Factors Shaping the Root-Associated Microbiome of Populus species

1. Factors Shaping the Root-Associated Microbiome of Populus species Christopher W. Schadt Oak Ridge National Laboratory & University of Tennessee - Knoxville

2. Plant-Microbe Interfaces Aim 1: Define Aim 2: Dissect fungi plants bacteria Natural Systems Components Interactions & Signaling

3. PMI Plant-Microbe Interactions: Genotype & Environment Interactions in Structuring the Populus Microbiome Caney Fork River, TN Populus deltoides (2009-2011 Campaigns) Populus trichocarpa (2012 & Beyond) Yadkin River, NC Studies focused on broad associations of microbiome patterns within natural riparian populations in the Southeast. Studies of common-garden GWAS populations that will allow examination of detailed genome-level associations between host & microbiome

4. Campaign 1: Caney Fork Study of Upland and Bottomland Root Endophyte and Rhizosphere Communities Objective: Assess the composition and variability within and between, endosphere and rhizosphere communities Gottel, NR, et al. (2011). Populus deltoides roots harbor distinct microbial communities within the endosphere and rhizosphere across contrasting soil types. Appl. Environ. Microbiol. 77, 5934-5944. N. Gottel H. Castro

5. Endosphere and rhizosphere communities are distinct regardless of origin from upland or bottomland sites Bacteria Endo|Rhizo Fungi Endo|Rhizo Fungi Hierarchical clustering of OTUs also shows most appear specialized to either the rhizosphere or endosphere niche, and are rarely in abundance in both PCA analysis shows distinct endophyte and rhizosphere communities regardless of upland or bottomland origin

6. Caney Fork River, TN Campaign 2: A P. deltoides regional study • Objectives • Verify the separation between the endosphere and rhizosphere microbial niches • Examination of seasonal effects on microbiomestructure • Test the effects of soil and host characteristics across a broad range of natural conditions Yadkin River, NC M. Shakya W. Muchero H. Castro

7. P. deltoides genotypes and soils from TN and NC are diverse, but covary by region • Soils exhibit a wide range of variation. pH, organic matter, nitrogen, and soil texture vary widely across the sites • SSR genotyping of trees shows a variation in population structure with division between samples originating from different regions Soil texture across sites Plant genotypes across sites

8. NMDS analyses of Caney Fork and Yadkin datasets show partitioning by sample type and region • Rhizosphere/Endosphere divide is confirmed • Variability between endophyte samples is extreme (but diversity low) • Structure according to regional origin and season is also significant

9. Disentangling the drivers of community patterns remain an elusive task • Variance partitioning between a suite of host and environmental factors explains only 33% to 56% of community-wide microbiome variation • Within Phyla & and dominant OTUs more significant trends are observed Bacteria Fungi

10. Common garden / GWAS populations of Populustrichocarpain the Pacific Northwest • 1100 variants of P. trichocarpacollected across it’s latitudinal range replicated in four common gardens in BC, OR and CA • >900 of these have been resequenced to at least 12X depth by JGI. W. Muchero G. Tuskan

11. 2012 CSP project examining associations of host characteristics and the root microbiome • Plant Growth • (Biomass) • Secondary Chemistry • (Lignin S:G) • Pathogen Susceptibility • (Meampsora infection rates) • Copy number variation • (Lectin Receptor Kinase)

12. Larger scale of analyses facilitated by new JGI collaboration via 2012 CSP program • Combination of large scale rRNA community profiling multiplexed on MiSeqIllumina system (all samples, all traits) • 8 genotypes selected at extremes for 4 traits (32 genotypes) • 32 genotypes w/ 3 replicates in 2 locations (192 trees) • Endophyte, rhizospshere & bulk soil samples (576 samples) • Bacterial, fungal & archaeal community (1728 rRNA profiles) • In depth metagenomic profiles using 32 lanes of HiSeqIllumina profiling (select sample/trait)

13. What is the genomic and functional diversity within Populus isolates? g-Proteobacteria S. Brown S. Utturkar D. Pelletier G. Bonito R. Vilgalys T. Karpinets b-Proteobacteria • Isolated 2800+ bacteria & 900+ fungi • 43 bacterial isolates have been sequenced from P. deltoideswith a concentration on Pseudomonas • 6 fungal isolates underway through various JGI programs a-Proteobacteria Bacteroidetes Bacilli

14. Pseudomonas spp. show high functional diversity

15. Often same types of endophytic isolates seen across Populus (ORNL) and Arabidopsis (Dangle) UNC ORNL Sur Paredes

16. Acknowledgements Schadt Lab at ORNL Migun Shakya Mike Robeson Zamin Yang Neil Gottel Hector Castro Marilyn Kerley ORNL Collaborators Jerry Tuskan Mitch Doktycz Dale Pelletier Mircea Podar Jessy L. Labbé Wellington Muchero Tatiana Karpinets Steven Brown Sagar Utturkar Duke University Rytas Villgalys Gregory Bonito Khalid Hameed Jay Krishnan Michael Gajdeczka Andrii Grygansky JGI Susannah Tringe Stephanie Malfatti Tijana Glavina del Rio INRA Francis Martin