in density; most appeared modest but adequate. Individual plants produced up to 12 tillers. C-In the pool experiment, the prevalence survey yielded 208 pool-level observations based on 2704 plant-level observations. We obtained 944 infection histories of individual leaves. Of Epichloë-inoculated plants tested for Epichloë so far, 44% tested positive; this is less than expected, but seems adequate. The pools largely prevented colonization by parasites except Puccinia and Magnaporthe grisea (cause of gray leaf spot on tall fescue). D-In the microbiome survey, we obtained 6,650,600 paired-end reads. DADA2 (see Other Products) detected 3659 unique sequencing variants. Data analysis is ongoing. E-In the mathematical model, the nullcline analysis indicated that the parasite species can compete both at the level of a leaf and at the level of the host population. 4-OUTCOMES We made substantial progress by establishing our proposed field experiment, conducting the second year of the seasonal spray experiment, and moving three other activities toward completion. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics and community genetic structure, O'Keeffe cultured fungi from symptomatic leaves of tagged tillers in the seasonal spray experiment. Cultures that, based on both morphology ITS, fell in the Rhizoctonia species complex are being sequenced by Carbone's group at multiple loci. 2-DATA For each culture, morphospecies was recorded. ITS sequences were obtained, and ongoing sequencing is yielding multi-locus haplotypes, including multiple alleles, if present, in a single individual. 3-RESULTS The seasonal spray experiment yielded 78 cultures of the target parasite taxon, the Rhizoctonia species complex. However, very few of the tagged tillers tested negative for Epichloë. 4-OUTCOMES This year's approach of using tagged tillers yielded a much larger sample of the target parasite taxon, but a surprising lack of hosts uninfected by Epichloë. These samples may not allow us to test effects of Epichloë, but should allow us to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses of the Rhizoctonia species complex to other parasite taxa. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, O'Keeffe and Jones computationally simulated RNA-seq of a fungus-infected plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using four different aligners. New in this reporting period: the fourth aligner (MapSplice2) was analyzed, and the manuscript was submitted, revised, then accepted. 2-DATA Data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome - as is common practice with non-model organisms - resulted in either a failure to map the reads (with two aligners) or (with the other two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis then demonstrated a solution that problem (O'Keeffe and Jones, in press).

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: OKeeffe, K.R., and C.D. Jones, in press. Challenges and Solutions for Analyzing Dual RNA-seq Data for Non-Model Host/Pathogen Systems. Methods in Ecology and Evolution.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: F.W. Halliday, J. Umbanhowar, C.E. Mitchell, in press. A host immune hormone modifies parasite species interactions and epidemics: insights from a field manipulation. Proceedings of the Royal Society B. (https://doi.org/10.1098/rspb.2018.2075).
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: F.W. Halliday, R.W. Heckman, P.A. Wilfahrt, C.E. Mitchell, in press. Past is prologue: Host community assembly and the risk of infectious disease over time. Ecology Letters. (https://doi.org/10.1111/ele.13176).


Progress 09/01/16 to 08/31/17

Outputs
Target Audience:During this reporting period, we reached at least three target audiences: fellow scientists, K-12 teachers, and middle school students. Specifically, among scientists, we reached plant biologists by publishing O'Keeffe et al. 2017 in Current Opinion in Plant Biology, and we reached ecologists by publishing Halliday et al. 2017 in Ecology Letters. Additionally, via outreach, we reached other target audiences, including both K-12 teachers and middle school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In terms of training, during this reporting period, this project trained 3 graduate students, 2 undergraduate students, 2 technical IT staff, and 3 research technicians who are recent college graduates planning to pursue graduate work in ecology. Each undergraduate and graduate student received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students additionally received training in publishing research in scientific journals. Additionally, all students and research technicians received mentoring on career development. Finally, UNC PhD student Kayleigh O'Keeffe completed training on science communication (IMPACTS, based on the NSF-funded program, Portal to the Public) provided by UNC's Morehead Planetarium and Science Center, the project's partner in outreach. In terms of professional development, during this reporting period, this project supported the following oral and poster presentations at conferences and universities. Invited presentations: Mitchell, C.E., 2017: Parasite species interactions across scales. Fudan University, Shanghai (School of Life Sciences). Mitchell, C.E., J. Umbanhowar, 2017. Parasite species interactions and epidemics. SIAM (Society for Industrial and Applied Mathematics) Central States Section Conference. Colorado State University. Mitchell, C.E., F.W. Halliday, K.R. O'Keeffe, A. Simha, J. Umbanhowar, 2017. Parasite species interactions across spatial and temporal scales. Ecology & Evolution of Infectious Diseases Conference. University of California - Santa Barbara. Contributed presentations: O'Keeffe, K. and C.E. Mitchell, 2017. Effects of endophyte symbiosis and pathogen intraspecific variation on the growth of a fungal plant pathogen. Ecological Society of America, Portland OR (Poster presentation). O'Keeffe, K., C.D. Jones, C.E. Mitchell, 2017. Challenges for analyzing dual RNA-seq data for non-model host/pathogen systems. Ecology & Evolution of Infectious Diseases Conference (15th Annual), University of California - Santa Barbara (Poster presentation). How have the results been disseminated to communities of interest?During the reporting period, the project's three outreach activities were all conducted by UNC PhD student Kayleigh O'Keeffe. SciREN (The Scientific Research and Education Network): Kayleigh designed a lesson plan for educators based on our research project. She then took the lesson plan to the 2016 SciREN Triangle event. At the event, approximately 30-35 NC teachers (chiefly middle school) approached her to discuss the lesson plan, expressing interest in adopting it for their classrooms. Skype-a-scientist: Kayleigh participated in Skype-a-scientist throughout the 2016-2017 school year. She Skyped with 3 middle school classes, each with 15-20 students, at a charter school in Kentucky, and discussed what it's like to be a scientist, how she got to be a PhD student at UNC working on plant disease, and what her research is on. Scientists of NC: Kayleigh was featured on this online project (modeled on the popular Humans of New York), which has hundreds of followers and has the goal of humanizing scientists. The feature discussed topics including how she chose her career path, some of the challenges of science, and how she spends her free time. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, and specifically testing the roles of individual plasticity, population genetics, and community structure. To do this, our main approach will be to conduct further experiments, both in growth chambers and in the field. We will extend the results of our experiments using observational data and mathematical models.

Impacts
What was accomplished under these goals? For the grass tall fescue, grown for hay, a field experiment demonstrated that a parasite that establishes first in a host population can obtain a head-start advantage over other parasites. Specifically, establishing first can allow that parasite to (1) infect leaves prior to other parasites, (2) thereby alter the composition of the leaf microbiome, and (3) ultimately suppress epidemics by other parasites. Which parasites had epidemics was determined by the order in which the parasites established. Therefore, which parasites have epidemics may be altered by factors that change the order in which parasites establish. Such factors could include weather, no-till farming, planting date, and timing of pesticide applications. This project provides a potentially general principle that may be applied to suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES This grant supported publication of Halliday et al. 2017 in Ecology Letters (above impact statement). To extend Halliday et al. 2017, UNC PhD students Fletcher Halliday and Kayleigh O'Keeffe attempted to manipulate the order in which parasites infected host populations (the "seasonal spray experiment"). For each of three focal parasites, a corresponding treatment sprayed replicate host populations (plots) with fungicide until that parasite's epidemic started. In the 64 plots, we deployed 1712 Epichloë-inoculated and Epichloë-free sentinel hosts in 3 cohorts. To execute our proposed field experiment manipulating Epichloë infection of replicate host populations, we moved forward on two fronts. (1) We planted a pilot experiment. (2) UNC PhD student Bradley Saul used a power analysis to estimate our ability to test the hypothesis that a defensive symbiont can indirectly protect symbiont-uninfected hosts from parasite transmission. To test the effects of Epichloë on Rhizoctonia epidemics, UNC undergraduate Brandon Wheeler, mentored by Kayleigh O'Keeffe, grew Epichloë-inoculated and Epichloë-free tall fescue in replicate kiddie pools, then inoculated them with Rhizoctonia. The purpose of the pools was to reduce transmission from outside each pool; to check this, additional pools were left uninoculated with Rhizoctonia. To investigate shifts in the leaf microbiome associated with infection by fungal parasites, we are using a metabarcoding approach. O'Keeffe and Halliday collected 420 leaf sections infected and uninfected by each of our three focal parasites from 60 gridded locations in the field. We are now extracting DNA; we will characterize the fungal microbiome on Illumina. To extend Halliday et al. 2017 by determining the conditions under which Colletotrichum and Rhizoctonia can suppress each other's epidemics, co-PD James Umbanhowar developed a mathematical model. 2-DATA In the seasonal spray experiment, we collected data on: (A) leaf-level parasite infection of each sentinel plant (weekly longitudinal survey), (B) infection of each sentinel plant by Epichloë (end-of-season harvest; preserved for testing), and (C) leaf-level parasite infection of resident plants (monthly), (D) sentinel plant root biomass and colonization by arbuscular mycorrhizal fungi (end-of-season harvest; shipped to collaborator Megan Rúa). In the pilot field experiment, we observed the density of fescue and other plant species. In the pool experiment, we collected data on the proportion of leaves infected by each parasite (weekly). At the end of the season, we also surveyed 5 leaves per plant for percent leaf area infected by each parasite. 3-RESULTS In the seasonal spray experiment, the surveys of infection of resident plants yielded 23,000 observations. The surveys of sentinel plants yielded 100,000 observations. In the pilot field experiment, the density of fescue was low relative other plant species. Therefore, we are conducting a pilot 2.0, and seeding earlier. Based on the power analysis, we will increase replication to at least 100 plots. The pools were highly effective at preventing colonization by all parasites except Puccinia. The weekly survey yielded 3900 observations. The severity survey yielded 1300 observations. This experiment also serves as a pilot for the parasite density - population growth experiments. In the metabarcoding survey, we collected data on cover of tall fescue at each of the 60 locations. In the mathematical model, if there is no soil transmission of Rhizoctonia, alternative stable states are possible. If there is soil transmission, its rate determines whether there is stable coexistence or whether Rhizoctonia excludes Colletotrichum. This non-spatial model can be expanded into a spatially explicit dynamical model. 4-OUTCOMES Halliday et al. 2017 described a major change in knowledge regarding the role of the microbiome in determining which parasites can have epidemics. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics, O'Keeffe cultured fungi from symptomatic leaves of sentinel plants in the seasonal spray experiment. O'Keeffe et al. 2017 synthesizes scientific understanding on population genetics and on phenotypic plasticity, arguing that studies directly integrating both areas are required. 2-DATA For each culture from the seasonal spray experiment, morphospecies was recorded. Amplicon sequencing will be used to distinguish fungal taxa. 3-RESULTS The seasonal spray experiment yielded 250 cultures. Cultures will be sequenced at multiple loci, and we will use coalescent models to characterize population genetic responses to microbial interactions. 4-OUTCOMES O'Keeffe et al. 2017 describes a change in knowledge regarding the scientific approaches required to understand the evolutionary consequences of interactions in the plant microbiome. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, O'Keeffe (with co-PD Jones) computationally simulated RNA-seq of a fungus infecting a plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using three different aligners. To test the importance of the order in which parasites infect a leaf, UNC undergraduate Anita Simha (mentored by O'Keeffe) conducted a growth chamber experiment (GCE1) manipulating the order of infection by Rhizoctonia and Colletotrichum. To test for genetic variation in microbial interactions, O'Keeffe conducted a growth chamber experiment (GCE2) in which plants were grown from endophyte-inoculated or endophyte-free seed and exposed to one of three isolates of Rhizoctonia. 2-DATA In the computational study of RNA-seq, data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. In both growth chamber experiments, data was collected on the length of each Rhizoctonia lesion (daily). 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome - as is common practice with non-model organisms - resulted in either a failure to map the reads (with one aligner) or (with two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. In GCE1, Colletotrichum facilitated Rhizoctonia. The strength of facilitation depended on order of arrival. In GCE2, the Rhizoctonia isolates differed in lesion growth rate, but this was not influenced by Epichloë. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis then demonstrated a solution that problem. Together, these results comprise a substantial change in knowledge (O'Keeffe and Jones, manuscript in preparation).

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: F.W. Halliday, J. Umbanhowar, C.E. Mitchell, 2017. Interactions among symbionts operate across scales to influence parasite epidemics. Ecology Letters 20(10):12851294. (http://dx.doi.org/10.1111/ele.12825).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: OKeeffe, K.R., Ignazio Carbone, C.D. Jones, C.E. Mitchell, 2017. Plastic potential: how the phenotypes and adaptations of pathogens are influenced by microbial interactions within plants. Current Opinion in Plant Biology 38:7883. (https://doi.org/10.1016/j.pbi.2017.04.014). (Invited).
  • Type: Websites Status: Published Year Published: 2016 Citation: http://fescuefungi.org/