Fig. 3: tRF^31Asp2 induces immunity in an AGO2-clade dependent manner.

a PR1 expression in local and distal leaves at the indicated times after tRF^31Asp2 (1 µM) or buffer infiltration was examined by qRT-PCR. b Bacterial growth in WT, dcl1-7, and ago2/3/7 pretreated with tRF^31Asp2, tRF^31Asp2_mSS, or mock at 0 and 3 dpi with Pst and Pst_avrRpt2; the mean±RSE (n = 12) from three independent experiments is presented. c tRF^31Asp2 promotes enhanced resistance to aphids. The green peach aphid (GPA) numbers at 5 dpi on WT and ago2/3/7 leaves were pretreated with tRF^31Asp2, tRF^31Asp2_mSS; untreated (No) and the buffer (PBS)-treated WT used as a control. Plants were initially infested with three adult apterous aphids/plant after 24 hpi. The same experiment was conducted twice with similar results. The mean ± SE (n = 7 to 10) is presented. d Predicted secondary structure of full-length Asp-2-tRNA^GTC and its 31 nt-long 5′-tRF (tRF^31Asp2) by RNAfold. e PR1 induction by tRF^31Asp2 and its mutants at 24 hpi was examined by qRT-PCR; relative transcription levels are presented as the mean ± SD (n = 3). Bar colors correspond to target sites in (d). f Antisense oligonucleotides or buffer were infiltrated into leaves 1 day before distal-infection with Pst_avrRpt2; PR1 was assessed at 1 dpi by qRT-PCR; the mean ± SD (n = 4). Three independent experiments were performed with similar results. g PR1 expression in distal leaves of short tandem target mimic (STTM) and empty vector (EV) lines, and WT at 24 hpi with Pst_avrRpt2 or mock was examined by qRT-PCR; naive is without any treatment; the mean ± SE (n = 4). h, i PR1 induction in dcl and ago mutants at 24 hpi responding to 1 (h) and 20 (i) µM of tRF^31Asp2 and its mock control. The mean ± SE (n ≥ 4) is presented. j Bacterial growth in WT, ago2-1, and AGO2 complementation lines (DDD and DAD are WT and a slicer mutant, respectively) in ago2-1 at 0 and 3 day-post-infection (dpi) with Pst_avrRpt2 with the mean ± SE (n = 4); rps2 is an ETI control. Significance for indicated pairs is noted; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 (t-test except for c where Tukey’s HSD test was used).