Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana

Abstract

Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

Keywords: HR, hypersensitive response; Hsp, heat shock protein; Hsp90; Nicotiana benthamiana; PCR, polymerase chain reaction; RAR1, required for Mla12 resistance 1; RT-PCR, reverse transcription-polymerase chain reaction; SGT1 suppressor-of-G2-allele-of-skp1; TTSS, type III secretion system; VIGS, virus-induced gene silencing.; plant immunity; qRT-PCR, quantitative real time polymerase chain reaction; required for Mla12 resistance 1; suppressor-of-G2-allele-of-skp1; virus-induced gene silencing.

Figure 1.

Creation of gene-silenced plants. Total RNA was isolated from Control (PVX), Hsp90-(PVX:Hsp90), RAR1-(PVX:RAR1) and SGT1-(PVX:SGT1) silenced plants. Expression values of Hsp70, Hsp90, RAR1 and SGT1 were estimated by qRT-PCR, and expressed as [Qty] after normalization with actin. Values represent the means and SD from triplicate experiments. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05).

Figure 2.

Effect of Hsp90, RAR1 and SGT1-silencing on bacterial wilt disease by inoculation with R. solanacearum. Control (PVX), Hsp90- (PVX:Hsp90), RAR1- (PVX:RAR1) and SGT1- (PVX:SGT1) silenced plants were infiltrated with R. solanacearum. (A) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (open squares) or silenced (solid squares) plants. Asterisks denote values significantly different from those ofcontrol plants (*; P < 0.05, t-test). (B) Characteristic symptoms in control and silenced plants. Photograph was taken 12 d after inoculation with R. solanacearum.

Figure 3.

Growth of Ralstonia solanacearum in Hsp90, RAR1 and SGT1-silenced plants Control (PVX), Hsp90- (PVX:Hsp90), RAR1- (PVX:RAR1) and SGT1- (PVX:SGT1) silenced plants were infiltrated with R. solanacearum (10⁸ CFU/ml). Bacterial population was determined by plating at specified time points. Values are means of 4 replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX controls (*; P < 0.05, t-test).

Figure 4.

Acceleration of PR genes expression in Hsp90 and SGT1-silenced plants in response to Ralstonia solanacearum infection. Total RNA was isolated from control (PVX) and Hsp90 (PVX:Hsp90) and SGT1 (PVX:SGT1)-silenced plants inoculated with R. solanacearum (10⁸ CFU/ml). Relative expression of PR-1a and PR-4 transcripts were normalized with actin and calculated as relative to the non-treated control. Values represent the means and SD from triplicate experiments. Asterisks denote valuessignificantly different from empty PVX controls (*; P < 0.05).