Supplementation and inhibition of P. microcarpus miR-8 alters colonization of E. grandis roots. (A) Relative Pmic_miR-8 levels in E. grandis mycorrhizal root tips treated with either mature, single-stranded Pmic_miR-8 (white bar), mature as Pmic_miR-8* (striped bar), or ds Pmic_miR-8 (light gray bar), as compared to treatment with a scrambled miRNA. We also tested E. grandis mycorrhizal root tips treated with either a single-stranded ZEN-tagged as Pmic_miR-8 inhibitor (i.e., repression; black bar) or a single-stranded as Pmic_miR-8 with designed bulge mismatch at nucleotides 10 to 11 (dark gray bar), as compared to a scrambled inhibitor sequence. All values are the result of three biological replicates, ±SE; * indicates significant difference from scrambled control (P < 0.05; Student’s t test). (B) Representative image of P. microcarpus mycorrhizal root tips on E. grandis showing a typical mycorrhizal phenotype. (C) Representative image of P. microcarpus mycorrhizal root tips on E. grandis treated with the Pmic_miR-8 inhibitor in the final week of fungal colonization. White arrows indicate mycorrhizal root tips that have senesced, and the root has recommenced growth. (D) Percent of E. grandis root tips in contact with P. microcarpus that are either colonized by the fungus (black bars) or that exhibit the beginning of colonization followed by senescence (light gray bars). Values are shown for either no spray treatment (No Treatment) or spray treated with scrambled miRNA (ssScrambled); as single-stranded, mature Pmic_miR-8 (ssmiR-8*); sense ds, mature Pmic_miR-8 (dsmiR-8); sense single-stranded, mature Pmic_miR-8 (ssmiR-8); scrambled as inhibitor (asScrambled); single-stranded, ZEN-tagged as Pmic_miR-8 inhibitor (asZEN); and single-stranded as Pmic_miR-8 with designed bulge mismatch at nucleotides 10 to 11 (asBulge). All values are based on six biological replicates, ±SE; */▵ indicates significant difference from scrambled control (P < 0.05; Student’s t test).