Jeff Dangl , UNC Chapel Hill Phil Hugenholtz , Susannah Tringe , JGI Ruth Ley, Cornell

1. Rhizosphere Grand Challenge Pilot Project Scott Clingenpeel Project Scientist, JGI Jeff Dangl, UNC Chapel Hill Phil Hugenholtz, Susannah Tringe, JGI Ruth Ley, Cornell

3. Specific Questions • Do plant roots assemble specific microbial rhizosphere and endophyte communities? • Do different mutants or natural genotypes within a plant species assemble different microbial communities? • Can the plant genes controlling these microbial communities be determined? • What does the microbial community offer the plant, and how does the plant communicate with its microbiota?

4. Approach to Answer the Questions • Grow multiple genotypes of Arabidopsis and corn (maize) in natural soils. • Characterize the rhizosphere and root endophyte microbial communities using pyrotags and iTAGs. • Characterize the function of the microbial communities using metagenomics and metatranscriptomics. • To focus on specific plant-microbe interactions by obtaining single cell genomes and plant transcriptomes.

5. Rhizosphere DNA isolation Obtaining Cells from Rhizosphere Remove soil to within 1mm radius from roots Spin Collection Tube. Transfer pellet to 1.5mL tube Place roots in sterile phosphate buffer with Silwet Shake 150 rpm, 30min; sonicate =rhizosphere Derek Lundberg, UNC collection tube

6. Microbial Community Characterization in Arabidopsis

7. Though soils differ, Arabidopsis rhizospheres reliably assemble several OTUs Col-0 n=8 MF Soil n=3 Mason Farm Nov. 2008 Mason Farm Jan. 2009 Col-0 n=17 MF Soil n=10 Col-0 n=3 CSoil n=3 Clayton Nov. 2008 Rank Abundance Curves, ranked by Mason Farm Nov. 2008 soil, showing the percent abundance of each major OTU in every sample From Dangl lab, April 15, 2010 Pyrotagger run

8. The rhizosphere of Arabidopsis missing efr1 have OTUs with different abundances than wild type There are a few OTUs that have different abundances in efr1fls2 plant. From Dangl lab, April 15, 2010 Pyrotagger run

9. Bray Curtis similarity of samples from three experiments, using OTUs above 0.5% in any sample Clayton Exp. 1 Nov. 2008 Mason Farm Exp. 1 Nov. 2008 Mason Farm Exp. 2 Jan. 2009 • Samples cluster by soil type or experiment date • Bulk soil clearly differs from rhizospheres • No separation of genotypes based on rhizospheres From Dangl lab, April 15, 2010 Pyrotagger run

10. Single Cell Genomics • Provides context for interpreting metagenomes and metatranscriptomes • Provides insight into metabolic capability of particular organisms that are enriched by the plant • This could begin to tell us what the plant is getting from the microbes and what they are getting from the plant

11. Methods • Fluorescently Activated Cell Sorting (FACS) • Multiple Displacement Amplification (MDA) of the single cell genome • Amplify 16S rRNA gene • Screen for organisms of interest • Sequence the MDA products of organisms of interest • Analyze the genomic data

12. Fluorescently Activated Cell Sorting (FACS) Flow Nozzle Laser Cell Flow Nozzle Side Scatter Photomultiplier tubes Laser Cell Forward Scatter Laser Cell Sorted Cells Fluorescence

13. Multiple Displacement Amplification (MDA) + Denature DNA Random Primers Amplification Bacterial Genome Lots of Copies!

14. Microbes to Focus On • 150 OTUs are enriched in the rhizosphere of wild type Arabidopsis by at least 2 fold over bulk soil

15. Chances of Getting What We Want • Getting a specific enriched OTU is unlikely without screening lots of cells • Most abundances are less than 1% of the community • Getting OTUs of interest is likely • Total abundance of rhizosphere-enriched OTUs where no genome exists in the same family is 21% • Variability means it can be useful to test multiple samples • Abundances between replicates can differ by several fold • High throughput process • If we test enough cells, eventually we should get what we want