addition, the amount of the DNA will affect the transfection
efficiency.
6. The incubation condition varies according to the purposes of
the experiments. Normally, fluorescent signal will appear after
4–6 h. Usually 12–24 h incubation is good for confocal
imaging.
7. Normally the protoplasts will float up to form a top layer after
10 min. A poor floating usually indicates poor protoplast condition and low transfection efficiency.
8. Low reconstruction quality of the SIM images may be due to
the low expression level of the fluorescent protein in protoplasts. Try to capture and reconstruct the protoplasts with
good expression.
Acknowledgements
Author contributions: Y.Z., C.J., Y.L., and L.J. designed the concept and the organization of the manuscript; Y.Z., C.J., and
Y.L. wrote the manuscript; L.J. edited the manuscript
We are grateful to the current and previous members of Prof Jiang’s
Laboratory for their contributions in setting up and improving the
above methods. This work was supported by grants from the
Research Grants Council of Hong Kong (G-CUHK403/17,
CUHK14130716, 14102417, 14100818, C4012-16E, C400217G, R4005-18, and AoE/M-05/12), the National Natural Science Foundation of China (31670179, and 91854201), and
CUHK Research Committee to L.J.
References
1. Denecke J, Aniento F, Frigerio L et al (2012)
Secretory pathway research: the more experimental systems the better. Plant Cell 24
(4):1316–1326
2. Kunzl F, Fruholz S, Fassler F et al (2016)
Receptor-mediated sorting of soluble vacuolar
proteins ends at the trans-Golgi network/early
endosome. Nat Plants 2(4):16017
3. Fruholz S, Fassler F, Kolukisaoglu U et al
(2018) Nanobody-triggered lockdown of
VSRs reveals ligand reloading in the Golgi.
Nat Commun 9:643
4. Shen JB, Zhao Q, Wang XF et al (2018) A
plant Bro1 domain protein BRAF regulates
multivesicular body biogenesis and membrane
protein homeostasis. Nat Commun 9:3784
5. Zhuang XH, Chung KP, Cui Y et al (2017)
ATG9 regulates autophagosome progression
from
the
endoplasmic
reticulum
in
Arabidopsis. Proc Natl Acad Sci U S A 114
(3):E426–E435
6. Lerich A, Langhans M, Sturm S et al (2011) Is
the 6 kDa tobacco etch viral protein a bona fide
ERES marker? J Exp Bot 62(14):5013–5023
7. Gao CJ, Yu CKY, Qu S et al (2012) The Golgilocalized Arabidopsis endomembrane Protein12 contains both endoplasmic reticulum
export and Golgi retention signals at its C terminus. Plant Cell 24(5):2086–2104
8. Cai Y, Zhuang XH, Gao CJ et al (2014) The
Arabidopsis Endosomal sorting complex
required for transport III regulates internal
vesicle formation of the Prevacuolar compartment and is required for plant development.
Plant Physiol 165(3):1328–1343
9. Wang XF, Cai Y, Wang H et al (2014) TransGolgi network-located AP1 gamma Adaptins
mediate Dileucine motif-directed vacuolar
164
Yonglun Zeng et al.
efficiency.
6. The incubation condition varies according to the purposes of
the experiments. Normally, fluorescent signal will appear after
4–6 h. Usually 12–24 h incubation is good for confocal
imaging.
7. Normally the protoplasts will float up to form a top layer after
10 min. A poor floating usually indicates poor protoplast condition and low transfection efficiency.
8. Low reconstruction quality of the SIM images may be due to
the low expression level of the fluorescent protein in protoplasts. Try to capture and reconstruct the protoplasts with
good expression.
Acknowledgements
Author contributions: Y.Z., C.J., Y.L., and L.J. designed the concept and the organization of the manuscript; Y.Z., C.J., and
Y.L. wrote the manuscript; L.J. edited the manuscript
We are grateful to the current and previous members of Prof Jiang’s
Laboratory for their contributions in setting up and improving the
above methods. This work was supported by grants from the
Research Grants Council of Hong Kong (G-CUHK403/17,
CUHK14130716, 14102417, 14100818, C4012-16E, C400217G, R4005-18, and AoE/M-05/12), the National Natural Science Foundation of China (31670179, and 91854201), and
CUHK Research Committee to L.J.
References
1. Denecke J, Aniento F, Frigerio L et al (2012)
Secretory pathway research: the more experimental systems the better. Plant Cell 24
(4):1316–1326
2. Kunzl F, Fruholz S, Fassler F et al (2016)
Receptor-mediated sorting of soluble vacuolar
proteins ends at the trans-Golgi network/early
endosome. Nat Plants 2(4):16017
3. Fruholz S, Fassler F, Kolukisaoglu U et al
(2018) Nanobody-triggered lockdown of
VSRs reveals ligand reloading in the Golgi.
Nat Commun 9:643
4. Shen JB, Zhao Q, Wang XF et al (2018) A
plant Bro1 domain protein BRAF regulates
multivesicular body biogenesis and membrane
protein homeostasis. Nat Commun 9:3784
5. Zhuang XH, Chung KP, Cui Y et al (2017)
ATG9 regulates autophagosome progression
from
the
endoplasmic
reticulum
in
Arabidopsis. Proc Natl Acad Sci U S A 114
(3):E426–E435
6. Lerich A, Langhans M, Sturm S et al (2011) Is
the 6 kDa tobacco etch viral protein a bona fide
ERES marker? J Exp Bot 62(14):5013–5023
7. Gao CJ, Yu CKY, Qu S et al (2012) The Golgilocalized Arabidopsis endomembrane Protein12 contains both endoplasmic reticulum
export and Golgi retention signals at its C terminus. Plant Cell 24(5):2086–2104
8. Cai Y, Zhuang XH, Gao CJ et al (2014) The
Arabidopsis Endosomal sorting complex
required for transport III regulates internal
vesicle formation of the Prevacuolar compartment and is required for plant development.
Plant Physiol 165(3):1328–1343
9. Wang XF, Cai Y, Wang H et al (2014) TransGolgi network-located AP1 gamma Adaptins
mediate Dileucine motif-directed vacuolar
164
Yonglun Zeng et al.