Transient assays in BY2 protoplast cells

6.4.1 The in vivo interaction between TGA2.2 and GRX480 in protoplasts

A modified two hybrid interaction assay was carried out in BY2 protoplasts, to determine the levels of interaction between TGA2.2 and GRX or GDM. The TGA2.2 (a homologue of TGA2 from N. tabacum) was expressed as a fusion protein with the Herpes simplex virus protein derived VP16 activation domain, while both GRX and GRX-DM were expressed as fusion proteins to the yeast derived Gal4 binding domain (Gal4BD). The expression of the effector proteins was driven by the HBT promoter. These interacting partners were co-transformed with a promoter-reporter construct containing the uidA gene (ß-glucoronidase or GUS) driven by a 4x-Gal4 promoter derived from the yeast system.

In a control transformation, only the GRX/Gal4BD and reporter constructs were transformed into BY2 protoplasts. The strength of the interaction could be measured quantitatively by the GUS assay. (Figure 6.4A)

On the average, the reporter assay measurements showed that GRX and TGA2.2 interact, with the GUS activity being 5 times more than the background due to GRX alone. GRX-DM on the other hand showed about a 2-fold weaker interaction with TGA2.2 compared to the wild type GRX. This result was repeated at least twice. In other control assays, TGA2.2VP/reporter alone, Gal4BD/reporter alone and G4BD-GRX/reporter alone showed only background activity, compared with the activity due to the interaction between GRX and TGA2.2 in the same assay (Abdallat, 2004). These results gave a hint that the active site cysteine residues of GRX may be important in their ability to interact with TGA transcription factors.

These results nevertheless assume that the levels of expression of both GRX and GRX-DM proteins are the same in the protoplast cells. This could nevertheless not be verified here, since there was no available antibody against the GRX.

(I)

Figure 6.4A. Interaction between TGA2.2 and GRX480 in protoplasts.

(I)An illustration of the two hybrid assay in protoplasts, where interaction between VP16AD-TGA2.2 and Gal4BD-GRX, is a prerequisite for transactivation, and GUS reporter gene expression in this assay.

(II)Plasmid constructs (above) and GUS activity (below) as a result of the interaction between the effectors.

10 µg of BT10 Gal4 GUS reporter gene plasmid were co-transfected with a total of 25 µg of effector plasmids (i.e. 12.5µg At1g28480 + 12.5 µg HBT-TGA2.2VP16 or 12.5µg HBTL/GAL4BD-GDM + 12.5 µg HBT-TGA2.2VP16 effector plasmids) into BY-2 protoplasts. After an overnight expression, the cells were assayed for ß-glucuronidase enzyme activity measured in [pmole/min/µg protein]. The standard deviation in each case is a result of three independent transformations.

6.4.2 GRX480 suppresses transcription from promoters containing the as-1 element in protoplasts

The effect of GRX and GRX-DM (GDM) on as-1::GUS inducible gene expression was also investigated in BY-2 protoplasts. The effector proteins GRX and GRX-DM were expressed in protoplasts, driven upstream by the HBT chimeric promoter. These were co-transformed with a promoter-GUS construct, which contains the as-1 element as the only regulatory element. A control plasmid containing only the HBTL promoter was also co-transformed with the as-1::GUS reporter construct. The relative GUS activity was measured after expression.

In every case observed over three times, and with independent plasmid preparations (also reported by Abdallat, 2004), the as-1::GUS reporter activity was always repressed in the presence of GRX. This was nevertheless not the case in the presence of GRX-DM, where the as-1::GUS reporter activity remained normal. (Figure 6.4B).

TATA GRX TATA

TATA GDM

TATA uidA

Effector plasmids

HBT

HBTlGRX HBT

HBT

HBT/GDM HBT

as-1::GUS as-1 Reporter

plasmid

0 200 400 600 800 1000

GUS activity

GDM - GRX

EFFECTORS

Figure 6.4B. GRX480 but not the double mutant represses the activity of the as-1 element in protoplasts.

Plasmid constructs are shown (above) and GUS reporter activity (below) as a result of the interaction between the effectors.

10 µg of as-1::GUS reporter plasmid were co-transfected with either 25 µg HBTL, 25 µg HBTL/GRX480 or 25 µg HBTL/GDM effector plasmids into BY-2 protoplasts. After overnight expression, the cells were assayed for β-glucuronidase enzyme activity measured in [pmole/min/µg protein].

The standard deviation in each case is a result from 3 independent transformations.

The effect observed on the as-1::GUS reporter activity was further investigated to find out how specific it was. Was it a general effect which GRX480 had on all promoters or was this a specific effect on a promoter containing the as-1 element as the only regulatory element. For this purpose, a control experiment was carried out using a construct containing the yeast Gal4 promoter, driving a downstream GUS reporter. These were co-transformed into BY-2 protoplasts, with constructs which lack or express the GRX, driven by the HBT promoter. The Gal4::GUS promoter reporter activity remained unchanged in the presence of GRX (Figure 6.4C), further solidifying the observation that the GRX protein represses only as-1::GUS mediated expression in protoplast.

Reporter

plasmid BT10 Gal4 GUS ^{4x UASGAL4} TATA GUS

TATA At1g28480 HBT TATA

HBTlGRX HBT

Effector plasmids HBT

GUS ASSAY

0 5 10 15 20 25 30 35 40

TREATMENTS

GUS Activity

-- GRX Effector

n=4

Figure 6.4C. GRX480 does not repress promoters that lack the as-1 element.

Plasmid constructs are shown (above) and GUS reporter activity (below) as a result of the interaction between the expressed GRX480 effector and the promoter reporter construct, after co-transformation into BY2protoplast cells.

10 µg of BT10 Gal4 GUS reporter gene plasmid were co-transfected with 25 µg HBTL or 25 µg HBTL/GRX480 effector plasmids into BY-2 protoplasts. After an overnight expression, the cells were assayed for ß-glucuronidase enzyme activity measured in [pmole/min/µg protein]. The standard deviation in each case is a result of four independent transformations.