7. Setting up and transforming a negative control ligation reaction with the same amount of linearized plasmid without insert
will provide an estimate of what percentage of colonies is due to
incompletely digested or empty re-ligated plasmid and thus
approximately how many colonies should be screened to identify tagged clones.
8. A single insertion (two stem-loops) in sense orientation, i.e.,
with the Csy4 cognate RNA recognition motifs in the (+) sense
vRNA, is sufficient for imaging. 2–8 stem-loops (1–4 insertions) are tolerated with little to no attenuation of virus infectivity, whereas the insertion of 12 stem-loops (6 insertions)
render the virus noninfectious.
9. Targeting the Csy4∗ reporter to the nucleus reduces nonspecific cytoplasmic fluorescence, but also reduces overall cytoplasmic signal. Therefore, either Csy4∗-NLS-GFP or Csy4∗-GFP
may be better suited depending on the experiment.
10. Utilizing the side of the needle tip like a scalpel can be helpful
to creating incisions that do not pierce entirely through
the leaf.
11. Avoid pressing too hard to prevent damage to the leaf. Additionally, face protection may be desired, as if too much pressure
is used the Agrobacterium suspension can spray back at
the user.
12. We find that PVX infection largely suppresses the expression of
agro-infiltrated constructs. Optimal imaging conditions, where
both virus and Csy4∗ reporter are present, are therefore most
easily found at the leading edge of the growing infection sites.
Using a silencing suppressor to boost the expression of Csy4∗
fusions makes it easier to find suitable imaging conditions.
Nevertheless, GFP-expressing cells can also be identified deeper within infection sites. Due to the extremely bright fluorescence of the PVX-expressed mCherry, they may not be visible
through the eye piece under UV illumination, but will be
apparent in confocal scanning mode.
13. We have found that the presence of an NLS significantly
reduces the expression of the Csy4∗ reporter in N. crassa;
therefore only the cloning of a cytoplasmic Csy4∗-GFP construct is described. With the primers in this protocol, a Gly 10
linker is inserted between Csy4∗ and mBasicGFP.
14. Transformation and subsequent integration into the fungal
genome are considerably more efficient when using linearized
DNA compared to circular plasmids.
15. SCR7 pyrazine suppresses nonhomologous end-joining
(NHEJ) repair of double-strand breaks, thereby significantly
increasing the efficiency of homologous recombination for the
176
David Burnett et al.
will provide an estimate of what percentage of colonies is due to
incompletely digested or empty re-ligated plasmid and thus
approximately how many colonies should be screened to identify tagged clones.
8. A single insertion (two stem-loops) in sense orientation, i.e.,
with the Csy4 cognate RNA recognition motifs in the (+) sense
vRNA, is sufficient for imaging. 2–8 stem-loops (1–4 insertions) are tolerated with little to no attenuation of virus infectivity, whereas the insertion of 12 stem-loops (6 insertions)
render the virus noninfectious.
9. Targeting the Csy4∗ reporter to the nucleus reduces nonspecific cytoplasmic fluorescence, but also reduces overall cytoplasmic signal. Therefore, either Csy4∗-NLS-GFP or Csy4∗-GFP
may be better suited depending on the experiment.
10. Utilizing the side of the needle tip like a scalpel can be helpful
to creating incisions that do not pierce entirely through
the leaf.
11. Avoid pressing too hard to prevent damage to the leaf. Additionally, face protection may be desired, as if too much pressure
is used the Agrobacterium suspension can spray back at
the user.
12. We find that PVX infection largely suppresses the expression of
agro-infiltrated constructs. Optimal imaging conditions, where
both virus and Csy4∗ reporter are present, are therefore most
easily found at the leading edge of the growing infection sites.
Using a silencing suppressor to boost the expression of Csy4∗
fusions makes it easier to find suitable imaging conditions.
Nevertheless, GFP-expressing cells can also be identified deeper within infection sites. Due to the extremely bright fluorescence of the PVX-expressed mCherry, they may not be visible
through the eye piece under UV illumination, but will be
apparent in confocal scanning mode.
13. We have found that the presence of an NLS significantly
reduces the expression of the Csy4∗ reporter in N. crassa;
therefore only the cloning of a cytoplasmic Csy4∗-GFP construct is described. With the primers in this protocol, a Gly 10
linker is inserted between Csy4∗ and mBasicGFP.
14. Transformation and subsequent integration into the fungal
genome are considerably more efficient when using linearized
DNA compared to circular plasmids.
15. SCR7 pyrazine suppresses nonhomologous end-joining
(NHEJ) repair of double-strand breaks, thereby significantly
increasing the efficiency of homologous recombination for the
176
David Burnett et al.