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Genetic Suppressor Screen Using an Inducible FREE1-RNAi Line to Detect ESCRT Genetic Interactors in Arabidopsis thaliana

  • Qiong Zhao
  • Ying Zhu
  • Wenhan Cao
  • Jinbo Shen
  • Yong Cui
  • Shuxian Huang
  • Liwen Jiang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1998)

Abstract

FREE1 (FYVE domain protein required for endosomal sorting 1), a newly identified component of endosomal sorting complex required for transport I (ESCRT I), plays multiple roles in regulating protein trafficking and organelle biogenesis in Arabidopsis. Similar to other ESCRT components, FREE1 is essential for plant growth and development because free1 mutant is seedling lethal. To identify key components that genetically interact with FREE1, we performed forward genetic suppressor screening using a dexamethasone (DEX)-inducible FREE1-RNAi line. Here we describe the detailed protocol of identifying novel FREE1 regulators using an inducible FREE1-RNAi line for the genetic suppressor screen. This protocol represents a whole procedure for identifying ESCRT genetic interactors in Arabidopsis thaliana.

Key words

ESCRT FREE1 Inducible RNAi EMS mutagenesis Suppressor Forward genetic screening Mapping 

Notes

Acknowledgments

This work was supported by grants from the Research Grants Council of Hong Kong (CUHK466313 and 14130716 and CUHK2/CRF/11G, C4011-14R, C4012-16E, and AoE/M-05/12), the National Natural Science Foundation of China (31270226 and 31470294), and the Shenzhen Peacock Project (KQTD201101) (to L.J.). The authors declare no conflict of interest.

References

  1. 1.
    Cui Y, Shen J, Gao C, Zhuang X, Wang J, Jiang L (2016) Biogenesis of plant prevacuolar multivesicular bodies. Mol Plant 9(6):774–786.  https://doi.org/10.1016/j.molp.2016.01.011CrossRefPubMedGoogle Scholar
  2. 2.
    Gao C, Zhuang X, Shen J, Jiang L (2017) Plant ESCRT complexes: moving beyond endosomal sorting. Trends Plant Sci 22(11):986–998.  https://doi.org/10.1016/j.tplants.2017.08.003CrossRefPubMedGoogle Scholar
  3. 3.
    Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y, Xia J, Jiang L (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24(21):2556–2563.  https://doi.org/10.1016/j.cub.2014.09.014CrossRefGoogle Scholar
  4. 4.
    Kolb C, Nagel MK, Kalinowska K, Hagmann J, Ichikawa M, Anzenberger F, Alkofer A, Sato MH, Braun P, Isono E (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167(4):1361–1373.  https://doi.org/10.1104/pp.114.253377CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Belda-Palazon B, Rodriguez L, Fernandez MA, Castillo MC, Anderson EA, Gao C, Gonzalez-Guzman M, Peirats-Llobet M, Zhao Q, De Winne N, Gevaert K, De Jaeger G, Jiang L, Leon J, Mullen RT, Rodriguez PL (2016) FYVE1/FREE1 interacts with the PYL4 ABA receptor and mediates its delivery to the vacuolar degradation pathway. Plant Cell.  https://doi.org/10.1105/tpc.16.00178
  6. 6.
    Gao C, Zhuang X, Cui Y, Fu X, He Y, Zhao Q, Zeng Y, Shen J, Luo M, Jiang L (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112(6):1886–1891.  https://doi.org/10.1073/pnas.1421271112CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Zhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L (2013) A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25(11):4596–4615.  https://doi.org/10.1105/tpc.113.118307CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhao Q, Gao C, Lee P, Liu L, Li S, Hu T, Shen J, Pan S, Ye H, Chen Y, Cao W, Cui Y, Zeng P, Yu S, Gao Y, Chen L, Mo B, Liu X, Xiao S, Zhao Y, Zhong S, Chen X, Jiang L (2015) Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants. J Genet Genomics 42(6):319–330.  https://doi.org/10.1016/j.jgg.2015.03.012CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Calcagno-Pizarelli AM, Hervas-Aguilar A, Galindo A, Abenza JF, Penalva MA, Arst HN Jr (2011) Rescue of Aspergillus nidulans severely debilitating null mutations in ESCRT-0, I, II and III genes by inactivation of a salt-tolerance pathway allows examination of ESCRT gene roles in pH signalling. J Cell Sci 124(Pt 23):4064–4076.  https://doi.org/10.1242/jcs.088344CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25(14):1754–1760.  https://doi.org/10.1093/bioinformatics/btp324CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079.  https://doi.org/10.1093/bioinformatics/btp352CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Galvao VC, Nordstrom KJ, Lanz C, Sulz P, Mathieu J, Pose D, Schmid M, Weigel D, Schneeberger K (2012) Synteny-based mapping-by-sequencing enabled by targeted enrichment. Plant J 71(3):517–526.  https://doi.org/10.1111/j.1365-313X.2012.04993.xCrossRefPubMedGoogle Scholar
  13. 13.
    Schneeberger K, Ossowski S, Lanz C, Juul T, Petersen AH, Nielsen KL, Jorgensen JE, Weigel D, Andersen SU (2009) SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat Methods 6(8):550–551.  https://doi.org/10.1038/nmeth0809-550CrossRefPubMedGoogle Scholar
  14. 14.
    Sun H, Schneeberger K (2015) SHOREmap v3.0: fast and accurate identification of causal mutations from forward genetic screens. Methods Mol Biol 1284:381–395.  https://doi.org/10.1007/978-1-4939-2444-8_19CrossRefPubMedGoogle Scholar
  15. 15.
    Tse YC, Mo B, Hillmer S, Zhao M, Lo SW, Robinson DG, Jiang L (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16(3):672–693.  https://doi.org/10.1105/tpc.019703CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chen L, Li F, Xiao S (2017) Analysis of plant autophagy. Methods Mol Biol 1662:267–280.  https://doi.org/10.1007/978-1-4939-7262-3_24CrossRefPubMedGoogle Scholar
  17. 17.
    Karahara I, Kang BH (2014) High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy. Methods Mol Biol 1080:147–157.  https://doi.org/10.1007/978-1-62703-643-6_12CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Qiong Zhao
    • 1
  • Ying Zhu
    • 1
  • Wenhan Cao
    • 1
  • Jinbo Shen
    • 1
  • Yong Cui
    • 1
  • Shuxian Huang
    • 1
  • Liwen Jiang
    • 1
    • 2
  1. 1.State Key Laboratory of Agrobiotechnology, Centre for Cell and Developmental Biology, School of Life SciencesThe Chinese University of Hong KongHong KongChina
  2. 2.CUHK Shenzhen Research InstituteThe Chinese University of Hong KongShenzhenChina

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