11. When generating ORF sensors carefully check that no in-frame
STOP codons are introduced into the linker region between
GFP and Renilla luciferase.
12. At this point the competent bacteria can be aliquoted into
1.5 mL tubes (100 μL/tube) and snap-frozen in liquid N 2 .
The frozen aliquots can be stored at À70
C and used for a few
months.
13. Either YEB-RTK or YEB-RTS plates are used. Rifampicin and
tetracycline select for the strain-specific markers of C58C1.
Kanamycin selection allows for the selection of the pBIN61based (here: AGO) vectors. Spectinomycin is used to select for
the pK7WG2D- or pK7WGF2-derived plasmids (miRNA
expression vectors, sensors).
14. Expression of the AGO proteins can be detected by either HA
or FLAG antibodies. However, in our hands the 3F10 anti-HA
antibody proved to be more sensitive than the M2 FLAG
antibody. For detection of AGO proteins stripping of the
membrane is not necessary. All N. benthamiana AGOs are
>100 kDa in their size; thus, none of the bands recognized
by the GFP antibody (GFP at ~33 kDa, GFP-Renilla luciferase
at ~66 kDa) interferes with their detection.
Acknowledgments
This work was supported by the National Research, Development
and Innovation Office, Hungary (K124705).
References
1. Voinnet O (2009) Origin, biogenesis, and
activity
of
plant
microRNAs.
Cell
136:669–687
2. Martinez de Alba AE, Elvira-Matelot E, Vaucheret H (2013) Gene silencing in plants: a
diversity of pathways. Biochim Biophys Acta
1829:1300–1308
3. Bologna NG, Voinnet O (2014) The diversity,
biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant
Biol 65:473–503
4. Yu Y, Jia T, Chen X (2017) The ‘how’ and
‘where’ of plant microRNAs. New Phytol
216:1002–1017
5. Merchante C, Stepanova AN, Alonso JM
(2017) Translation regulation in plants: an
interesting past, an exciting present and a
promising future. Plant J 90:628–653
6. Fa ´tyol K, Ludman M, Burgya ´n J (2016) Functional dissection of a plant Argonaute. Nucleic
Acids Res 44:1384–1397
7. Llave C, Xie Z, Kasschau KD, Carrington JC
(2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA.
Science 297:2053–2056
8. Franco-Zorrilla JM, Valli A, Todesco M,
Mateos I, Puga MI, Rubio-Somoza I,
Leyva A, Weigel D, Garcia JA, Paz-Ares J
(2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat
Genet 39:1033–1037
9. Allen E, Xie Z, Gustafson AM, Carrington JC
(2005) microRNA-directed phasing during
trans-acting siRNA biogenesis in plants. Cell
121:207–221
10. Braman J, Papworth C, Greener A (1996) Sitedirected mutagenesis using double-stranded
plasmid DNA templates. Methods Mol Biol
57:31–44
11. Bombarely A, Rosli HG, Vrebalov J, Moffett P,
Mueller LA, Martin GB (2012) A draft
genome sequence of Nicotiana benthamiana
An In Vivo Reporter System for RNA Silencing
449
STOP codons are introduced into the linker region between
GFP and Renilla luciferase.
12. At this point the competent bacteria can be aliquoted into
1.5 mL tubes (100 μL/tube) and snap-frozen in liquid N 2 .
The frozen aliquots can be stored at À70
C and used for a few
months.
13. Either YEB-RTK or YEB-RTS plates are used. Rifampicin and
tetracycline select for the strain-specific markers of C58C1.
Kanamycin selection allows for the selection of the pBIN61based (here: AGO) vectors. Spectinomycin is used to select for
the pK7WG2D- or pK7WGF2-derived plasmids (miRNA
expression vectors, sensors).
14. Expression of the AGO proteins can be detected by either HA
or FLAG antibodies. However, in our hands the 3F10 anti-HA
antibody proved to be more sensitive than the M2 FLAG
antibody. For detection of AGO proteins stripping of the
membrane is not necessary. All N. benthamiana AGOs are
>100 kDa in their size; thus, none of the bands recognized
by the GFP antibody (GFP at ~33 kDa, GFP-Renilla luciferase
at ~66 kDa) interferes with their detection.
Acknowledgments
This work was supported by the National Research, Development
and Innovation Office, Hungary (K124705).
References
1. Voinnet O (2009) Origin, biogenesis, and
activity
of
plant
microRNAs.
Cell
136:669–687
2. Martinez de Alba AE, Elvira-Matelot E, Vaucheret H (2013) Gene silencing in plants: a
diversity of pathways. Biochim Biophys Acta
1829:1300–1308
3. Bologna NG, Voinnet O (2014) The diversity,
biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant
Biol 65:473–503
4. Yu Y, Jia T, Chen X (2017) The ‘how’ and
‘where’ of plant microRNAs. New Phytol
216:1002–1017
5. Merchante C, Stepanova AN, Alonso JM
(2017) Translation regulation in plants: an
interesting past, an exciting present and a
promising future. Plant J 90:628–653
6. Fa ´tyol K, Ludman M, Burgya ´n J (2016) Functional dissection of a plant Argonaute. Nucleic
Acids Res 44:1384–1397
7. Llave C, Xie Z, Kasschau KD, Carrington JC
(2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA.
Science 297:2053–2056
8. Franco-Zorrilla JM, Valli A, Todesco M,
Mateos I, Puga MI, Rubio-Somoza I,
Leyva A, Weigel D, Garcia JA, Paz-Ares J
(2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat
Genet 39:1033–1037
9. Allen E, Xie Z, Gustafson AM, Carrington JC
(2005) microRNA-directed phasing during
trans-acting siRNA biogenesis in plants. Cell
121:207–221
10. Braman J, Papworth C, Greener A (1996) Sitedirected mutagenesis using double-stranded
plasmid DNA templates. Methods Mol Biol
57:31–44
11. Bombarely A, Rosli HG, Vrebalov J, Moffett P,
Mueller LA, Martin GB (2012) A draft
genome sequence of Nicotiana benthamiana
An In Vivo Reporter System for RNA Silencing
449