2.4 Agro-Infiltration
of N. benthamiana
with Csy4∗
Expression Plasmids
1. A. tumefaciens AGL1 strains transformed with pGWB405.
Csy4∗-NLS-GFP or pGWB405.Csy4∗-GFP plasmids (see
Subheading 3.1).
2. Glycerol stock of A. tumefaciens AGL1 strain transformed with
a plasmid for expression of the Tomato bushy stunt virus p19
silencing suppressor, e.g., pDGB3alpha2_35S:P19:Tnos
(GB1203) (Addgene #68214 [19]).
3. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL spectinomycin and 50 μg/mL rifampicin.
4. LB liquid medium (see Subheading 2.1, item 11) containing
50 μg/mL kanamycin and 50 μg/mL rifampicin.
5. 28
C Shaking incubator.
6. Infiltration buffer: 10 mM morpholino ethane sulfonic acid
(MES), 10 mM MgCl 2 , 67 mM acetosyringone.
7. UV spectrophotometer.
8. 1 mL Syringes.
9. 25G Needle.
2.5 Visualization of
Tagged PVX vRNA in N.
benthamiana Leaf
Epidermal Cells
1. Upright confocal laser scanning microscope equipped with
10Â long-distance and 40Â water-dipping lenses, and capable
of detecting GFP and mCherry.
2. Glass slide.
3. Double-sided tape.
4. Leaf prepared as per Subheadings 3.3 and 3.4.
2.6 Construction of
Csy4∗ Expression
Plasmid for RNA
Imaging in N. crassa
1. pDONR221.Csy4∗ (see Subheading 3.1), template for Csy4∗.
2. pLS3 [20], template for mBasicGFP.
3. pAB261 [21] (see Note 3), template for expression vector
backbone.
4. Oligonucleotide primers Csy4-IF-F (5
0 -CAGCACATCAACC
GTCAAAGATATCATGGACCATTACCTCGAC-3
0 ), Csy4IF-R (5
0 -GCCTCCGCCTCCGCCTCCGCCGCCTCCGCC
GAACCAAGGAACGAAACC-3
0 ), GFP-IF-F (5
0 -GGCGG
AGGCGGCGGAGGCGGAGGCGGAGGCATGGTGAGCAA
GGGCG-3
0 ), GFP-IF-R (5
0 -GTCGACGGTATCGATAAGC
TTTTACTTGTACAGCTCGTCCATGC-3
0 ), pAB261-lin-F
(5
0 -AAGCTTATCGATACCGTC-3
0 ), and pAB261-lin-R (5
0 -
GATATCTTTGACGGTTGATG-3
0 ).
5. High-fidelity DNA polymerase.
6. 10 mM dNTP mixture.
7. PCR thermocycler.
8. Components for agarose gel electrophoresis (see Subheading
2.2, item 11).
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David Burnett et al.
of N. benthamiana
with Csy4∗
Expression Plasmids
1. A. tumefaciens AGL1 strains transformed with pGWB405.
Csy4∗-NLS-GFP or pGWB405.Csy4∗-GFP plasmids (see
Subheading 3.1).
2. Glycerol stock of A. tumefaciens AGL1 strain transformed with
a plasmid for expression of the Tomato bushy stunt virus p19
silencing suppressor, e.g., pDGB3alpha2_35S:P19:Tnos
(GB1203) (Addgene #68214 [19]).
3. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL spectinomycin and 50 μg/mL rifampicin.
4. LB liquid medium (see Subheading 2.1, item 11) containing
50 μg/mL kanamycin and 50 μg/mL rifampicin.
5. 28
C Shaking incubator.
6. Infiltration buffer: 10 mM morpholino ethane sulfonic acid
(MES), 10 mM MgCl 2 , 67 mM acetosyringone.
7. UV spectrophotometer.
8. 1 mL Syringes.
9. 25G Needle.
2.5 Visualization of
Tagged PVX vRNA in N.
benthamiana Leaf
Epidermal Cells
1. Upright confocal laser scanning microscope equipped with
10Â long-distance and 40Â water-dipping lenses, and capable
of detecting GFP and mCherry.
2. Glass slide.
3. Double-sided tape.
4. Leaf prepared as per Subheadings 3.3 and 3.4.
2.6 Construction of
Csy4∗ Expression
Plasmid for RNA
Imaging in N. crassa
1. pDONR221.Csy4∗ (see Subheading 3.1), template for Csy4∗.
2. pLS3 [20], template for mBasicGFP.
3. pAB261 [21] (see Note 3), template for expression vector
backbone.
4. Oligonucleotide primers Csy4-IF-F (5
0 -CAGCACATCAACC
GTCAAAGATATCATGGACCATTACCTCGAC-3
0 ), Csy4IF-R (5
0 -GCCTCCGCCTCCGCCTCCGCCGCCTCCGCC
GAACCAAGGAACGAAACC-3
0 ), GFP-IF-F (5
0 -GGCGG
AGGCGGCGGAGGCGGAGGCGGAGGCATGGTGAGCAA
GGGCG-3
0 ), GFP-IF-R (5
0 -GTCGACGGTATCGATAAGC
TTTTACTTGTACAGCTCGTCCATGC-3
0 ), pAB261-lin-F
(5
0 -AAGCTTATCGATACCGTC-3
0 ), and pAB261-lin-R (5
0 -
GATATCTTTGACGGTTGATG-3
0 ).
5. High-fidelity DNA polymerase.
6. 10 mM dNTP mixture.
7. PCR thermocycler.
8. Components for agarose gel electrophoresis (see Subheading
2.2, item 11).
162
David Burnett et al.