A. tumefaciens. We obtained good results with pCLEAN-S48,
but any other helper plasmid that carries the pSA replicase gene
intended to act in trans upon the pSA replication origin should
work just as well.
9. Discard any colonies that appear in plates too early because they
will most probably contain plasmids with deleted versions of
the virus. Remember that the pGTEVΔNIb derivative is a
plasmid with a large insert (more than 10 kbp in most clones),
and good colonies will necessarily grow slowly in the plates
with three antibiotics. The grow rate depends on the length of
the recombinant clone, which is reason why 5–7-day window is
given.
10. Kanamycin is the critical antibiotic to select the recombinant
A. tumefaciens. There is no need to add rifampicin and tetracycline to the liquid culture.
11. Optic density is not critical, although an interval between 0.2
and 1 is recommended to harvest bacteria under the best
physiological conditions.
12. We found that 5 mL Eppendorf tubes were most convenient in
this step. Centrifuge for 5 min at 10,000 rpm to sediment
A. tumefaciens.
13. If the recombinant TEV clone is infectious, a single infiltration
in one leaf is enough. However, we recommend infiltrating two
or three leaves per plant until efficient infectivity is confirmed.
14. Once again, the exact day in which symptoms appear will
depend on the complexity of the recombinant virus.
Acknowledgments
This work was supported by the Ministerio de Ciencia, Innovacio ´ n
y Universidades (Spain) grant BIO2017-83184-R (AEI/FEDER
UE).
References
1. Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice
WP (2002) Making an ally from an enemy:
plant virology and the new agriculture. Annu
Rev Phytopathol 40:45–74. https://doi.org/
10.1146/annurev.phyto.40.021102.150133
2. Hefferon K (2017) Plant virus expression vectors: a powerhouse for Global Health. Biomedicine
5(3).
https://doi.org/10.3390/
biomedicines5030044
3. Pasin F, Menzel W, Daro ` s JA (2019) Harnessed viruses in the age of metagenomics and
synthetic biology: an update on infectious
clone assembly and biotechnologies of plant
viruses. Plant Biotechnol J. https://doi.org/
10.1111/pbi.13084
4. Krenek P, Samajova O, Luptovciak I,
Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: principles, methods
and applications. Biotechnol Adv 33(6 Pt
2):1024–1042. https://doi.org/10.1016/j.
biotechadv.2015.03.012
5. Majer
E,
Llorente
B,
Rodrı ´guezConcepcio ´ n M, Daro `s JA (2017) Rewiring
Potyvirus Vectors to Produce Carotenoids
311
but any other helper plasmid that carries the pSA replicase gene
intended to act in trans upon the pSA replication origin should
work just as well.
9. Discard any colonies that appear in plates too early because they
will most probably contain plasmids with deleted versions of
the virus. Remember that the pGTEVΔNIb derivative is a
plasmid with a large insert (more than 10 kbp in most clones),
and good colonies will necessarily grow slowly in the plates
with three antibiotics. The grow rate depends on the length of
the recombinant clone, which is reason why 5–7-day window is
given.
10. Kanamycin is the critical antibiotic to select the recombinant
A. tumefaciens. There is no need to add rifampicin and tetracycline to the liquid culture.
11. Optic density is not critical, although an interval between 0.2
and 1 is recommended to harvest bacteria under the best
physiological conditions.
12. We found that 5 mL Eppendorf tubes were most convenient in
this step. Centrifuge for 5 min at 10,000 rpm to sediment
A. tumefaciens.
13. If the recombinant TEV clone is infectious, a single infiltration
in one leaf is enough. However, we recommend infiltrating two
or three leaves per plant until efficient infectivity is confirmed.
14. Once again, the exact day in which symptoms appear will
depend on the complexity of the recombinant virus.
Acknowledgments
This work was supported by the Ministerio de Ciencia, Innovacio ´ n
y Universidades (Spain) grant BIO2017-83184-R (AEI/FEDER
UE).
References
1. Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice
WP (2002) Making an ally from an enemy:
plant virology and the new agriculture. Annu
Rev Phytopathol 40:45–74. https://doi.org/
10.1146/annurev.phyto.40.021102.150133
2. Hefferon K (2017) Plant virus expression vectors: a powerhouse for Global Health. Biomedicine
5(3).
https://doi.org/10.3390/
biomedicines5030044
3. Pasin F, Menzel W, Daro ` s JA (2019) Harnessed viruses in the age of metagenomics and
synthetic biology: an update on infectious
clone assembly and biotechnologies of plant
viruses. Plant Biotechnol J. https://doi.org/
10.1111/pbi.13084
4. Krenek P, Samajova O, Luptovciak I,
Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: principles, methods
and applications. Biotechnol Adv 33(6 Pt
2):1024–1042. https://doi.org/10.1016/j.
biotechadv.2015.03.012
5. Majer
E,
Llorente
B,
Rodrı ´guezConcepcio ´ n M, Daro `s JA (2017) Rewiring
Potyvirus Vectors to Produce Carotenoids
311