Biology Reference
In-Depth Information
4
Notes
1. The p19 protein suppresses silencing of plant genes that func-
tion against bacterial infection, thereby promoting suffi cient
expression of the transformed genes. ATL31 is an Arabidopsis
ubiquitin ligase and 14-3-3 is its target protein. GFP is used
for infection and expression control as it is extremely stable
and is not degraded signifi cantly by 26S Proteasome.
2. Acetosyringone is a phenolic compound produced by plants,
especially in wound tissue, which activates the Agrobacterium
infection and increases the transformation effi ciency.
3. This buffer allows effi cient degradation of poly-ubiquitinated
protein by 26S Proteasome. ATP and MgCl
2
are required for
the ATPase activity of the RPT subunit in 19S RP.
4. As an alternative to MG132, clasto-Lactacystin
-lactone or
Epoxomicin can be used as the proteasome inhibitor.
5. The number and suspended volume of the Agrobacterium
used for the infi ltration assay should be adjusted to increase the
effi ciency of each protein expression.
6. The incubation time is also adjusted to increase the effi ciency
of detection of the transformed protein under experimental
conditions. Liu et al evaluated the effect of incubation length
and expression effi ciency [
8
]. In addition, if the transformed
protein has specifi c functions in terms of plant defense response,
the fact that the Agrobacterium treatment stimulates plant
immune responses should be taken into consideration.
7. Tsuda et al established the effi cient transient expression system
in
Arabidopsis thaliana
with an effector gene
AvrPto
[
13
].
8. The protein amount in the extracted sample before reaction
(0 h) is quantifi ed and 3
β
g proteins for the SDS-PAGE/WB
analysis are used. Use the same volume of digested sample
(2 h) as the sample (0 h).
9. GFP intensity with anti-GFP antibody can be used for normal-
ization as a non-degraded protein. In addition, point-mutated
ATL31 (ATL31C143S), which abolishes ubiquitin ligase activ-
ity is used as a more accurate negative control. Since tobacco
plants may have a ubiquitin ligase that is homologous to the
transiently expressed protein, the expressed ubiquitin ligase
activity can be evaluated against the target protein compared
with that of the mutated control, which is defi cient in this
activity [
7
].
10. An accumulation of ubiquitinated proteins can be also com-
pared when plants are grown under various growth conditions;
for example, under metal defi cient conditions [
14
].
μ
