All samples were transported back to the lab at ambient temperature and refrigerated at 4 degrees Celsius for 24 hours prior to DNA extraction. Nucleic acid extraction Three hundred milliliters

of sterile distilled water were added to each ziplocked bag of plant parts and samples, which was sonicated for 6 minutes to disrupt cells and knock organisms from biofilms or other protective habitat associated with plant organs. This wash was centrifuged and DNA was extracted from the resulting pellet using the Promega Wizard® Genomic DNA purification Kit (Cat.# NSC23766 A1120) (Promega Corporation, Madison, WI) following the extraction protocol for Gram-positive bacterial species. 16S rRNA gene amplicon preparation PCR products designed to target the V2 region of 16S rRNA genes were Emricasan order amplified for Roche pyrosequencing (454) using Roche Fusion Primer A, key (TCAG), and MIDs (Multiplex identifiers for 24 individual samples) and the 27F universal primer: 5’ CGT ATC GCC TCC CTC GCG CCATCAGAGA GTT TGA TCC TGG CTC AG 3’ Reverse primer 533R was used with Roche Fusion Primer B, key, and no mids: 5’ CTA TGC

GCC TTG CCA GCC CGC TCAG CGA GAG ATA C TTA CCG CGG CTG CTG GCA C 3’ PCR fragments were cleaned (fragments under 300 bases were removed) using AMPure XP from Beckman Coulter Genomics (Danvers, Massachusetts) at a ratio of 60 μl of AMPure beads to 100 μl PCR product. Remaining PCR fragments were run on the Agilent Bioanalyzer 2100, using the High Sensitivity lab-on-a-chip Reagents (Agilent Technologies, Inc., Santa Clara, CA) to ensure that smaller fragments had been removed prior to emulsion PCR preparation. 18S rRNA gene amplicon preparation EF4 5’GGAAGGGRTGTATTTATTAG 3’ and Fung5 5’GTAAAAGTCCTGGT TCCCC 3’ [10] with 24 MIDs and Roche Fusion Primer adaptors A and B. PCR fragments were cleaned (removal

of fragments under 300 bases) using AMPure XP at a ratio of 60 μl of AMPure beads to 100 μl PCR product. Resulting PCR fragments were run on the Bioanalyzer 2100 using to ensure that smaller fragments had been removed prior to emulsion PCR preparation. Metagenome heptaminol preparation Four independent replicates from each plant organ were pooled to create one representative metagenome for each of the 6 regions: Top Leaves, Flowers, Fruits, Stems, Bottom Leaves, and Roots. DNA was sheared using the Covaris S2 (Woburn, Massachusetts) set for 200 cycles per burst, Duty cycle= 5%, Intensity= 3, for a total of 80 seconds. Emulsion PCR To allow optimal amplification in emulsion, 16S and 18S rRNA gene amplicons were p38 MAPK inhibitor diluted to estimate .3 copies of DNA per bead. Sheared whole genome shotgun (WGS) DNA for metagenomes was diluted to estimate between 3 and 9 copies per bead. Emulsion PCR and breaking and enriching was performed using the Lib-A MV kit for FLX Titanium pyrosequencing from Roche Diagnostics Corp. (Indianapolis, IN) according to the manufacturer’s specifications.