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The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato

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The plant protein ARGONAUTE1 (AGO1) functions in multiple RNA-silencing pathways, including those of microRNAs, key regulators of growth and development. Genetic analysis of ago1 mutants with informative defects has provided valuable insights into AGO1’s biological functions. Tomato encodes two AGO1 homologs (SlAGO1s), but mutants have not been described to date. To analyze SlAGO1s’ involvement in development, we confirmed that both undergo decay in the presence of the Polerovirus silencing suppressor P0 and produce a transgenic responder line (OP:P0HA) that, upon transactivation, expresses P0 C-terminally fused to a hemagglutinin (HA) tag (P0HA) and destabilizes SlAGO1s at the site of expression. By crossing OP:P0HA with a battery of driver lines, constitutive as well as organ- and stage-specific SlAGO1 downregulation was induced in the F1 progeny. Activated plants exhibited various developmental phenotypes that partially overlapped with those of Arabidopsis ago1 mutants. Plants that constitutively expressed P0HA had reduced SlAGO1 levels and increased accumulation of miRNA targets, indicating compromised SlAGO1-mediated silencing. Consistent with this, they exhibited pleiotropic morphological defects and their growth was arrested post-germination. Transactivation of P0HA in young leaf and floral organ primordia dramatically modified corresponding organ morphology, including the radialization of leaflets, petals and anthers, suggesting that SlAGO1s’ activities are required for normal lateral organ development and polarity. Overall, our results suggest that the OP:P0HA responder line can serve as a valuable tool to suppress SlAGO1 silencing pathways in tomato. The suppression of additional SlAGOs by P0HA and its contribution to the observed phenotypes awaits investigation.

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Polerovirus silencing suppressor


Solanum lycopersicum


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We thank Véronique Ziegler-Graff, Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg, France, for the P0 plasmid. We thank Hanita Zemach and Eduard Belausov for excellent technical assistance in histology and microscopy, respectively. This work was supported by a grant from the Chief Scientist of the Israel Ministry of Agriculture and Rural Development no. 256-0828-10 to T.A. This is contribution no. 116/2012 series, from the Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel.

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Correspondence to Tzahi Arazi.

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Supplementary Figure S3.Transgenic plant validation. a Schematic representation of the binary constructs used for transgenic expression of P0HA under the OP promoter and LhG4 under the 35S promoter. Restriction enzymes used for cloning are indicated. Eight copies of the lac operator sequence linked to a minimal Cauliflower mosaic virus (CaMV) 35S promoter (OP-TATA), cauliflower mosaic virus 35S promoter (35S), octopine synthase terminator (3’ OCS), selectable kanamycin-resistance marker (nptII), nopaline synthase promoter (pNOS), nopaline synthase terminator (3’ NOS). RB – right border; LB – left border. b Genomic DNA PCR analysis of representative tomato OP:P0HA reporter lines and 35S:LhG4 driver line. Specificity of primers used is indicated on the right. (TIFF 361 kb)


Supplementary Figure S4.Effects of leaf-specific P0HA expression on cotyledon development. Control (FIL:LhG4) and P0HA-expressing genotypes are indicated. Scale bar = 5 mm. (TIFF 524 kb)


Supplementary Figure S5.Specific P0HA expression increases the levels of miRNA-target transcripts in the expressing tissue. Quantitative RT-PCR analysis of selected miRNA-target transcripts from the indicated genotypes in apices collected from a 1-month-old seedling (a) and 2–3 mm buds (b). Primers were designed around the corresponding miRNA complementary site. TIP41 expression values were used for normalization. Data are means ± SE of two biological replicates, each measured in triplicate. Asterisks indicate statistically significant difference compared to control as determined by Student’s t test (P ≤ 0.05). (TIFF 68 kb)


Supplementary Figure S6.Cross sections of red fruit of the control (OP:P0HA) and indicated genotypes. Scale bar = 1 mm. (TIFF 1525 kb)

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Hendelman, A., Kravchik, M., Stav, R. et al. The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato. Planta 237, 363–377 (2013).

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