Set 531 to win following Australia's declaration before play resumed on Sunday's day four, England have reached 4-143 at tea.
Debutant Ben Stokes is yet to score and 22-year-old Root is on 66 after Pietersen was out to his long-time tormentor Peter Siddle for 53.
Ian Bell, England's other star batsman, also fell in the middle session for six.
Mitchell Johnson claimed his eighth victim of the match when Alastair Cook was caught at deep backward square leg for one in the second over of the innings.
With nine wickets in Australia's victory in last month's Brisbane Test and 7-40 in the first innings in Adelaide, tearaway Johnson is in the form of his life at 32.
Cook plundered Australia's attack for 766 runs at 127.66 in the 2010/11 series, but so far this time has tallied 82 at 20.50.
His choice of shot on Sunday, opting to hook the fastest bowler in world cricket so early in the innings, was unusual and an indication the tourists are looking rattled.
Siddle removed Michael Carberry (14), who was also caught on the leg-side boundary.
The paceman also claimed Pietersen's wicket for the second time in the match and the ninth time in his career, as the tall right-hander inside-edged onto his stumps at 3-131.
Pietersen's 99-ball innings included two fours and three sixes and was a more patient knock than his four in the first dig, when he arrogantly tried to clip a ball through short mid-wicket on the charge with two fielders placed there for the shot.
Three consecutive maidens preceded Pietersen's wicket as Australia built pressure on Sunday.
Root and Pietersen added 111 for the third wicket on Sunday after England had been rocked by two early wickets.
Twelve runs after Pietersen's exit, Bell was out in comical circumstances for a player of his ability as the right-hander hit a rank full toss from legspinner Steve Smith to mid-on.
Australia declared on Sunday morning on their overnight score of 3-132 in their second innings.
England trailed by 398 runs on the first innings after they were bowled out for 172, their third successive sub-200 total in the series.
The world record for the highest successful fourth-innings run chase is the 7-418 by West Indies against Australia in Antigua in May 2003.