Extended Data Fig. 1: Validation of the root fractionation protocol and assessment of primer amplification bias.

a, Protocol to fractionate four microbial niches across a distance gradient from bulk soil to roots’ interior. Roots of A. thaliana grown in their natural environments were briefly washed (1) to separate loosely attached soil particles from the root surface (rhizosphere, RS). After a second washing step, roots were vigorously washed with detergent (three times) to capture microbes that tightly adhere to the root surface (2). The resulting washes were then filtered through a 0.22 µM membrane (rhizoplane, RP). Finally, surface sterilization of root samples by consecutive EtOH and NaClO washes enriched the final root sample in microbial root endophytes (3). b, Validation of the fractionation protocol (depicted in panel a) was performed by printing root washes (left panel) and washed roots (right panel) on 50% Tryptic Soy Agar medium. Sequential detergent washes efficiently release microbes from the root surface and further root surface sterilization prevents the growth of rhizoplane-associated microbes (Wilcoxon rank sum test, P < 0.01). All three detergent steps were combined and filtered to prepare the RP fraction (light green dots, left panel). c, Comparison of bacterial (left panel) and fungal (right panel) classes profiled with the V3V4 and V5V7 regions of the bacterial 16 s rRNA gene, and the ITS1 and ITS2 of the fungal ITS. The correlation between the RA of each microbial class is shown (Pearson correlation: P < 0.001).