Acknowledgments
We thank the microscopy sections of Serveis Centrals de Suport a la
Investigacio ´ Experimental, University of Valencia (SCSIE).
F.A. was supported by the Ministerio de Economı ´a y Competitividad (grant no BFU2016-76607P) and Generalitat Valenciana
(ISIC/2013/004, GVACOMP2014-202). V.V. was supported by
a Nigel Groome/Oxford Brookes University 150th Anniversary
Studentship. C.B.-S. and J.S.-S. are recipients of a fellowship from
the Ministerio de Ciencia (FPU program) and an EMBO shortterm fellowship.
References
1. Hwang H-H, Yu M, Lai E-M (2017) Agrobacterium-mediated plant transformation: biology
and applications. The Arabidopsis Book 15:
e0186
2. Marion J, Bach L, Bellec Y, Meyer C, Gissot L,
Faure JD (2008) Systematic analysis of protein
subcellular localization and interaction using
high-throughput transient transformation of
Arabidopsis seedlings. Plant J 56:169–179
3. Li J-F, Park E, von Arnim AG, Nebenfu ¨h A
(2009) The FAST technique: a simplified Agrobacterium-based transformation method for
transient gene expression analysis in seedlings
of Arabidopsis and other plant species. Plant
Methods 5:6. https://doi.org/10.1186/
1746-4811-5-6
4. Wu H-Y, Liu K-H, Wang Y-C, Wu J-F, Chiu
W-L, Chen C-Y, Wu S-H, Sheen J, Lai E-M
(2014) AGROBEST: an efficient Agrobacterium-mediated transient expression method for
versatile gene function analyses in Arabidopsis
seedlings. Plant Methods 10:19. https://doi.
org/10.1186/1746-4811-10-19
5. Wang Y-C, Yu M, Shih P-Y, Wu H-Y, Lai E-M
(2018) Stable pH suppresses defense signaling
and is the key to enhance Agrobacteriummediated transient expression in Arabidopsis
seedlings. Sci Rep 8:1–9
6. Koncz C, Schell J (1986) The promoter of
TL-DNA gene 5 controls the tissue-specific
expression of chimaeric genes carried by a
novel type of Agrobacterium binary vector.
Mol Gen Genet 204:383–396
7. Sparkes IA, Runions J, Kearns A, Hawes C
(2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature
Protocols 1 (4):2019–2025
8. Montesinos JC, Langhans M, Sturm S,
Hillmer S, Aniento F, Robinson DG, Marcote
MJ (2013) Putative p24 complexes in Arabidopsis contain members of the delta and beta
subfamilies and cycle in the early secretory
pathway. J Exp Bot 64:3147–3167
9. Martinie `re A, Lavagi I, Nageswaran G et al
(2012) Cell wall constrains lateral diffusion of
plant plasma-membrane proteins. PNAS
109:12805–12810
10. Pereira C, Pereira S, Satiat-Jeunemaitre B, Pissarra J (2013) Cardosin A contains two vacuolar sorting signals using different vacuolar
routes in tobacco epidermal cells. Plant J
76:87–100
11. Subramoni S, Nathoo N, Klimov E, Yuan Z-C
(2014) Agrobacterium tumefaciens responses
to plant-derived signaling molecules. Front
Plant Sci 5:322. https://doi.org/10.3389/
fpls.2014.00322
Seedling Transformation
155
We thank the microscopy sections of Serveis Centrals de Suport a la
Investigacio ´ Experimental, University of Valencia (SCSIE).
F.A. was supported by the Ministerio de Economı ´a y Competitividad (grant no BFU2016-76607P) and Generalitat Valenciana
(ISIC/2013/004, GVACOMP2014-202). V.V. was supported by
a Nigel Groome/Oxford Brookes University 150th Anniversary
Studentship. C.B.-S. and J.S.-S. are recipients of a fellowship from
the Ministerio de Ciencia (FPU program) and an EMBO shortterm fellowship.
References
1. Hwang H-H, Yu M, Lai E-M (2017) Agrobacterium-mediated plant transformation: biology
and applications. The Arabidopsis Book 15:
e0186
2. Marion J, Bach L, Bellec Y, Meyer C, Gissot L,
Faure JD (2008) Systematic analysis of protein
subcellular localization and interaction using
high-throughput transient transformation of
Arabidopsis seedlings. Plant J 56:169–179
3. Li J-F, Park E, von Arnim AG, Nebenfu ¨h A
(2009) The FAST technique: a simplified Agrobacterium-based transformation method for
transient gene expression analysis in seedlings
of Arabidopsis and other plant species. Plant
Methods 5:6. https://doi.org/10.1186/
1746-4811-5-6
4. Wu H-Y, Liu K-H, Wang Y-C, Wu J-F, Chiu
W-L, Chen C-Y, Wu S-H, Sheen J, Lai E-M
(2014) AGROBEST: an efficient Agrobacterium-mediated transient expression method for
versatile gene function analyses in Arabidopsis
seedlings. Plant Methods 10:19. https://doi.
org/10.1186/1746-4811-10-19
5. Wang Y-C, Yu M, Shih P-Y, Wu H-Y, Lai E-M
(2018) Stable pH suppresses defense signaling
and is the key to enhance Agrobacteriummediated transient expression in Arabidopsis
seedlings. Sci Rep 8:1–9
6. Koncz C, Schell J (1986) The promoter of
TL-DNA gene 5 controls the tissue-specific
expression of chimaeric genes carried by a
novel type of Agrobacterium binary vector.
Mol Gen Genet 204:383–396
7. Sparkes IA, Runions J, Kearns A, Hawes C
(2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature
Protocols 1 (4):2019–2025
8. Montesinos JC, Langhans M, Sturm S,
Hillmer S, Aniento F, Robinson DG, Marcote
MJ (2013) Putative p24 complexes in Arabidopsis contain members of the delta and beta
subfamilies and cycle in the early secretory
pathway. J Exp Bot 64:3147–3167
9. Martinie `re A, Lavagi I, Nageswaran G et al
(2012) Cell wall constrains lateral diffusion of
plant plasma-membrane proteins. PNAS
109:12805–12810
10. Pereira C, Pereira S, Satiat-Jeunemaitre B, Pissarra J (2013) Cardosin A contains two vacuolar sorting signals using different vacuolar
routes in tobacco epidermal cells. Plant J
76:87–100
11. Subramoni S, Nathoo N, Klimov E, Yuan Z-C
(2014) Agrobacterium tumefaciens responses
to plant-derived signaling molecules. Front
Plant Sci 5:322. https://doi.org/10.3389/
fpls.2014.00322
Seedling Transformation
155