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
Part of the Methods in Molecular Biology book series (MIMB, volume 1998)


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 



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.


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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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