Abstract
For perennial woody plants, it is important to establish an efficient transformation method to facilitate gene functional studies and protein subcellular localization assays. Therefore, calluses are ideal materials for these purposes. Here, we optimized a series of over-expression vectors with different fluorescent protein tags and antibiotic selections, providing the possibility to generate transgenic callus expressing two different fusion proteins at the same time. On the other hand, a series of RNAi (RNA interference) vectors were also modified by introducing free eGFP or mCherry as reporters to facilitate visual screening. With the help of these improvements, stable transgenic lines expressing an autophagosome marker, GFP-FhATG8a (Autophagy 8a) were generated, and further studies showed that autophagosomes were more abundant during NaCl treatment or phosphate deficiency. By knocking down the expression of a key autophagy gene, FhATG5, the resulting callus was more susceptible to salt, indicating that autophagy is essential for stress response in citrus. In summary, our study provided an effective system for callus-base transformation to study gene function, and this new approach was successfully used to study the activity of autophagy in citrus as an example.
Key message
We optimized the over-expression and RNAi vectors for citrus callus transformation and established an effective strategy for gene functional studies and protein subcellular localization assays in citrus.
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Data availability
All materials and data generated in the current study are available from the corresponding author on reasonable request.
Abbreviations
- AS:
-
Acetosyringone
- ATG:
-
Autophagy
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- eGFP:
-
Enhance green fluorescent protein
- EV:
-
Empty vector
- mCherry:
-
Monomeric cherry
- MCS:
-
Multiple cloning site
- MT:
-
Murashge-tucker medium
- PCR:
-
Polymerase chain reaction
- qRT-PCR:
-
Real-time quantitative PCR
- RNAi:
-
RNA interference
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Acknowledgements
This work was supported by the National Key Research and Development Program (2019YFD1000103), NSFC grants (no.31772281, 91854102), Foundation of Hubei Hongshan Laboratory (2021hszd016) to P.W. We thank Saiyu Cao, Kun Wang (Huazhong Agricultural University) for their help in this work.
Funding
National Key Research and Development Program,2019YFD1000103,Pengwei Wang,National Natural Science Foundation of China,31772281,Pengwei Wang,91854102,Pengwei Wang
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PW: designed and supervised the project, EG: performed experiments and wrote the manuscript, YZ: helped experiments and tissue culture; WG: provided citrus materials and gave valuable suggestions throughout the study.
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Communicated by Goetz Hensel.
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Gao, E., Zhao, Y., Guo, W. et al. Optimized plasmids for callus transformation and functional studies of autophagy in citrus. Plant Cell Tiss Organ Cult 152, 507–516 (2023). https://doi.org/10.1007/s11240-022-02423-0
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DOI: https://doi.org/10.1007/s11240-022-02423-0