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

2. Natural SystemsComponents Aim 1: DefineAim 2: Dissect plantsfungi bacteria Interactions & Signaling Plant-Microbe Interfaces

3. Caney Fork River, TN Yadkin River, NC PMI Plant-Microbe Interactions: Genotype & Environment Interactions in Structuring the Populus Microbiome Populus deltoides (2009-2011 Campaigns) Populus trichocarpa (2012 & Beyond) 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. 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. Objective: Assess the composition and variability within and between, endosphere and rhizosphere communities Campaign 1: Caney Fork Study of Upland and Bottomland Root Endophyte and Rhizosphere Communities N. GottelH. Castro

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

6. Caney Fork River, TN Yadkin River, NC Campaign 2: A P. deltoides regional study Objectives Verify the separation between the endosphere and rhizosphere microbial niches Examination of seasonal effects on microbiome structure Test the effects of soil and host characteristics across a broad range of natural conditions M. ShakyaW. 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 sitesPlant genotypes across sites

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

9. 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 Disentangling the drivers of community patterns remain an elusive task Fungi Bacteria

10. Common garden / GWAS populations of Populus trichocarpa in the Pacific Northwest 1100 variants of P. trichocarpa collected 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. G. Tuskan W. Muchero

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

12. Larger scale of analyses facilitated by new JGI collaboration via 2012 CSP program Combination of large scale rRNA community profiling multiplexed on MiSeq Illumina 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 HiSeq Illumina profiling (select sample/trait)

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

14. Pseudomonas spp. show high functional diversity TREEECO-UNITSTRAINAntiQSSidProtPhosIAA(ug/ml)NO3MHB B1BendophytePseudomonas GM172high++-0.2+neg B1BendophytePseudomonas GM21neg +--2.4-o B4ArhizospherePseudomonas GM491neg+--0.1-o B4ArhizospherePseudomonas GM481neg++-0.1-+ B2BendophytePseudomonas GM331neg+-+1.0-+ U2ArhizospherePseudomonas GM741neg+--1.0-o B4BendophytePseudomonas GM55neg +-+1.5-o B3BendophytePseudomonas GM411neg+++1.5++ B1BendophytePseudomonas GM181low+++0.6-+ B4BendophytePseudomonas GM502low+++1.0-o U3BendophytePseudomonas GM1021neg-+0.9-o U2BendophytePseudomonas GM792neg++-2.2++ B2BendophytePseudomonas GM303med+++-0.9-o B4BendophytePseudomonas GM244neg+++-1.5-neg B1BendophytePseudomonas GM163neg++++1.4-neg B2ArhizospherePseudomonas GM251neg+++0.8-o soil Pseudomonas fluorescens PfO1ND U2BendophytePseudomonas GM801neg+++-0.9-o B4BendophytePseudomonas GM60neg ++-0.9+o U1BendophytePseudomonas GM671neg+--1.0+o U2BendophytePseudomonas GM781neg+-+1.3-o Cotton rootPseudomonas fluorescens Pf52ND+++0.1neg U3ArhizospherePseudomonas GM841neg+-+1.7-o endophytePseudomonas putida W619ND pathogenPseudomonas syringae DC3000ND

15. ORNL Often same types of endophytic isolates seen across Populus (ORNL) and Arabidopsis (Dangle) UNC 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 JGI Susannah Tringe Stephanie Malfatti Tijana Glavina del Rio Duke University Rytas Villgalys Gregory Bonito Khalid Hameed Jay Krishnan Michael Gajdeczka Andrii Grygansky INRA Francis Martin