Skip to main content
SpringerLink
Log in

The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

AGO1:

ARGONAUTE1

miRNA:

MicroRNA

PO:

Polerovirus silencing suppressor

Tomato:

Solanum lycopersicum

References

  • Alvarez J, Smyth DR (1999) CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS. Development 126:2377–2386

    PubMed  CAS  Google Scholar 

  • Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151

    Article  PubMed  CAS  Google Scholar 

  • Arazi T, Talmor-Neiman M, Stav R, Riese M, Huijser P, Baulcombe DC (2005) Cloning and characterization of micro-RNAs from moss. Plant J 43:837–848

    Article  PubMed  CAS  Google Scholar 

  • Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102:11928–11933

    Article  PubMed  CAS  Google Scholar 

  • Baumberger N, Tsai CH, Lie M, Havecker E, Baulcombe DC (2007) The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation. Curr Biol 17:1609–1614

    Article  PubMed  CAS  Google Scholar 

  • Berger Y, Harpaz-Saad S, Brand A, Melnik H, Sirding N, Alvarez JP, Zinder M, Samach A, Eshed Y, Ori N (2009) The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves. Development 136:823–832

    Article  PubMed  CAS  Google Scholar 

  • Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J 17:170–180

    Article  PubMed  CAS  Google Scholar 

  • Bortolamiol D, Pazhouhandeh M, Marrocco K, Genschik P, Ziegler-Graff V (2007) The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing. Curr Biol 17:1615–1621

    Article  PubMed  CAS  Google Scholar 

  • Bowman JL, Smyth DR (1999) CRABS CLAW, a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development 126:2387–2396

    PubMed  CAS  Google Scholar 

  • Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190

    Article  PubMed  CAS  Google Scholar 

  • Chen X (2009) Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25:21–44

    Article  PubMed  Google Scholar 

  • Csorba T, Lozsa R, Hutvagner G, Burgyan J (2010) Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1. Plant J 62:463–472

    Article  PubMed  CAS  Google Scholar 

  • Fernandez AI, Viron N, Alhagdow M, Karimi M, Jones M, Amsellem Z, Sicard A, Czerednik A, Angenent G, Grierson D et al (2009) Flexible tools for gene expression and silencing in tomato. Plant Physiol 151:1729–1740

    Article  PubMed  CAS  Google Scholar 

  • Garcia D (2008) A miRacle in plant development: role of microRNAs in cell differentiation and patterning. Semin Cell Dev Biol 19:586–595

    Article  PubMed  CAS  Google Scholar 

  • Giovannoni J (2001) Molecular biology of fruit maturation and ripening. Annu Rev Plant Physiol Plant Mol Biol 52:725–749

    Article  PubMed  CAS  Google Scholar 

  • Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207

    Article  PubMed  CAS  Google Scholar 

  • Hunter C, Sun H, Poethig RS (2003) The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr Biol 13:1734–1739

    Article  PubMed  CAS  Google Scholar 

  • Jack T, Brockman LL, Meyerowitz EM (1992) The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68:683–697

    Article  PubMed  CAS  Google Scholar 

  • Jay F, Wang Y, Yu A, Taconnat L, Pelletier S, Colot V, Renou JP, Voinnet O (2011) Misregulation of AUXIN RESPONSE FACTOR 8 underlies the developmental abnormalities caused by three distinct viral silencing suppressors in Arabidopsis. PLoS Pathog 7:e1002035

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jones L, Keining T, Eamens A, Vaistij FE (2006) Virus-induced gene silencing of Argonaute genes in Nicotiana benthamiana demonstrates that extensive systemic silencing requires Argonaute1-like and Argonaute4-like genes. Plant Physiol 141:598–606

    Article  PubMed  CAS  Google Scholar 

  • Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428:81–84

    Article  PubMed  CAS  Google Scholar 

  • Kidner CA, Martienssen RA (2005) The role of ARGONAUTE1 (AGO1) in meristem formation and identity. Dev Biol 280:504–517

    Article  PubMed  CAS  Google Scholar 

  • Lanet E, Delannoy E, Sormani R, Floris M, Brodersen P, Crete P, Voinnet O, Robaglia C (2009) Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell 21:1762–1768

    Article  PubMed  CAS  Google Scholar 

  • Lifschitz E, Eviatar T, Rozman A, Shalit A, Goldshmidt A, Amsellem Z, Alvarez JP, Eshed Y (2006) The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc Natl Acad Sci USA 103:6398–6403

    Article  PubMed  CAS  Google Scholar 

  • Lynn K, Fernandez A, Aida M, Sedbrook J, Tasaka M, Masson P, Barton MK (1999) The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126:469–481

    PubMed  CAS  Google Scholar 

  • Mandel MA, Gustafson-Brown C, Savidge B, Yanofsky MF (1992) Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature 360:273–277

    Article  PubMed  CAS  Google Scholar 

  • Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C et al (2008) Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133:116–127

    Article  PubMed  CAS  Google Scholar 

  • Moore I, Galweiler L, Grosskopf D, Schell J, Palme K (1998) A transcription activation system for regulated gene expression in transgenic plants. Proc Natl Acad Sci USA 95:376–381

    Article  PubMed  CAS  Google Scholar 

  • Moussian B, Schoof H, Haecker A, Jurgens G, Laux T (1998) Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J 17:1799–1809

    Article  PubMed  CAS  Google Scholar 

  • Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni I, Pekker I et al (2007) Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Genet 39:787–791

    Article  PubMed  CAS  Google Scholar 

  • Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez GR, Moyseenko JB, Robbins MD, Morejon NH, Francis DM, van der Knaap E (2010) Tomato analyzer: a useful software application to collect accurate and detailed morphological and colorimetric data from two-dimensional objects. J Vis Exp. doi:10.3791/1856

    PubMed  Google Scholar 

  • Sato S, Tabata S, Hirakawa H, Asamizu E, Shirasawa K, Isobe S, Kaneko T, Nakamura Y, Shibata D, Aoki K et al (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635–641

    Article  CAS  Google Scholar 

  • Shalit A, Rozman A, Goldshmidt A, Alvarez JP, Bowman JL, Eshed Y, Lifschitz E (2009) The flowering hormone florigen functions as a general systemic regulator of growth and termination. Proc Natl Acad Sci USA 106:8392–8397

    Article  PubMed  CAS  Google Scholar 

  • Shani E, Burko Y, Ben-Yaakov L, Berger Y, Amsellem Z, Goldshmidt A, Sharon E, Ori N (2009) Stage-specific regulation of Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins. Plant Cell 21:3078–3092

    Article  PubMed  CAS  Google Scholar 

  • Stav R, Hendelman A, Buxdorf K, Arazi T (2010) Transgenic expression of tomato bushy stunt virus silencing suppressor P19 via the pOp/LhG4 transactivation system induces viral-like symptoms in tomato. Virus Genes 40:119–129

    Article  PubMed  CAS  Google Scholar 

  • Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y (2008) The mechanism selecting the guide strand from small RNA duplexes is different among Argonaute proteins. Plant Cell Physiol 49:493–500

    Article  PubMed  CAS  Google Scholar 

  • Talmor-Neiman M, Stav R, Frank W, Voss B, Arazi T (2006) Novel micro-RNAs and intermediates of micro-RNA biogenesis from moss. Plant J 47:25–37

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  • Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17:49–63

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13:350–358

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H, Mallory AC, Bartel DP (2006) AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1. Mol Cell 22:129–136

    Article  PubMed  CAS  Google Scholar 

  • Xiao H, Radovich C, Welty N, Hsu J, Li D, Meulia T, van der Knaap E (2009) Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape. BMC Plant Biol 9:49

    Article  PubMed  Google Scholar 

  • Zhang X, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ, Chua NH (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20:3255–3268

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, 50250, Bet Dagan, Israel

    Anat Hendelman, Michael Kravchik, Ran Stav & Tzahi Arazi

  2. Department of Life Sciences, Ben Gurion University, 84105, Beer-Sheva, Israel

    Moriyah Zik & Nitsan Lugassi

Authors
  1. Anat Hendelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Kravchik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ran Stav
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Moriyah Zik
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nitsan Lugassi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tzahi Arazi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tzahi Arazi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (XLSX 12 kb)

Supplementary material 2 (DOCX 17 kb)

425_2012_1778_MOESM3_ESM.tif

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)

425_2012_1778_MOESM4_ESM.tif

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)

425_2012_1778_MOESM5_ESM.tif

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)

425_2012_1778_MOESM6_ESM.tif

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

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hendelman, A., Kravchik, M., Stav, R. et al. The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato. Planta 237, 363–377 (2013). https://doi.org/10.1007/s00425-012-1778-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-012-1778-8

Keywords

Search

Navigation