A nat-siRNA is induced by Pst (avrRpt2). (A) Detection of the nat-siRNA by Northern blot analysis. The nat-siRNA sequence is shown under the panel. Small RNA was extracted from the leaves harvested at 15 hpi of Pst (2 × 10⁷ cfu/ml) carrying various avr genes and an oligonucleotide probe complementary to the siRNA was used. DNA probe was used for detecting U6 RNA for measuring the relative abundance (RA) (shown below). Ethidium bromide-stained tRNA is also shown as a loading control. (B and C) Relative expression levels of ATGB2 (1B) and PPRL (1C) as measured by real-time RT-PCR. The expression levels of AtGB2 and PPRL were normalized to that of ubiquitin. (D) Northern blot analysis shows reduced ATGB2 and nat-siRNAATGB2 expression levels in Salk_083103 homozygous line upon Pst (avrRpt2) (2 × 10⁷ cfu/ml) challenge comparing to the WT Col-0. The levels of actin and U6 were used for quantification and loading controls. (E) Relative mRNA level of PPRL in Salk_083103 homozygous line. The expression levels in untreated WT Col-0 were used as 100 % and standard deviations were plotted from three replicates (B, C, and E).

Accumulation of nat-siRNAATGB2 depends on DCL1, HYL1, and RDR6, and also requires HEN1, NRPD1a, and SGS3. (A) Northern blot analysis of nat-siRNAATGB2 in various Pst (avrRpt2)-treated small RNA biogenesis mutants and their corresponding WT controls. MiR171 and U6 RNA was used as controls. U6 level was used for quantification. (B) Relative PPRL mRNA levels in sgs3, dcl1-9, hyl1, rdr6, nrpd1a, and their corresponding WT controls after Pst (avrRpt2) infection. The expression levels were normalized to that of ubiquitin. The expression level in untreated WT Col-0 was used as 100%. Standard deviations were plotted from three replicates. (C) Transient coexpression of PPRL and ATGB2 in N. benthamiana. Agrobacterium GV3101 harboring PPRL was coinfiltrated with GV3101 carrying full-length (F) or only the overlapping region (O) of ATGB2 constructs into 3-week-old N. benthamiana leaves. The expression of AtGB2 was induced by Dex at 2 dpi, and tissue was harvested after 24 h of induction. The expression of PPRL was measured at both RNA and protein levels by semiquantitative RT-PCR (Top) and Western blot analysis (Sigma anti-FLAG, 1:2,000 dilution, Middle), respectively. Actin was used as a control for RT-PCR. The Rubisco large subunit from a gel that was run in parallel was stained with Coomassie blue for Western blot loading control. nat-siRNAATGB2 is detected after the induction of either full-length or overlapping region of ATGB2. (Bottom) tRNA was used as a loading control.

Accumulation of nat-siRNAATGB2 is controlled by RPS2 and some components of the disease resistance signaling pathway. Northern blot analysis of nat-siRNAATGB2 (A) and ATGB2 (B) was performed on Pst (avrRpt2)-treated defense-signaling mutants and WT Col-0 plants. U6 RNA was used for small RNA quantification. (C) Relative quantification of PPRL expression in defense signaling mutants by real-time RT-PCR analysis. PPRL expression level was normalized to that of ubiquitin. The expression levels in untreated WT Col-0 were used as 100%. Standard deviations were plotted from three replicates.

Overexpression of PPRL attenuates RPS2-mediated resistance in Arabidopsis plants. (A) Western blot analysis of transgenic Arabidopsis plants overexpressing PPRL (Sigma anti-FLAG, 1:2,000 dilution). Shown is the Rubisco large subunit from a gel that was run in parallel and stained with Coomassie blue. Two lines with high (line 32) or low (line 33) expression level of PPRL were selected for phenotypic analysis. (B) PPRL overexpression line displays delayed HR. Picture of line 32 was taken at 16 hpi of Pst (avrRpt2) (2 × 10⁷ cfu/ml). (C) PPRL overexpression lines exhibit reduced electrolyte leakage. Plants treated with 10 mM MgCl₂ and Pst (avrRpt2) (1 × 10⁷ cfu/ml) were measured at 0 and 24 hpi. Error bars represent standard deviation of four replicates. Similar results were obtained from two independent experiments. (D) PPRL overexpression lines display enhanced pathogen growth of Pst (avrRpt2). Bacterial growth was measured at 0 and 4 dpi of Pst carrying EV, avrRpt2 or avrRpm1 (2 × 10⁵ cfu/ml). Error bars represent standard deviation of five replicates. Similar results were obtained in three independent experiments. (E) Pst (avrRpt2) accumulates to a higher level in rdr6 and hyl1, but not in dcl3, as compared with that in the corresponding WT C24, No, or Col-0, respectively. No difference was observed in the growth of Pst (avrRpm1) between the mutants rdr6, hyl1, or dcl3 and their corresponding WT control. Pathogen growth was measured at 0 and 4 dpi. Similar results were obtained in two independent experiments.

Model for nat-siRNAATGB2 biogenesis and function. Components in red are required for nat-siRNAATGB2 formation. RISC, RNA-induced silencing complex.