Table 1.

Synergism among Acidic Activators and Class B HSFs

No.	Activator	GUS Activity (Rfu)a	Synergism (Fold Activity)
Group A: Acidic activators tested with LpHsfB1
1	LpHsfA1	45.8 ± 0.9	7.8×
2	LpHsfA2	42.7 ± 1.0	5.0×
3	HsfA1xGal4AD	44.3 ± 4.5	10.0×
4	HsfA1xVP16AD	51.0 ± 7.3	2.5×
Group B: Class B HSFs tested with LpHsfA1
5	HsfB1xNtB1CTD	52.0 ± 2.5	12.5×
6	HsfB1xGmB1CTD	64.4 ± 1.1	9.5×
7	HsfB1xAtB1CTD	5.0 ± 0.7	NDb

To evaluate the synergistic activation of GUS expression, tobacco protoplasts were transformed and tested in the four standard combinations of reporter and activators (Figure 1B, arrows). Results are given only for combination 4 (i.e., 0.25 μg of acidic activator coexpressed with 0.75 μg of class B HSF). In group A, 0.75 μg of LpHsfB1 was cotransformed with 0.25 μg of acidic activator, for example, tomato HsfA1 and HsfA2 as well as the two indicated fusion proteins of tomato HsfA1 (amino acids 23 to 394; see arrow in Figure 1A) with the yeast Gal4 and the viral VP16 activation domains. In group B, 0.25 μg of LpHsfA1 was cotransformed with 0.75 μg of the indicated fusion proteins of LpHsfB1 (amino acids 1 to 198) with the CTDs of tobacco (Nt), soybean (Gm), and Arabidopsis (At) HsfB1. For details of the fusion constructs, see Supplemental Table S1 online. Calculation of synergism is based on the following formula (exemplified for GUS activities in samples 1, 2, and 6 in Figure 1B): GUS(6) − GUS(1)/GUS(2) − GUS(1) = 11-fold.

^aRfu, relative fluorescence units.

^bND, not determined.